Cell Fusions: Regulation and Control

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Cell Fusions

Lars-Inge Larsson Editor

Cell Fusions Regulation and Control

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Editor Lars-Inge Larsson Faculty of Life Sciences Division of Cell Biology University of Copenhagen Gronnegaardsvej 7 1870 Frederiksberg C Denmark [email protected]

ISBN 978-90-481-9771-2 e-ISBN 978-90-481-9772-9 DOI 10.1007/978-90-481-9772-9 Springer Dordrecht Heidelberg London New York © Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The fusion between the sperm and the egg marks the beginning of life and a number of subsequent cell fusions are needed to form the placenta, the skeleton, the skeletal muscles and part of our immune defence system. Cell fusions are also needed during adulthood, e.g. to regulate our calcium homeostasis. Apart from the fusion between the sperm and the egg, all of these fusions occur between cells of the same type. Such homotypic fusions generate multinucleated cells that have lost the ability to propagate but which, through their large sizes, are more powerful than single cells. This is well illustrated by skeletal muscle fibers, which are derived from fusions of multiple stem cells and can reach 1/2 m in length. Similarly, giant cells of the immune system and osteoclasts, degrading bone tissue, reach their powerful abilities of phagocytosis from the fusion of smaller progenitor cells (macrophages/monocytes). Heterotypic fusions occur between cells of different lineages and may result in cells with proliferative ability. If such fusions occur between haploid gametes, an embryo results. However, if heterotypic fusions occur between diploid or aneuploid cells, the mitotic spindle apparatus may encounter problems of sorting the supernumerary chromosomes. This may result in genomic instability and in cell death or cancer. Accordingly, cell fusion is a process that should be entered with even more care than marriage since a divorce (defuse?) is impossible. Nevertheless, as we shall learn from this volume, heterotypic cell fusions may play roles in repair of damaged tissues and may be put to use for production of monoclonal antibodies and for boosting the immune system against cancer cells. In this book, we learn of mechanisms regulating and controlling cell fusions. An important aspect in normal physiology is the matter of self recognition mechanisms, which ensure that, in the healthy individual, homotypic fusions predominate. As we shall see from the contributions on fertilization, placentation, macrophages/osteoclasts and skeletal muscle development, multiple mechanisms are involved. At the turn of the millennium, a new player entered the fusion game. It emerged that we have adopted certain fusion genes from ancient viral infections and have used them in the cell fusion machinery. The initial chapters in this book are therefore devoted to an in-depth discussion of the viral fusion machinery and how cells may utilize these proteins for their own use. This is intellectually and conceptually a challenging area in modern biology since it shortcuts traditional Darwinian

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evolution with infections. Astonishingly, similar but distinct retroviral genes have been adopted by primates, rabbits, mice and, possibly, sheep, for cell fusions. Mouse knock-down experiments indicate that a mouse endogenous retroviral gene is needed for placentation, probably because it made pre-existing fusion genes redundant. In addition to the new “viral” players, model organisms like Drosophila and Caenorhabditis have made considerable impact on our understanding of the cell fusion machinery. These mechanisms as well as methodological aspects have been dealt with in an excellent recent volume edited by Chen and are referred to here in the context of homotypic cell fusions and in the FuRMAS structure in Chapter 6. In the final chapters, we address the question of heterotypic cell fusions. Such fusions seem to gain momentum as main players in tissue repair and stem cell therapy as well as in immune therapy of tumors. In both of these settings heterotypic fusions appear to do good whereas the backside of the coin, cancer, is addressed in the last two chapters. As captain on this voyage I have had the pleasure to be able to recruit a crew consisting of the most eminent specialists in this evolving field. They have helped set sails and chartered our path from viruses over normal development to cancer and treatment. My sincerest thanks go to them, to the representatives of the shipping company, Meran Owen and Tanja van Gaans, who patiently awaited the sight of sails against the horizon, and to my family; Benedikte, Nina and Blackie, who have served as “stowaways” on board the vessel whilst the log-book was edited. Lars-Inge Larsson

Contents

1 Regulation and Control of Cell–Cell Fusions . . . . . . . . . . . . . Lars-Inge Larsson

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2 Retroviruses and Cell Fusions: Overview . . . . . . . . . . . . . . . Anders L. Kjeldbjerg, Shervin Bahrami, and Finn Skou Pedersen

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3 Retroviral Membrane Fusions: Regulation by Proteolytic Processing and Cellular Factors . . . . . . . . . . . . . . . . . . . . Yoshinao Kubo 4 A Comparative Portrait of Retroviral Fusogens and Syncytins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Philippe Pérot, Cécile Montgiraud, Dimitri Lavillette, and François Mallet 5 Syncytins: Molecular Aspects . . . . . . . . . . . . . . . . . . . . . Hungwen Chen and Mei-Leng Cheong 6 Role of the Actin Cytoskeleton Within FuRMAS During Drosophila Myoblast Fusion and First Functionally Conserved Factors in Vertebrates . . . . . . . . . . . . . . . . . . . Susanne-Filiz Önel, Christine Dottermusch, Angela Sickmann, Detlev Buttgereit, and Renate Renkawitz-Pohl 7 Role of CD9 in Sperm-Egg Fusion and Its General Role in Fusion Phenomena . . . . . . . . . . . . . . . . . . . . . . . . . . Natsuko Kawano, Yuichiro Harada, Keiichi Yoshida, Mami Miyado, and Kenji Miyado

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8 Gamete Binding and Fusion . . . . . . . . . . . . . . . . . . . . . . Young-Joo Yi, Shawn W. Zimmerman, and Peter Sutovsky

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9 Mechanisms Regulating Human Trophoblast Fusion . . . . . . . . Berthold Huppertz and Martin Gauster

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Contents

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Macrophage Fusion: The Making of a New Cell . . . . . . . . . . . Agnès Vignery

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Molecules Regulating Macrophage Fusions . . . . . . . . . . . . . Takeshi Miyamoto and Toshio Suda

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Current Progress Towards Understanding Mechanisms of Myoblast Fusion in Mammals . . . . . . . . . . . . . . . . . . . Grace K. Pavlath

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The Endogenous Envelope Protein Syncytin Is Involved in Myoblast Fusion . . . . . . . . . . . . . . . . . . . . Bolette Bjerregaard, Jan Fredrik Talts, and Lars-Inge Larsson

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Cell Fusion and Stem Cells . . . . . . . . . . . . . . . . . . . . . . Alain Silk, Anne E. Powell, Paige S. Davies, and Melissa H. Wong

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Cell Fusion and Dendritic Cell-Based Vaccines . . . . . . . . . . . Jianlin Gong and Shigeo Koido

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Cancer Cell Fusion with Myeloid Cells: Implications for Energy Metabolism in Malignant Hybrids . . . . . . . . . . . . Rossitza Lazova, Ashok K. Chakraborty, and John M. Pawelek

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Cell–Cell Fusions and Human Endogenous Retroviruses in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reiner Strick, Matthias W. Beckmann, and Pamela L. Strissel

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Shervin Bahrami Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark, [email protected] Matthias W. Beckmann Department of Gynaecology and Obstetrics, Laboratory for Molecular Medicine, University-Clinic Erlangen, D-91054 Erlangen, Germany, [email protected] Bolette Bjerregaard Division of Cell Biology, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark, [email protected] Detlev Buttgereit Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany, [email protected] Ashok K. Chakraborty Department of Dermatology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520-8059, USA, [email protected] Hungwen Chen Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, [email protected] Mei-Leng Cheong Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, [email protected] Paige S. Davies Department of Dermatology, Knight Cancer Institute, Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR 97239, USA, [email protected] Christine Dottermusch Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany, [email protected] Martin Gauster Institute of Cell Biology, Histology and Embryology, Medical University Graz, Austria, [email protected] Jianlin Gong Department of Medicine, Boston University Medical School, Boston, MA 02118, USA, [email protected]

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Yuichiro Harada Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan, [email protected] Berthold Huppertz Cell Biology, Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, 8010 Graz, Austria, [email protected] Natsuko Kawano Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan, [email protected] Anders L. Kjeldbjerg Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark, [email protected] Shigeo Koido Department of Internal Medicine, Institute of Clinical Medicine and Research, The Jikei University School of Medicine, Tokyo, Japan, [email protected] Yoshinao Kubo Department of AIDS Research, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan, [email protected] Lars-Inge Larsson Division of Cell Biology, Anatomy, Cell Biology and Genetics, IBHV, Faculty of Life Sciences, University of Copenhagen, 1809 Frederiksberg C, Denmark, [email protected] Dimitri Lavillette INSERM, U758, 69007 Lyon, France, [email protected] Rossitza Lazova Department of Dermatology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520-8059, USA, [email protected] François Mallet Ecole Normale Supérieure de Lyon, 69007 Lyon, France, [email protected] Mami Miyado Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan, [email protected] Kenji Miyado Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan, [email protected] Takeshi Miyamoto Department of Orthopedic Surgery, Keio University School of Medicine, Tokyo 160-8582, Japan; Keio Kanrinmaru Project, Keio University School of Medicine, Tokyo 160-8582, Japan, [email protected] Cécile Montgiraud Université de Lyon, UCB-Lyon1, IFR128, 69007 Lyon, France, [email protected] Susanne-Filiz Önel Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany, [email protected] Grace K. Pavlath Department of Pharmacology, Emory University, Atlanta, GA 30322, USA, [email protected]

Contributors

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John M. Pawelek Department of Dermatology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520-8059, USA, [email protected] Finn Skou Pedersen Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark, [email protected] Philippe Pérot Laboratoire Commun de Recherche Hospices Civils de Lyon – bioMérieux, Cancer Biomarkers Research Group, Centre Hospitalier Lyon Sud, 69495 Pierre Bénite cedex, France, [email protected] Anne E. Powell Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239, USA, [email protected] Renate Renkawitz-Pohl Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany, [email protected] Angela Sickmann Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany, [email protected] Alain Silk Department of Dermatology, Knight Cancer Institute, Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR 97239, USA, [email protected] Reiner Strick Department of Gynaecology and Obstetrics, Laboratory for Molecular Medicine, University-Clinic Erlangen, D-91054 Erlangen, Germany, [email protected] Pamela L. Strissel Department of Gynaecology and Obstetrics, Laboratory for Molecular Medicine, University-Clinic Erlangen, D-91054 Erlangen, Germany, [email protected] Toshio Suda Department of Cell Differentiation, Keio University School of Medicine, Tokyo 160-8582, Japan, [email protected] Peter Sutovsky Division of Animal Science, University of Missouri-Columbia, Columbia, MO 65211, USA; Departments of Obstetrics, Gynecology & Women’s Health, University of Missouri-Columbia, Columbia, MO 65211, USA, [email protected] Jan Fredrik Talts Division of Cell Biology, Faculty of Life Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark, [email protected] Agnès Vignery Departments of Orthopaedics and Cell Biology, Yale School of Medicine, New Haven, CT 06510, USA, [email protected] Melissa H. Wong Department of Dermatology, Knight Cancer Institute, Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR 97239,

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USA; Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, OR 97239, USA, [email protected] Young-Joo Yi Division of Animal Science, University of Missouri-Columbia, Columbia, MO 65211, USA; Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, South Korea, [email protected] Keiichi Yoshida Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan, [email protected] Shawn W. Zimmerman Division of Animal Science, University of Missouri-Columbia, Columbia, MO 65211, USA, [email protected]

Chapter 1

Regulation and Control of Cell–Cell Fusions Lars-Inge Larsson

Abstract Cell fusions are important to fertilization, fetal development and homeostasis. Retroviruses infect cells by fusing with them and recent data suggest that mammals may have adopted the retroviral fusion machinery for their own use and combined it with numerous other factors controlling cell specificity and self recognition, motility-migration, filopodia formation, signaling and membrane organization. The multifactorial aspect of the process is suggested to create a certain amount of wobble so that, in specific disease states, heterotypic cell fusions may occur. Professional phagocytes, which specialize in recognizing and eliminating injured or dying cells, appear to be particularly prone to fusion. Such fusions may be useful for repairing damaged tissues and have been harnessed in immune therapy against cancer but may also contribute to disease development and progression. Keywords Cancer · cell-cell fusion · HERV · microdomains · retrovirus · syncytin Abbreviations ADAM ASCT BMDC CD CRISP DC-STAMP env ERM F-actin FuRMAS GCM HERV

A disintegrin and a metalloprotease Alanine, serine and cysteine selective transporters Bone marrow-derived cells Cluster of differentiation Cysteine-rich secretory protein Dendritic cell-specific transmembrane protein Envelope Ezrin-radixin-moesin Filamentous (polymerized) actin Fusion restricted myogenic adhesive structure Glial cells missing Human endogenous retrovirus

L.-I. Larsson (B) Division of Cell Biology, Anatomy, Cell Biology and Genetics, IBHV, Faculty of Life Sciences, University of Copenhagen, 1809 Frederiksberg C, Denmark e-mail: [email protected] L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_1, C Springer Science+Business Media B.V. 2011

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LTR MFR NSF PCD PKA RANK RANKL SIRP-α SNAP SNARE t-SNARE v-SNARE

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Long terminal repeat Macrophage fusion receptor N-ethylmaleimide-sensitive factor Programmed cell death Protein kinase A Receptor activator of NFκ B RANK ligand Signal regulatory protein-alpha Soluble NSF attachment protein SNAP receptors Target-SNARE Vesicle-SNARE

Cell–cell fusions are important to the beginning of life, to the sculpturing of the new individual and to the maintenance of muscle strength, immune responses and calcium homeostasis in the adult. Additionally, cell fusions participate in tissue repair and may be important to cancer development, progression and therapy. Not surprisingly, fusions must be tightly controlled in order to ensure that only the appropriate cells fuse with their right partners at the right time. Much knowledge of such regulation comes from studies of model organisms like Caenorhabditis elegans and, as so many times before, the little worm and its associates have provided us with a wealth of important data. Much of this, as well as many methodological aspects has been summarized in an admirable recent volume, edited by Chen (2008). In the present volume, we interrogate what mechanisms that may be important to cell fusions in mammals and what mechanisms that may be shared with lower organisms, including viruses. Additionally, the importance of cell fusions to the pathogenesis as well as treatment of diseases is addressed. Retroviruses, which infect cells by fusing with them, are discussed first. Retroviral infections often induce fusions between cells in the infected individual and recent data suggest that multiple mammalian species may have adopted the retroviral fusion machinery for their own use (Chapters 2 and 4). Interestingly, different species seem to have adopted different, but basically similar, fusion machineries from different types of retroviruses. In doing so they seem to have bypassed millions of years of evolution and have taken advantage of the higher mutation rates found in viral genomes. For this reason, mammalian cells may possess cell–cell fusion mechanisms unrelated to those detected in lower invertebrates. Retroviral membrane fusions are subject to a number of controlling factors as reviewed in Chapter 3. Some of these relate to proteolytic processing e.g. of the cytoplasmic tail and others to insertions into rafts/microdomains and interactions with cell adhesion molecules and the actin cytoskeleton – regulatory events also recognized in mammalian cell–cell fusions. In the process of forming new virus particles, retroviruses get their genetic information integrated into the host DNA. It is, indeed, a humbling thought that over 8% of our genome represents identifiable remnants of ancient infections. Most of this lies in ruins because our cells have found little use for the information

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encoded by viral genomes. Thus, most of these sequences have mutated or undergone other changes resulting in transcriptionally inactive mumbo-jumbo. However, certain sequences, including envelopes (env), which encode the viral proteins which makes fusion – and, hence, infection – possible, have been highly conserved, suggesting that our bodies have adopted them to specific functions (Chapters 2 and 4). At the turn of the millennium, two reports provided data suggesting that the retrovirally encoded transmembrane protein, syncytin (later renamed syncytin-1) could be important to trophoblast cell–cell fusions, which are needed for forming a functioning placenta in primates, including man (Blond et al. 2000, Mi et al. 2000). Syncytin is an env protein, which was derived from a retroviral infection that entered our genome over 25 million years ago and which has been highly conserved (Chapter 4). The fusion mechanism employed by syncytin is very similar to that used by retroviruses for infection and may be inhibited by specific peptide sequences interfering with this mechanism (Chapters 2 and 5). Later, syncytin was implicated in mediating fusions between cancer cells and endothelial cells, between cancer cells themselves and in fusions between skeletal muscle-forming myoblasts (Bjerregaard et al. 2006, Strick et al. 2007, Chapters 13 and 17). Recent data also indicate an involvement of syncytin in fusions needed to form osteoclasts (Soe K, Andersen TL, HoboltPedersen AS, Bjerregaard B, Larsson LI, Delaisse JM: submitted). Thus, syncytin may possibly participate in multiple types of cell–cell fusions in humans but, as we shall see shortly, there are many players in this game. We believe that a plethora of molecules are important for determining diverse aspects of cell fusions, including specific cell migration, cell–cell recognition, signaling events, cytoskeletal and membrane microdomain organization, filopodia/nanotube formation, and, eventually, the membrane fusion event itself. In fact, syncytin is the best candidate for a true cell–cell fusogen that, so far, has been detected in primate cells (Oren-Suissa and Podbilewicz 2007). Other retroviral envelope proteins, encoded for by other endogenous retroviral sequences, are expressed in other mammals, including rabbits, sheep and mice (Dunlap et al. 2006, Dupressoir et al. 2005, Heidmann et al. 2009). Importantly, a knock-down of murine syncytin-A results in defect placentation and prevents successful reproduction (Dupressoir et al. 2009). If confirmed, this seems to suggest that the reproduction of modern mice depends upon an accidental retroviral infection acquired some 20–25 million years ago! One way to reconcile this with traditional evolution is to assume that the accidentally acquired viral env gene was so much more efficient in fusing cells so that it made a preexisting endogenous fusogen redundant. So far, the syncytin family of proteins, which, in man, includes syncytin-1, -2 and the fusogenic retroviral envelope protein P(b) are the only bona fide membrane fusogens detected (Chapters 2 and 4). The founding member, syncytin-1 has been implicated in a number of diseases including placental disorders, multiple sclerosis and cancer (Chapters 4, 5, 9 and 17). Expression of syncytin-1 and -2 are subject to a number of regulations as described in Chapters 4 and 5. Especially, the transcription factor GCM1 (glial cells missing) appears to play an important role in such regulation and GCM1 is regulated by cAMP and protein kinase A (PKA) – factors known to regulate trophoblast cell fusions (Chapter 5). Importantly, the degree of methylation of the syncytin-1

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and -2 promoters affects their regulation by GCM1 and the possibility that GCM1 itself may affect methylation in the 5 -LTR of the syncytin-2 promoter in MCF-7 breast cancer cells is raised in Chapter 5. Syncytins may represent cell-unspecific fusogens with the specific aim of making membranes fuse, whereas other proteins guarantee the necessary cell-specificity and/or may serve migratory, scaffolding or signaling roles. The syncytins possess receptors, which are needed for activating the fusion machinery itself but which also represent rather ubiquitously expressed transporters. Thus, the receptors confer little cell-specificity but may form part of a larger protein scaffold, which contains other regulators conferring cell specificity. Additionally, as pointed out in Chapter 4, syncytins have many other and potentially non-redundant functions and it is possible that these molecules have many more surprises in store for us! Membrane fusion is triggered upon binding of syncytin-1 to its receptors, the neutral amino acid transporters ASCT1 and 2. Whether such binding also may trigger other cellular events remains to be seen. The situation with respect to fusion of intracellular membranes is quite different. Here, a complicated machinery of proteins controlling the v- and the t-SNARE system have been unraveled and have been found to constitute targets for various poisons and medications (Rothman 1994). This machinery controls both membrane fusions and its organelle specificity. It seems that we have a long way to go before similar models can be constructed for cell–cell fusions. Against this background, a volume such as the present may serve a useful purpose by emphasizing differences but also similarities in the machinery that make some cells fuse with their appropriate partners. As hinted to above, it is likely that cell–cell fusions depend upon multiple molecular interactions. In fact, membrane microdomains and the actin cytoskeleton as well as cell adhesion factors play decisive roles in cell–cell fusions. Thus, again we have a parallel to the mechanisms regulating retroviral fusions as discussed in Chapter 3. In Drosophila a specific structure, the FuRMAS (fusion restricted myogenic adhesive structure) – a synapse-like structure which may integrate cell fusion with cell adhesion, signaling and actin regulation exists (Chapter 6). As also discussed in this chapter, similar mechanisms may operate in e.g. zebrafish and mice. In fact, synapse-like structures, incorporating activated signaling molecules, F-actin and ezrin-radixin-moesin (ERM) proteins, which link transmembrane proteins to the actin cytoskeleton, are also observed at sites of contact between breast cancer cells known to undergo spontaneous fusion (Larsson, unpublished data). Much data suggests that the plasma membrane is subdivided into several distinct microdomains or rafts, which i. a. differ in protein and cholesterol composition (Simons and Ikonen 1997). Such rafts have been implicated in viral infection (Chapter 3) as well as in cell–cell recognition and signaling. Tetraspanins are proteins known to participate in the organization of the plasma membrane into microdomains and some tetraspanins, in particular CD9 and CD81, have been implicated in regulation of cell–cell fusions (Duelli et al. 2005, Miyado et al. 2000, Parthasarathy et al. 2009, Weng et al. 2009). CD 9 and CD81 may also bind to the actin cytoskeleton via EWI and ERM proteins (Sala-Valdés et al. 2006). Interestingly, recent data suggest that CD9 increases GCM1 expression via the

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cAMP/PKA signaling pathway, resulting in the increase in syncytin expression (Muroi et al. 2009). The roles of CD9 and the interacting protein, IZUMO with respect to fertilization are discussed in Chapters 7 and 8. Interestingly, recent data show that small CD9-containing vesicles, resembling exosomes, can be released from eggs. Moreover, such exosome-like vesicles may induce fusion between spermatozoa and eggs lacking CD9, which otherwise are refractory to fusion. These data reinforce the central role of CD9 in sperm-egg cell fusions and emphasize that analogous functions may exist in other cell–cell fusion systems. Interestingly, CD9 and CD81 positive exosomes have been implicated also in cancer–cancer cell fusions in an in-vitro model (Duelli et al. 2005) and in HIV-induced syncytia formation (Weng et al. 2009). Exosomes are potentially interesting mediators of fusions as they are released from cells and, at least in theory, could fuse receptive bystander cells. This mechanism would be analogous to the viral “fusion from without”, discussed in Chapter 2. Much more studies are needed before we know whether exosomes indeed participate in “fusion from without” mechanisms under physiological circumstances. As with all other cell–cell fusions, sperm-egg cell fusions most likely depend upon multiple interacting proteins. Additional molecules that may participate in sperm-egg fusions, including v- and t-SNARE-like proteins, ADAM, CRISP and MN9, are discussed in Chapters 7 and 8. Overall, Chapters 6–8 provide a useful input into the complexities and potential redundancies that may exist in cell– cell fusions and underline the importance of membrane organization and protein scaffolding for fusions. Chapter 9 emphasizes the potential role of molecules involved in programmed cell death (PCD) like caspase-8 and phosphatidylserine in trophoblast cell fusions. Phosphatidylserine is a membrane phospholipid, which normally faces the cytosolic side of the plasma membrane. In cells destined to undergo PCD, phosphatidylserine is exposed to the outside of the plasma membrane. This is considered an “appetizer” for phagocytosis so that the dying cell can be cleared without traces or inflammatory reactions (Zhou 2007). Interestingly, phosphatidylserine is also flipped out on the outside of the plasma membrane during cell–cell fusion and experiments show that antibodies binding to phosphatidylserine can inhibit cell–cell fusion (reviewed in Chapter 9). Intriguingly, similarities between cell–cell fusions and PCD do not end here. A PCD-inducing enzyme, caspase-8, may also play a role in cell–cell fusions in the placenta. These intriguing data are placed in a comprehensive context of other molecules implicated in trophoblast cell–cell fusions and potential methodological issues regarding e.g. syncytin antibody specificities and labeling preferences in Chapter 9. Chapters 10 and 11 discuss mechanisms which regulate fusions between macrophages/monocytes to form giant cells and osteoclasts, including factors like MFR/SIRPα-CD47, CD200-CD200R, CD44, DC-STAMP and RANK/RANKL. These mechanisms are of enormous potential not least in view of the frequency of osteoporosis – a disease characterized by osteoclast hyperactivity. We learn how signaling factors like cytokines elicit intracellular signaling cascades, which ultimately stimulate osteoclast formation and bone resorption. This is guided by self recognition factors, which make the fusions cell-specific. However, osteoclasts

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may also fuse with neoplastic cells like myeloma cells (Andersen et al. 2007). In Chapter 16, Lazova et al. gives numerous examples of how macrophage-like cells may fuse with cancer cells and provide new data on the potential importance of autophagy – an apoptosis-related path to cell death – for melanoma cell fusions. This disease-associated promiscuity of macrophage-cancer cell fusions could point to some kind of wobble mechanism in the specificity of cell–cell fusions and, potentially, macrophage-like, bone marrow-derived cells (BMDCs) may travel round the body, willing to fuse with and, thus, repair cells facing demise. Incidentally, this draws some interesting parallels to Chapter 9 regarding the potential role of factors regulating PCD in cell–cell fusions. Notably, phosphatidylserine, exposed on the surface of apoptotic cells attracts macrophages bearing specific receptors for this membrane phospholipid (Zhou 2007). The role of macrophages and other BMDCs in tissue repair and maintenance in the context of stem cell therapy is dealt with in Chapter 14. Again, the question arises as to how heterotypic cell–cell fusions may become triggered by diseases. Fortunately for our good looks, heterotypic fusions probably only occur in disease states and the intricate cell–cell recognition mechanisms described in Chapters 10 and 11 would normally ensure appropriate cell-specificities of fusions. As emphasized above, cell–cell fusions are probably regulated by both cell-specific and cell-unspecific factors and changes in the expression profiles of these factors may result in cell–cell liaisons, which normally are forbidden. The complexities and intricacies of cell fusions are well illustrated by fusion of myoblasts to generate skeletal muscle fibers (Chapters 6, 12 and 13). As presented by Grace Pavlath in Chapter 12, changes in myoblast elongation, migration, adhesion and cytoskeleton characterize such fusions and are regulated by a myriad of factors which are being unraveled one by one (see Tables 1–5 in Chapter 12, which elegantly orders all of these factors according to functions during the fusion process). Some of these factors are needed for the fusion per se whereas others regulate muscle differentiation. Such effects are important to distinguish when the complicated machinery of skeletal muscle formation is sorted out. Chapter 13 by Bjerregaard et al. introduces syncytin-1 as a additional new player in myoblast fusions. Chapters 14 and 15 introduce cell fusions in the therapeutic and pathogenetic scenery. In Chapter 14, Silk et al. describes that BMDCs are capable of fusing heterotypically with different types of stem cells and considers the physiologic and pathologic implications of the high degree of fusogenicity of stem cells. Heterotypic fusions between stem cells and other cells will result in hybrids that exhibit nuclear reprogramming in a context that may be useful for normal tissue repair but also may induce chromosomal aberrations, aneuploidy, in activation of tumor suppressor genes or activation of oncogenes and genomic instability in the context of cancer (Duelli et al. 2007). With the exception of the sperm-egg fusion, physiologic cell–cell fusions are homotypic since they occur between cell types of the same lineage. However, fusions between different cells – although of the same lineage – are seen during skeletal muscle formation. In Drosophila, such fusions occur between founder cells and fusion competent myoblasts and, in mammals, fusions between myoblasts resulting in nascent myotubes are followed by

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fusions between myotubes and myoblasts. Moreover, during skeletal muscle repair, fusions occur between satellite cells and mature muscle fibers (Chapters 6 and 12). Homotypic fusions usually result in cells, which are multinucleated and without proliferative ability. In the last chapters, heterotypic fusions are considered. Such fusions have been shown to occur in vitro as well as in vivo and incontrovertible evidence for this is presented in Chapters 14, 16 and 17. Heterotypic fusions result in initial formation of heterokaryons, containing two or more nuclei. Such heterokaryons formed by tumor-host cell fusion are able to undergo a subsequent round of cell division which dissolves the individual nuclear envelopes and, thus, mixes the genomes (Mortensen et al. 2004). The ensuing daughter cells are synkaryons, containing one nucleus each, which stores the mixed parental genomes. During division of polyploid cells, formed by fusion, there is a high risk of missorting and damaging of chromosomes and it has been considered that such events may predispose to increased malignancy or to PCD (Duelli et al. 2007). In Chapter 17 Lazova et al. provides numerous examples of how fusions between e.g. macrophages and melanoma cells may lead to hybrids of increased malignancy that express markers characterizing both parental cells. The best example of stable inheritance of parental characteristics by hybrids are hybridoma cells (Kohler and Milstein 1975), which have found widespread use for unlimited production of monoclonal antibodies. Another promising way of exploiting cell fusions is introduced by Gong in Chapter 15, where the production of hybrids between dendritic cells (macrophages which are very good at presenting antigens to the immune system) and cancer cells is described. Such hybrids are used clinically and experimentally for harnessing the immune response towards malignant tumors. The technique has so far produced useful results in animal models and shown promise in clinical settings. In this chapter we depart from the discussion of spontaneous hybrid formation and are taught different methods for creating artificial hybrids between dendritic cells and cancer cells. It may be a sobering thought that hybrid yields can be very low even when all sails are set for inducing them! Moreover, in this setting hybrids do good by instructing the immune system to fight cancer. Tumor cell hybrids are also the focus of the Chapter 17 by Strick et al., who provide a historic overview and lists many examples of different human tumors containing multinucleated cells that may have formed either by fusion or endomitosis (defect cytokinesis). Finally, an overview of the expression of human endogenous retroviral (HERV) sequences, including syncytin, in different human tumors is presented with indications for future studies. Together, the chapters contained in the present volume present an overview of factors that have actions that transcend the cell specificity of cell–cell fusions as well as factors which confer cell specificity. It is hoped that it will enhance understanding of this multifactorial process, which include many components involving cell–cell recognition, migration, signaling, cytoskeletal reorganization, “synapseformation” and lowering the energy level for lipid bilayer fusion. By bringing all of these experts together I am trying to propagate the theory that we should look for both similarities and dissimilarities between the fusion processes in different cell types. Finally, the final chapters also address the consequences of cell–cell fusion

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L.-I. Larsson

as a means to repair damaged tissue, as a possible danger for carcinogenesis and cancer progression but also as an aid to immune therapy of cancer, at least when exercised under controlled conditions. Acknowledgements Work by the author presented herein was supported by the Danish MRC, FTP and Lundbeck foundation.

References Andersen TL, Boissy P, Sondergaard TE et al (2007) Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer-host partnership? J Pathol 211:10–17 Bjerregaard B, Holck S, Christensen IJ et al (2006) Syncytin is involved in breast cancerendothelial cell fusions. Cell Mol Life Sci 63:1906–1911 Blond JL, Lavillette D, Cheynet V et al (2000) An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol 74:3321–3329 Chen EH (2008) Cell fusion. Overviews and methods. Methods Mol Biol 475:1–421 Duelli DM, Hearn S, Myers MP et al (2005) A primate virus generates transformed human cells by fusion. J Cell Biol 171:493–503 Duelli DM, Padilla-Nash HM, Berman D et al (2007) A virus causes cancer by inducing massive chromosomal instability through cell fusion. Curr Biol 17:431–437 Dunlap KA, Palmarini M, Varela M et al (2006) Endogenous retroviruses regulate periimplantation placental growth and differentiation. Proc Natl Acad Sci USA 103:14390–14395 Dupressoir A, Marceau G, Vernochet C et al (2005) Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae. Proc Natl Acad Sci USA 102:725–730 Dupressoir A, Vernochet C, Bawa O et al (2009) Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene. Proc Natl Acad Sci USA 106:12127–12132 Heidmann O, Vernochet C, Dupressoir A et al (2009) Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new “syncytin” in a third order of mammals. Retrovirology 6:107 Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495–497 Mi S, Lee X, Li X, Veldman GM et al (2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403:785–789 Miyado K, Yamada G, Yamada S et al (2000) Requirement of CD9 on the egg plasma membrane for fertilization. Science 287:321–324 Mortensen K, Lichtenberg J, Thomsen PD et al (2004) Spontaneous fusion between cancer cells and endothelial cells. Cell Mol Life Sci 61:2125–2131 Muroi Y, Sakurai T, Hanashi A et al (2009) CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway. Reproduction 138:945–951 Oren-Suissa M, Podbilewicz B (2007) Cell fusion during development. Trends Cell Biol 17:537–546 Parthasarathy V, Martin F, Higginbottom A et al (2009) Distinct roles for tetraspanins CD9, CD63 and CD81 in the formation of multinucleated giant cells. Immunology 127:237–248 Rothman JE (1994) Mechanisms of intracellular protein transport. Nature 372:55–63 Sala-Valdés M, Ursa A, Charrin S et al (2006) EWI-2 and EWI-F link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixin-moesin proteins. J Biol Chem 281:19665–19675

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Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572 Strick R, Ackermann S, Langbein M et al (2007) Proliferation and cell–cell fusion of endometrial carcinoma are induced by the human endogenous retroviral Syncytin-1 and regulated by TGFbeta. J Mol Med 85:23–38 Weng J, Krementsov DN, Khurana S et al (2009) Formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells. J Virol 83:7467–7474 Zhou Z (2007) New phosphatidylserine receptors: clearance of apoptotic cells and more. Dev Cell 13:759–760

Chapter 2

Retroviruses and Cell Fusions: Overview Anders L. Kjeldbjerg, Shervin Bahrami, and Finn Skou Pedersen

Abstract Retroviruses are a large and diverse group of enveloped animal viruses. A metastable envelope protein (ENV) on the surface of virus particles harbors a machinery for receptor-dependent fusion of biological membranes as needed for viral entry. The basic mechanism that drives fusion is widely conserved among different groups of retroviruses, whereas the precise signals that trigger the activation of this machinery vary. The exact same processes that drive viral entry may also mediate cell–cell fusion in a receptor-dependent manner. Such fusion events that may lead to the formation of giant multinucleated cells have been widely observed in cultured cells exposed to retroviruses. However, their possible contribution to the spread and pathogenesis of retroviral infections in man and animals is unclear. By way of their mode of replication via a DNA-intermediate that is stably integrated in the chromosomal DNA of the host cell, retroviruses may also establish germ-line infections that can be vertically transmitted from parents to offspring. Such remnants of retroviral infections of our ancestors constitute 8% of the human genome. Some of these human endogenous retroviruses of more than 25 million years of age have selectively maintained the coding capacity for functional envelope proteins, which provides strong evidence that these envelope genes have been co-opted to serve a beneficial function for their host. Currently, three of these old envelope genes have been found to encode proteins that can mediate cell–cell fusions and at least two of the envelope proteins have been implicated in the generation of a multi-nucleated layer of cells in the placenta. Keywords Retrovirus · exogenous retrovirus · endogenous envelope · entry receptor · receptor interference · fusion inhibitors · HERV Abbreviations AIDS ALV

Acquired immune deficiency syndrome Avian leukosis virus

F.S. Pedersen (B) Department of Molecular Biology, Aarhus University, DK-8000 Aarhus C, Denmark e-mail: [email protected] L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_2, C Springer Science+Business Media B.V. 2011

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ASCT DC-SIGN enJSRV ENV ERV FeLV GLUT1 HA HERV HIV-1 HTLV-1 HYAL2 ISU JSRV LTR mCAT-1 MFSD2 MMTV MLV MPMV Mya NHR CHR ORF Pit PRR RBD RSV SFV SIV Smit-1 SNP SP SU TM WDSV

A.L. Kjeldbjerg et al.

Alanine, serine and cysteine selective transporters Dendritic cell-specific ICAM-3-grabbing nonintegrin Endogenous JSRV Envelope protein Endogenous retrovirus Feline leukemia virus Glucose transporter 1 Hemagglutinin Human endogenous retrovirus Human immunodeficiency virus type 1 Human T-cell lymphotropic virus type 1 Hyaluronidase 2 Immunosuppressive domain Jaagsiekte sheep retrovirus Long terminal repeat Mouse cationic amino acid transporter Major facilitator superfamily domain containing 2 Mouse mammary tumor virus Murine leukemia viruse Mason-Pfizer monkey virus Million years ago Coiled-coil – N- and C-terminal heptad repeat Open reading frame Sodium-dependent phosphate symporter Proline-rich region Receptor-binding domain Rous sarcoma virus Simian foamy virus Simian immunodeficiency virus Sodium-dependent myo-inositol transporter 1 Single nucleotide polymorphism Signal peptide Surface subunit Transmembrane subunit Walleye dermal sarcoma virus

Contents 2.1 Introduction . . . . . . . . . . . . . . . . . . 2.2 Basic Features of the Retroviral Fusion Machinery 2.2.1 Entry . . . . . . . . . . . . . . . . . . 2.2.2 Receptors . . . . . . . . . . . . . . . . 2.2.3 Interference . . . . . . . . . . . . . . . 2.2.4 Membrane Fusion . . . . . . . . . . . .

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2.2.5 Fusion Inhibitors . . . . . . . . . . . . . . . . . 2.2.6 The Significance of the Coiled Coil Structures . . . 2.2.7 Control Mechanism for Fusion Activation . . . . . 2.2.8 Retroviral Fusion of Cells . . . . . . . . . . . . . 2.3 Fusion Control in Different Groups of Retroviruses . . . . 2.4 Endogenous Retroviruses and Cell Fusion . . . . . . . . 2.4.1 Origin and Classification of Endogenous Retroviruses 2.4.2 Evolutionary View of HERV env Genes . . . . . . 2.4.3 Structural Composition of HERV Envelope Proteins . 2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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2.1 Introduction Retroviruses comprise a large and diverse group of viruses whose main characteristic is that their genomic information is transmitted in the form of two copies of an RNA molecule that upon infection of a target cell is copied into a DNA molecule, which in turn will be integrated into the host genome. As all viruses, retroviruses are dependent on the host cell machinery for replication. The replication cycle of a retrovirus (Fig. 2.1a) begins by entry of a viral particle into an appropriate cell, at which stage a DNA copy of the genomic RNA is completed. The DNA copy enters the nucleus and becomes integrated into the host genome. In some viral species, degradation of the nuclear membrane during cell division is necessary for the access of the viral integration machinery to the host genome. After integration, the provirus (as the integrated viral DNA is called) will remain a part of the cellular genome, which will be inherited by the daughter cells. At this stage the transcription/translation machinery of the host cell is employed to produce more viruses (Fig. 2.1a). All retroviruses contain three vital genes: gag, which encodes the structural proteins, pol, which encodes the enzymes and env, which encodes the envelope protein (Fig. 2.1b). Gag and pol encode polyproteins that form the particle which eventually buds off the producer cell. Once inside a budded virion, these polyproteins are cleaved by the viral protease enzyme to yield the individual and functional proteins/enzymes in a process called maturation. Some retroviruses such as human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, harbor additional genes that encode proteins with regulatory roles. If the integration event happens in cells of the germ line, the provirus will become an integrated part of every cell of the offspring. Hence a large part of the vertebrate genomes are comprised of remains of retroviral integrations that are vertically transmitted as endogenous retroviruses (ERVs). This chapter reviews the basic mechanisms whereby retroviruses mediate the controlled fusion of two biological membranes needed for retroviral entry, and illustrate how these same mechanisms may drive cell–cell fusion. These sections are mainly based upon features of the retroviral genus of gammaretroviruses, including prototypic murine leukemia viruses (MLVs).

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Fig. 2.1 Panel a Schematic representation of a retroviral replication cycle. The first steps are entry by attachment to a surface receptor (step 1) followed by fusion (step 2). The subsequent reactions of reverse transcription (step 3) and integration (step 4) depend upon viral enzymes whereas the later steps of transcription, RNA splicing, and translation (step 5) that make the building blocks for new viruses depend upon the macromolecular synthesis machinery of the host cell. Finally, viral RNA and proteins are packed into particles that bud off the host cell (step 6) to yield a new virus particle surrounded by a lipid bilayer envelope. The maturation (step 7) that involves proteolytic cleavage by a virus-specific protease is needed for infectivity of the virus particle. Panel b Genetic map of an integrated retrovirus in the DNA form. Indicated are the three protein-encoding genes gag, pol, and env as well as nucleic acids motifs needed for the retroviral replication cycle

Then, examples are provided of the diversity of regulation of membrane fusion among retroviruses, and subsequently the involvement of retroviral infection in causing cell–cell fusion is discussed. In the final part of the chapter the fusiogenic properties of envelope genes of human endogenous retroviruses (HERVs) are summarized and their possible beneficial role for their human hosts discussed.

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2.2 Basic Features of the Retroviral Fusion Machinery 2.2.1 Entry Since retroviruses are enveloped viruses (which means that the virus particle is surrounded by a lipid membrane), the viral core is topologically in the same space as the cellular cytoplasm. Therefore fusion of the viral and cellular membranes is sufficient to grant the virus access to the inside of the cell. Fusion of lipid membranes does not happen spontaneously, and an active mechanism for fusion is needed. In the case of retroviruses, this is provided by the envelope protein, which is responsible for both binding of the virus to the appropriate target cell and fusion of the viral and cellular membranes. The specificity of the binding of the envelope protein to its cellular receptor is a major determinant of the viral tropism. This specificity is found in one of the two subunits that make the envelope protein, the surface subunit or SU. The other subunit, the transmembrane subunit or TM is anchored in the viral membrane by a transmembrane helix and is responsible for the fusion of the membranes upon binding of SU to its receptor. Both subunits are products of a single viral gene, which is cleaved by a host cell protease in the endoplasmic reticulum. The envelope protein is a trimer held together by interaction of the TM subunit.

2.2.2 Receptors Most viral receptors are membrane-integral proteins in the plasma membrane, encoded by the host cells, while a few viruses such as jaagsiekte sheep retrovirus (JSRV) use protein receptors anchored in the plasma membrane by lipid anchors (Miller 2003, 2008). Retroviruses, even closely related ones, use different cellular receptors. For example, murine leukemia viruses can be divided into six subgroups, ecotropic, polytropic, amphotropic, 10A1, xenotropic and M813, based on their receptor usage. Most viral receptors have several predicted transmembrane helices and in the case of MLVs function as transporters. Ecotropic MLVs utilize the mouse cationic amino acid transporter (mCAT-1) receptor and are unable to infect non-murine cells (Albritton et al. 1989, 1993, Wang et al. 1991). Amphotropic viruses use the sodiumdependent phosphate symporter Pit-2 (Kavanaugh et al. 1994) and are able to infect cells from a variety of species including humans. The 10A1 virus, closely related to the amphotropic viruses, uses two different receptors: both the amphotropic Pit-2 and the homologous Pit-1 receptors (Miller and Miller 1994) and has a similarly wide tropism. Xenotropic and polytropic viruses utilize the same receptor XPR !1 (Battini et al. 1999, Tailor et al. 1999, Yang et al. 1999) but differ in species tropism. A relatively new member of the MLV family (M813) isolated from the Southeast Asian rodent Mus cervicoloruses a sodium-dependent myo-inositol transporter 1 (Smit-1) as the cellular receptor (Hein et al. 2003, Prassolov et al. 2001). Other retroviruses, such as HIV-1, utilize a receptor for binding to the cells, but need a co-receptor for entry. The primary receptor for HIV is CD4, the natural ligand

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for the major histocompatibility complex class II molecule and an important factor in the generation of immune responses (Dalgleish et al. 1984, Klatzmann et al. 1984). Expression of CD4 mediates binding of the HIV virus to cells but is not sufficient for HIV infection (Chesebro et al. 1990). Other membrane proteins besides CD4, also called co-receptors, are necessary for HIV entry. Several receptors for chemokines act as co-receptors for HIV, the most important ones are CXCR4 and CCR5 (Alkhatib et al. 1996, Choe et al. 1996, Endres et al. 1996).

2.2.3 Interference Interaction of the viral envelope protein and a cellular receptor is absolutely vital for entry of the virus. Virus-infected cells are resistant to re-infection by the viruses utilizing the same receptor. A receptor can be blocked through interaction with a viral envelope protein that is expressed inside the cell (Fig. 2.2). The envelope protein binds to the receptor on the plasma membrane, thereby blocking it from interaction with viruses in the environment. This phenomenon, called receptor interference or superinfection resistance, is used by retroviruses to protect their host cells from invasion by other viruses. Mostly as a result of the aforementioned receptor interference, similar viruses have evolved to use either different receptors or even different sites on the same receptor protein. One interesting group is the polytropic/xenotropic murine leukemia viruses which belong to the gammaretroviral group. All of these viruses use the XPR1 protein as the entry receptor, but are sensitive to small variations found on the XPR1s from different species. For example the xenotropic viruses are unable to use the murine version of this receptor, but are fully functional when encountering receptors from other species. For example a xenotropic-like virus has

Fig. 2.2 Blocking of retroviral entry by receptor interference. Panel a A retrovirus binds via the envelope protein (red) to its cognate receptor (black) as the first step in the entry process. Panel b Envelope protein (red) expressed in the target cell blocks the receptor (black) and prevents entry of a new virus

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been implicated in prostate cancer in humans (Dong et al. 2007, Urisman et al. 2006). On the other hand polytropic viruses have a broad species tropism and can use both murine and non-murine XPR1 receptors. Interestingly, these viruses show non-reciprocal interference with each other, meaning that they can block each other with different efficiencies and even bind to different parts of the same receptor protein (Van Hoeven and Miller 2005). Few mutations in the envelope protein of polytropic MLVs can have dramatic effect on the species tropism and interference pattern of the virus (Bahrami et al. 2004). In the lentivirus HIV-1, blocking of the receptor is more elaborate. In HIV infection, receptor/envelope complexes are retained in the endoplasmic reticulum, and the viral accessory proteins Nef and Vpu stimulate the degradation of the CD4 receptor, thus the receptor expression on the cell surface is diminished (Crise and Rose 1992, Fackler and Baur 2002, Levesque et al. 2003, Willey et al. 1992a, b). Knowledge of envelope/receptor interaction has been utilized to design coreceptor blockers, a class of anti-HIV entry inhibitors that have affinity for HIV co-receptors (Shaheen and Collman 2004).

2.2.4 Membrane Fusion Membrane fusion is an integral part of many important biological activities, such as intracellular transport, endo/exocytosis or fertilization of an egg by sperm. The machineries that mediate these processes, although different, share some important characteristics, dictated by the chemistry of lipids, the main component of biological membranes (White et al. 2008) Lipid molecules of biological membranes are amphipathic. They contain a hydrophilic, usually charged head group and a hydrophobic tail of hydrocarbons. Upon exposure to water, the hydrophobic tails cluster together in what is essentially a hydrophobic environment, while the hydrophilic head groups are oriented towards water. In biological membranes, two layers of parallelly oriented lipid molecules with their tails pointing towards each other form a lipid bilayer. This arrangement constitutes the thermodynamically most stable conformation of amphipathic molecules by exposing the hydrophilic head of the molecules to the aqueous environment, while masking the hydrophobic parts from it. Fusing two membranes necessarily involves bringing two lipid bilayers into close proximity. This involves expulsion of water from hydrophilic surfaces and opposing the electrostatic repulsion from between equal charges, both of which are energetically unfavorable processes. For this reason, membrane fusion is not a spontaneous process and requires input of free energy. One of the best studied membrane fusion processes is that mediated by the influenza envelope protein hemagglutinin (HA). Unlike retroviral envelope proteins, the crystal structure of the hemagglutinin protein is known both in pre and post fusion states (Bullough et al. 1994, Wilson et al. 1981). Because of striking structural similarities between HA and retroviral ENV, it is feasible to use HA-mediated fusion as a model for retroviruses. Similarities include the presence of two subunits in both HA and ENV, one responsible for binding the receptor (HA1 and SU) and

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one for mediating fusion (HA2 and TM) (Katen et al. 2001, McClure et al. 1990, Nussbaum et al. 1993). While structural deviance between HA1 and SU reflects the usage of greatly different molecules as receptors, the very similar structures of HA2 and TM reflect the functional requirements that both proteins must fulfill in order to facilitate membrane fusion. HA2, like TM, has a hydrophobic stretch of amino acids at its amino-terminus followed by a heptad repeat region known to form coiled coils. Coiled coil structures are intertwined alpha helices stabilized through hydrophobic interactions on the contact side of each helix. This hydrophobic contact surface is made of the third and seventh amino acids in a repetitive sequence. The twisting of an α-helix brings these amino-acid residues to the same surface of the helix in the secondary structure. Thus

Fig. 2.3 Membrane fusion by the hemagglutinin protein (HA) of influenza virus. Panel a shows the structure of the trimer of HA2 at neutral and acidic pH, respectively. Panel b is a schematic diagram that illustrates steps of the fusion process mediated by structural changes in the influenza hemagglutinin caused by reduction in pH. Step I, The HA1 subunit binds to its receptor, wherafter the virus is internalized by endocytosis. Step II, Low pH in the endosome triggers the structural changes in HA2 shown in panel a, which leads to insertion of the amino-terminal fusion peptide into the target membrane. Step III, The helices of HA2 forld back on themselves and bring the two target membranes in close proximity. Step IV, hemifusion, which allows mixing of lipids in the two outer leaflets, but not the innter leaflets. Step V, the creation of a fusion pore through which the virus can enter the cytosol. See text for details and color schemes

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the primary structure of coiled coil helices is characterized by repeated hydrophobic amino acids at the third and seventh positions often referred to as a heptad repeat (Creighton 1996). As will be discussed shortly, activation of the fusion machinery must be very precisely timed. In the case of HA, fusion is triggered by the acidic pH encountered in endosomes, while most retroviruses are not dependent on pH change (Skehel et al. 1982, White et al. 2008). Fusion mediated by the influenza HA involves several sequential and extensive conformational changes induced by acidic pH (Fig. 2.3). At neutral pH the carboxyterminal portion of the HA2 heptad repeat regions (Fig. 2.3, cyan) form a coiled coil which folds back onto itself so that the amino-terminal portion (Fig. 2.3, yellow) is oriented towards the viral membrane and the fusion peptides (Fig. 2.3, black) are buried in the trimeric structure. Infection initiates when the virus binds to the target cell and is internalized into the endosomes where the pH is lower. This change in pH induces a dramatic conformational change in the HA2 protein so that the loop structure (green) that connects the amino and carboxy-terminal helices in the prefusion structure assumes a helical structure (Fig. 2.3). This extends the triple helix and inserts the hydrophobic fusion peptide into the target membrane. The process is followed by formation of a six-bundle helix where the carboxy-terminal helices fold back to form the so-called “hairpins”. This pulls the membranes together to form a hemifusion state (where only the outer leaflets of the membranes are fused) followed by full fusion and formation of a pore through which the viral core can enter the cytoplasm (Hunter 1997, White et al. 2008).

2.2.5 Fusion Inhibitors This detailed insight into the mechanism of the fusion has given rise to the rational design of fusion inhibitors. One example is the anti-HIV drug Enfuvirtide. Enfuvirtide or T20 is a peptide corresponding to the carboxy-terminal portion of the triple-helix of HIV-1 envelope protein. As illustrated in Section 2.2.4, this portion of the helix will ultimately fold back into the triple-helix structure to form the stable six-bundle helix conformation that brings the membranes close together. When added into the environment, it can interact with the intermediate (the long triple helix) conformation and inhibit formation of the final six bundle helix, thus inhibiting viral entry (Fig. 2.4) (Greenberg et al. 2004). By the same principle, specific peptide inhibitors of fusion have been made for other retroviruses (Chang et al. 2004, Lamb et al. 2008, Netter et al. 2004).

2.2.6 The Significance of the Coiled Coil Structures Membrane fusion is accompanied by conformational changes, which result in formation of coiled coil structures and hairpins. Triple-hairpin structures are remarkably stable structures, dissociating at temperatures around 90◦ C (Fass and Kim

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Fig. 2.4 The point of action of peptide inhibitors of retroviral fusion. The peptide inhibitor (red) is dervied from the carboxy-terminal heptad repeat (light grey) and has affinity for the aminoterminal heptad repeat (dark grey). The inhibitor blocks the association of the two heptad repeats and thereby hinders the transition from the triple-helix structure to the six-helix bundle as needed for fusion

1995, Lu et al. 1995), which suggests that a large amount of free energy is released upon their formation. Presumably the released energy is necessary to bring the membranes into close proximity and drive their destabilization which ultimately results in fusion (Bentz and Mittal 2000, White 1992). Consequently, the fusion trigger is a “one-time-only” event for every molecule: The change in the free energy of the reaction is too large for any equilibrium to exist between the “fusion-potent” and “fusion-active” conformations of the fusion proteins. Thus the fusion trigger must be timed precisely. In the case of viral fusion it must occur after the binding of the virion to target cells. As mentioned in Section 2.2.4, the trigger can be either a pH change after internalization or conformational changes in the binding subunit of the viral fusion protein upon interaction with the receptor.

2.2.7 Control Mechanism for Fusion Activation One such mechanism that is involved in control of fusion activation in gammaretroviruses is a disulfide isomerization that occurs when the envelope binds to its receptor. In these viruses, the SU and TM subunits are bound together by a disulfide bridge. One of the cysteine residues involved is found in a CXXC motif reminiscent of the active sites of protein-disulfide isomerases. In order for the fusion process to go forth, it is necessary that these two subunits dissociate. This happens when

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binding to the receptor presumably results in a conformational change that brings a crucial histidine residue in the right position so that a disulfide isomerase potential in the SU subunit is activated. The result is that the inter-subunit disulfide bond is broken, and one formed between the two cysteines in the CXXC motif, resulting in the dissociation of the SU from TM (Li et al. 2007, Wallin et al. 2004). This dissociation step can be facilitated by Ca2+ depletion (Wallin et al. 2004). Another interesting control mechanism found in gammaretroviruses and some other retroviruses is the so-called R-peptide. As mentioned before, the envelope protein is found interacting with the receptors on the surface of the cells where it is expressed (Fig. 2.2). Yet this interaction does not result in triggering the fusion machinery, in which case the energy stored in the envelope protein would be wasted. The reason is that the cytoplasmic tail of envelope molecules, the R-peptide, locks the fusion machinery and prohibits the cascade of conformational changes, probably by distorting the TM trimers by pulling them out of position through the R-peptides affinity for membrane lipids. Cleavage of the R-peptide during maturation of the viral particles by the viral protease releases the pressure on the individual TM subunits and enables them to form the triple helix bundle necessary for fusion (Olsen and Andersen 1999). Interestingly, addition of the R-peptide to fusion proteins of non-retroviral origin has a similar effect (Li et al. 2006).

2.2.8 Retroviral Fusion of Cells The exact same machinery that mediates the fusion of a viral and a cellular membrane may also cause fusion of cells in a receptor-dependent manner leading to the formation of giant multi-nucleated syncytia (Fig. 2.5). Similar to retroviral entry, cell fusion can also be inhibited by receptor interference caused by expression of an

Fig. 2.5 A syncytium of HEK 293 cells caused by murine leukemia virus (unpublished result from the authors’ laboratory)

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envelope protein. Hence, fusion of two cells may require that one of them is noninfected and thereby has an accessible receptor. Two distinct processes have been identified, fusion from within and fusion from without (Andersen 1994, Siess et al. 1996). Fusion from within is driven by viral envelope gene expression after integration of the provirus, which results in expression of the envelope protein on the cell surface whereas fusion from without is driven by envelope proteins on virus particles. In contrast to fusion from within, fusion from without does not require viral gene expression in the target cell and can be observed during the first hours after addition of virus to an uninfected cell culture (Andersen 1994). Not unexpectedly, deletion of the R-peptide enhances the efficiency of cell–cell fusion by MLV (Rein et al. 1994). Newer results indicate that envelope-receptor interaction may serve to guide and establish 3D-patterns of cell–cell contacts and suggest that such contacts involving fusiogenic envelope proteins may lead to the formation of intercellular bridges (Jin et al. 2009, Sherer and Mothes 2008). Other than abundance and post-translational modification of envelope protein and receptor, the factors that contribute to the large variability in fusion potential among cell lines are poorly understood. One contributing factor is the presence of other molecules that facilitate cell–cell interactions (Ceccaldi et al. 2006, Pantaleo et al. 1991). Retroviruses are reported to bud from lipid rafts rich in cholesterol as also reflected by the lipid composition of the membrane enveloping the virus (Metzner et al. 2008). It is conceivable that certain membrane patches may be more amenable to fusion than others, and that such variation also contributes to variability in fusiogenicity among cells (Lorizate et al. 2009). XC-cells are a rat-muscle tumor-derived cell line that is particularly susceptible to the induction of syncytia by ecotropic MLVs (Klement et al. 1969). In these cells fusion from within is efficient even in the case of envelope proteins that have retained the R-peptide. It has been suggested that the fusion of XC-cells may involve a cellular factor with a role in membrane fusion in myogenesis (Kubo et al. 2003).

2.3 Fusion Control in Different Groups of Retroviruses The activation of the metastable envelope protein of retroviruses is tightly regulated. It is not surprising that there has been a strong evolutionary selection for such precise regulation in diverse biological settings since premature activation would lead to loss of viral infectivity. While the overall membrane-fusion machinery is quite similar, the exact modes of control, which are now beginning to be uncovered, show wide specialization for different groups of retroviruses. Such control mechanisms also impact on the regulation of the cell–cell fusiogenic activity of retroviral envelope proteins in the plasma membrane of an infected cell. The family Retroviridae comprises seven genera, alpharetroviruses, betaretroviruses, gammaretroviruses, deltaviruses, epsilonretroviruses, lentiviruses, and spumaviruses (Fig. 2.6). While alpharetroviruses are restricted to birds and epsilonretroviruses to fish, the remaining five genera are all represented in

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Fig. 2.6 Classification of the seven genera of Retroviridae and their three classes of related human endogenous retroviruses (HERVs), class I, class II, and class III. Avian leukosis virus (ALV), Rous sarcoma virus (RSV), mouse mammary tumor virus (MMTV), Mason-Pfizer monkey virus (MPMV), jaagsiekte sheep retrovirus (JSRV), human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), human T-cell lymphotropic virus type I (HTLV-1), simian foamy virus (SFV), walleye dermal sarcoma virus (WDSV), murine leukemia virus (MLV), feline leukemia virus (FeLV)

mammals. If endogenous retroviruses (ERVs) are included together with their normal (exogenous) counterparts all of those five genera, betaretroviruses, gammaretroviruses, deltaretroviruses, lentiviruses and spumaviruses are represented in primates including humans. Cell–cell fusion has been documented for all genera of the family Retroviridae with the possible exception of epsilonretroviruses. The functional organization of envelope protein of selected exogenous retroviruses is shown in Fig. 2.7. The regulation of the membrane-fusion machinery of envelope proteins of gammaretroviruses has been intensively studied as described in Section 2.2. The prototype virus of this group is murine leukemia virus, but the same principles hold for closely related viruses of other species such as cats and primates. There is no unifying overall picture as to the requirement for acidic pH during the entry step of gammaretroviral infections, however there is ample evidence that several gammaretroviral envelope proteins can mediate cell–cell fusion at neutral pH (Andersen 1994). As mentioned, one hallmark of gammaretroviral envelope proteins is the covalent linkage of SU and TM by a disulfide bond and the dissociation of SU from TM as a result of a disulfide isomerization reaction during the steps towards membrane fusion. Another characteristic feature of this group, the negative regulation

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Fig. 2.7 Schematic organization of envelope motifs involved in the regulation of the fusion. C(X)6 CC, grey line, involved in a disulfide bond between TM and the CXXC motif in SU. SP: Signal peptide; SPH: histidine motif; RBD: Receptor-binding domain; PRR: Proline-rich region; FP: Fusion peptide. ISU: immunosuppressive domain (black box); NHR CHR: Coiled-coil – amino- and carboxyl-terminal heptad repeat structures (green); Transmembrane domain (gray box). The disulfide partner in SU of ALV is not identified. HIV-1 contains a C(X)6 C motif, however, it is not known whether it is involved in a disulfide bond. In HTLV only one heptad repeat is identified

of fusion by the carboxy-terminal R-peptide in the cytoplasmic tail of TM, which is cleaved off by the viral protease after budding, is also shared by Mason-Pfizer monkey virus, a member of the betaretroviruses genus which has been widely used experimentally to trigger cell–cell fusion (Duelli et al. 2005). The genus of deltaretroviruses includes the important human pathogen human T-cell lymphotropic virus type 1 (HTLV-1) as well as related virus isolates from simians and humans (Lairmore and Franchini 2007). HTLV-1 is associated with adult T-cell leukemia and other severe human diseases. Progression to clinical disease generally takes decades following infection and there is a lack of knowledge on the mode of virus replication and onset of disease. HTLV-1 particles are poorly infectious and believed to spread mainly through specialized cell to cell contacts termed virological synapses (Igakura et al. 2003). The envelope protein of HTLV-1 has a disulfide bridge between SU and TM and undergoes disulfide isomerization after activation by a process dependent upon similar motifs to those of MLV (Li et al. 2008) (see Fig. 2.7). The entry receptor for HTLV-1 was recently found to be the glucose transporter 1 (GLUT1) (Manel et al. 2003). GLUT1 expression is not limited to T-lymphocytes, which are the primary target for adult T-cell leukemia, and it is likely that HTLV-1 may also infect other cells in vivo. HTLV-1 is an efficient inducer of syncytia in cultured cells (Paré et al. 2005) in a process that takes place at neutral pH and requires the viral envelope protein and the receptor. Additional cell– cell interactions may facilitate fusion by HTLV-1. It has been found that the dendritic cell-specific lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) will facilitate fusion with HTLV-1 infected T-cells as a result of interaction of this

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lectin with ICAM molecules on the T-cells (Ceccaldi et al. 2006). It is not known to what extent cell fusion is triggered by HTLV-1 infections in man, and if cell fusion plays a role in viral pathogenesis. A unique mechanism of fusion activation in two steps that use a combination of receptor-binding and low pH for activation was first discovered for avian leukosis virus (ALV) of the alpharetroviruses group (Barnard et al. 2006, Mothes et al. 2000). In contrast to other retroviruses, the fusion protein of ALV is not located at the very amino-terminus of TM (Fig. 2.7). The first step in the entry process takes place after envelope binding to the receptor at neutral pH at the cell surface. Receptor binding leads to a conformational change in the envelope protein that exposes the fusion peptide to become inserted into the target membrane. At this stage the receptor-primed intermediate conformation of the envelope protein is susceptible to the action of a fusion-inhibitory peptide derived from the carboxy-terminal heptad repeat of TM (Netter et al. 2004). However, progression to full fusion requires acidic pH. In accordance with this model cell–cell fusion mediated by the envelope protein of ALV also requires as well the cognate receptor as acidic pH. Related mechanisms in which both receptor interaction and acidic pH are required for activation of the fusion machinery have recently been proposed for the betaretroviruses mouse mammary tumor virus (MMTV) (Wang et al. 2008) and jaagsiekte sheep retrovirus (JSRV) (Côté et al. 2009). For both of these viruses cell–cell fusion was shown to require receptor presence as well as acidic pH. Lentiviruses such as HIV-1 infect only specialized target cells by an entry process that requires interaction with a receptor as well as a co-receptor as outlined earlier in this chapter. The SU and TM subunits of HIV are not linked by a disulfide bond (Fig. 2.7). However, a host cell disulfide isomerase is implicated in fusion mediated by HIV (Papandreou et al. 2010). The fusion machinery of HIV does not need low pH for activation. The exact subcellular location where the fusion takes place has been a matter of debate, however it is now believed that receptor binding takes place at the plasma membrane and that most fusion events happen after endocytosis, but before acidification of the endosome (Miyauchi et al. 2009). HIV-1 is an inducer of cell fusion in cultured cells that express the cognate receptor and co-receptor (Ji et al. 2006). The regulation of cell–cell fusion has the same hallmarks as that of infection by HIV-1, i.e. it takes place at neutral pH and it can be inhibited by coreceptor blockers and fusion inhibitors (Ji et al. 2009). However, in some instances additional cell–cell interaction may have an impact on fusion activity (Pantaleo et al. 1991). Progression of an HIV-1 infection often leads to a shift in co-receptor usage from CCR5 to CXCR4 as a result of mutations that cause amino-acid changes in the V3-loop of SU (Shankarappa et al. 1999). The emerging CXCR4-tropic viruses are very efficient in the induction of syncytia and it has been proposed that killing of lymphocytes by cell fusion (Perfettini et al. 2005) contributes to disease progression, but this issue remains unresolved. The retroviral genus of spumaviruses or foamy viruses includes many isolates from mammals including primates. There is no clear association of any disease with infection by spumaviruses. Their mode of replication exhibits several distinctive features from that of other retroviruses (Linial 2007). Structural and functional aspects

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of the envelope protein of a foamy virus have been investigated, but no information on the identification of a cellular receptor for foamy virus entry is available (Duda et al. 2006). Replicating foamy viruses cause the appearance of syncytia and are highly cytopathic in cell culture. Efficient formation of syncytia requires the presence of viral capsid as well as envelope protein, presumably reflecting a need for a gag-envelope interaction (Pietschmann et al. 2000). Entry of foamy viruses has been suggested to involve a low-pH step and formation of syncytia can in fact be triggered by a short exposure to acidic pH (Picard-Maureau et al. 2003).

2.4 Endogenous Retroviruses and Cell Fusion 2.4.1 Origin and Classification of Endogenous Retroviruses If retroviral infection has taken place in the germ line, the provirus will be transmitted vertically throughout generations as an endogenous retrovirus (ERV). Each independent germ line infection event will define a novel ERV family, whose copy number may increase over an evolutionary time span, either by intracellular retrotransposition (Sverdlov 1998) or by extracellular re-infection (Belshaw et al. 2004), following the normal replication cycle of a retrovirus. Chromosomal duplication may also increase the copy number of an ERV family (Boeke and Stoye 1997, Kjeldbjerg et al. 2008). Each novel germ line infection may thereby result in a few to several hundred genomic copies throughout evolution (Löwer et al. 1996). However, the rate of ERV amplification is highest shortly after the primary infection, and decreases during evolution. This may be caused by either transcriptional inactivation of viral RNA due to proviral DNA methylation (Lavie et al. 2005, O’Neill et al. 1998), loss of viral receptor affinity which prevents reinfection (Boeke and Stoye 1997), or inactivation of the provirus as a consequence of insertions, deletions, accumulated mutations or editing during retro-transposition (Esnault et al. 2005, 2006). Recombination between the long terminal repeat (LTR) regions the form a direct repeat at the termini of the provirus can result in the excision of internal viral sequences, leaving a single LTR in the genome, termed solo LTR (Löwer et al. 1996, Roeder and Fink 1980, Rotman et al. 1984). Altogether sequences related to retroviruses represent approximately 8% of the human genome (Lander et al. 2001). Endogenous retroviruses (ERVs) are classified somewhat differently from exogenous retroviruses. ERVs are divided into three classes depending on their similarity to exogenous retroviruses (Fig. 2.6), originally based on endogenous retrovirus found in humans (HERV). The HERVs, which are related to gammaretroviruses, are classified as class I, the betaretroviruses related ERVs as class II, and the spumaviruses related ERVs within class III. Endogenous elements are also found in the epsilon-, alpha- and lentiviruses genus but none are human associated (Fig. 2.6) (Gilbert et al. 2009, Goff 2007, Herniou et al. 1998). ERV classification was originally based on sequence similarity between the primer binding site (PBS) in the ERV locus and the tRNA used to prime the reverse transcription process in the host (e.g.

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HERV-W has a PBS matching W; tryptophan) (Boeke and Stoye 1997), however this classification is inconsistent for some ERV groups, because two ERV primed by the same tRNA do not necessarily belong to the same phylogenetic family as classified by nucleic acid homology of other parts of their genomes. The majority of the HERVs invaded the human genome at least 25 million years ago (mya) (Boeke and Stoye 1997, Shih et al. 1991), after splitting of Old World and New World monkey lineages approximately 43 mya (Steiper and Young 2006), however the oldest identified HERV-loci seem to be approximately 100 million years (my) old (Katzourakis and Tristem 2005). Nevertheless members of the HERV-K family have been active in the human genome less than 100,000 years ago as demonstrated by insertional polymorphisms (Belshaw et al. 2005, Medstrand and Mager 1998, Turner et al. 2001). However, no present-day replication-competent HERVs have been described, even though fully intact members of the HERV-K group have been reported, encoding all the viral genes (Turner et al. 2001). HERV-K related virus-like particle have been observed in germ-cell tumours (Bieda et al. 2001, Boller et al. 1983, Kurth et al. 1983), and while the HERV-K113 locus was shown to be capable of producing intact particles of retroviral morphology in cell lines derived from human germ-cell tumours (Boller et al. 2008), they were not found to be infectious. Nevertheless, trans-complementation and recombination of human HERV-K loci can generate functional and infectious HERV-K virus particles, indicating that human cells still have the potential to produce infectious retrovirus particles (Dewannieux et al. 2006, Lee and Bieniasz 2007). In contrast, other mammalian species such as mouse, cat and pig, naturally active ERVs still exist that produce infectious particles (Boeke and Stoye 1997).

2.4.2 Evolutionary View of HERV env Genes Since infection by retroviruses is usually harmful, evolutionary selection may neutralize endogenized retroviruses of infected germ lines. Nevertheless, some ERV loci still contain an open reading frame for one or more viral genes. A survey of the human genome identified 29 viral env genes with an ORF longer than 500 amino acids (Villesen et al. 2004, de Parseval et al. 2003), however, some of them appear to be pol-env fusion proteins, leaving 19 real env genes within the human genome. The 19 env genes represent 10 distinct HERV families (Fig. 2.8). Transcriptome analysis shows that all 19 envelope genes are expressed in healthy tissues, with the placenta as the organ having the maximum expression level of numerous envelope genes (Aagaard et al. 2005, Blaise et al. 2005, de Parseval et al. 2003). The env genes of the youngest family HERV-K, which is not represented in our primate relatives, are also the most abundant, represented by six full-length envelope genes. Three different families are represented by the three oldest envelope genes found in all simian primates, syncytin 2, EnvPb1, and ENV-V2 (Fig. 2.8) (Aagaard et al. 2005, Blaise et al. 2003, Kjeldbjerg et al. 2008). Characteristic of these ancient HERV envelope genes is that the envelope genes are the only preserved viral genes in an otherwise degenerated HERV locus (Fig. 2.9). Altogether, the conservation and expression of the envelope genes have led to speculation that HERV envelope genes

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Fig. 2.8 Evolutionary view of HERV envelope loci. a Detection of HERV solo LTRs (Hughes and Coffin 2004). b Insertionally polymorphic. c Nonsense polymorphic sites in human population (de Parseval et al. 1998) and not detected in gorilla. d Carboxy-terminal truncation. e Homologue locus in R. macaque found on chromosome 2, and a locus is identified in New World monkeys (de Parseval et al. 2005), but is not preserved

Fig. 2.9 Proviral organization of the HERV-Pb1 locus. The env gene is preserved while the gag and pol genes are broken down by insertions and deletions (Indel). Moreover, human reading frame destroying SNPs are found only outside env

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have been co-opted as a bona fide gene beneficial to the host. Hypothetical beneficial functions of such a gene include (i) protecting the fetus due to immunomodulatory properties via an immunosuppressive domain located in the TM subunit of the envelope (Mangeney et al. 2001, 2007), (ii) preventing present-day retroviral infections by inhibiting cell entry of related exogenous retroviruses that use a common surface receptor through receptor interference, as discussed in Section 2.2 (Best et al. 1997, Ponferrada et al. 2003), or (iii) providing cell–cell fusions by the mechanism discussed earlier in this chapter. In particular three HERV envelope proteins can induce cell–cell fusion in vitro, the HERV-W envelope gene syncytin 1 (Blond et al. 2000, Mi et al. 2000), the HERV-FRD envelope syncytin 2 (Blaise et al. 2003), and the HERV-Pb envelope EnvPb1 (Blaise et al. 2005) (Table 2.1). All three are candidates for having a beneficial function, because they are evolutionarily conserved and have undergone amino acid-conserving (purifying) selection during primate evolution (Aagaard et al. 2005, Blaise et al. 2003, Bonnaud et al. 2004). Syncytin 1 belongs to the HERV-W family and entered the genome of the common ancestor of the hominoids and Old World monkeys about 30 mya, however, it is inactivated in Old World monkeys (Fig. 2.8) (Bonnaud et al. 2005, Cáceres and Thomas 2006, Mallet et al. 2004). A systematic comparison of paralogue HERV-W env sequences reveals a syncytin 1-specific 12-bp deletion in the carboxy-terminal end that results in a four-amino-acid shortened cytoplasmic tail in the TM subunit of syncytin 1. This deletion is critical for the fusion activity of syncytin 1, by allowing syncytin 1 to mediate cell–cell fusion (Bonnaud et al. 2004), which might have played a major role in the domestication of syncytin 1 to become a bona fidegene. The deletion furthermore indicates that there may be sequences in the cytoplasmic tail of syncytin 1 that modulate its fusiogenicity in a way reminiscent of the function of the R-peptide in gammaretroviruses (see Section 2.2.7) (Ragheb and Anderson 1994, Yang and Compans 1996). However, R-peptide- like sequences cannot be identified in the cytoplasmic tail of syncytin 1, neither could any retroviral protease cleavage site be found (Blond et al. 2000). Additionally, a syncytin 1 orthologue gene is active in all hominoid species, and none of the single-nucleotide-polymorphisms (SNPs) in the human genome spoil its fusiogenic properties (de Parseval et al. 2005). The two other fusiogenic HERV envelope proteins syncytin 2 and EnvPb1 (Table 2.1) belong to the HERV-FRD and HERV-Pb families, respectively. Together with the non-fusiogenic ENV-V2 envelope they are the oldest preserved envelope

Table 2.1 HERV envelopes with cell–cell fusion capability Fusiogenic HERV-envelope

Classification

Expression

Receptor

Evolutionary age (my)

Syncytin 1 Syncytin 2 EnvPb1

Class I Class I Class I

Placenta Placenta Low expression in many tissues

ASCT1 ASCT2 MFDS2 Not known

20 40 40

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genes in the human genome. These entered the simian genome about 40 mya in a common ancestor to the Old and New World monkeys. Additionally, the fusiogenic potential of all simian syncytin 2 orthologues has been preserved throughout evolution, and no human SNPs alter the open reading frames of syncytin 2 and EnvPb1 (Aagaard et al. 2005, Blaise et al. 2003, Kjeldbjerg et al. 2008, de Parseval et al. 2005). The HERV-K family also contains a functional envelope protein, however, it does not seem to be capable of mediating cell–cell fusion, and only functions as a fusion protein during infection of pseudotyped virus particles (Dewannieux et al. 2005). The highest expression levels of HERV envelope genes are found in the placenta, where among others the two fusiogenic HERV envelope proteins syncytin 1 and syncytin 2 are highly expressed (Malassiné et al. 2005, 2007, Mi et al. 2000, de Parseval et al. 2003). This pattern may reflect a physiological role of the HERV envelope proteins in mediating cell–cell fusion in placenta by generating the multinuclear syncytiotrophoblast layer, which forms a barrier between the mother and the fetus. In fact, inhibition of syncytin 1 in cytotrophoblasts leads to a decrease in cell fusion in vitro (Frendo et al. 2003). The hypothesis of syncytin 1 and syncytin 2 involvement in syncytiotrophoblast formation is further supported by the finding that the receptors ASCT2 utilized by syncytin 1 and MFSD2 utilized by Syncytin 2 are expressed in cytotrophoblasts and syncytiotrophoblast (Esnault et al. 2008, Hayward et al. 2007, Lavillette et al. 2002). Another proposed function of ERV envelope proteins relate to immunosuppressive properties of the envelope protein, However only syncytin 2 among the syncytin genes has been shown to have an immunosuppressive activity (Mangeney et al. 2007). Three other HERV envelope genes also contain immunosuppressive activities: EnvPb1, ENV-V2 and ERV3 (Mangeney et al. 2007), among which ENV-V2 and ERV3 (Blaise et al. 2005, Kato et al. 1987) are expressed in placenta. Yet the impact of the immunosuppressive activity of HERV envelope genes in placenta needs to be studied further. While the HERV envelope genes, presumed to take part in the formation of the syncytiotrophoblast and protection of the fetus from the maternal immune system, are restricted to the simian species, the placental mammals originated more than 100 mya (Springer et al. 2003). However, endogenization of ERV envelope genes involved in the development of placenta seems to have taken place in a striking show of convergent evolution in placental mammals. For example, mice have acquired two placenta-specific ERV envelope genes named syncytin A and syncytin B, which, like the human syncytins, are both fusiogenic (Dupressoir et al. 2005) and predominantly expressed in the placenta. Only one of the murine syncytins has immunosuppressive properties (Mangeney et al. 2007). Knockout of the syncytin A gene prevents formation of a syncytial layer of trophoblast cells and results in miscarriage (Dupressoir et al. 2009). Additionally, two other placental mammals also seem to have endogenized an ERV env gene utilized in placenta development. The endogenous jaagsiekte sheep retrovirus (enJSRV) env gene in sheep is expressed in the beginning of day 12 in trophoblasts, whereas its receptor hyaluronidase 2 (HYAL2) is expressed from day 16, but only in the binucleate cells

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and the syncytiotrophoblast (Dunlap et al. 2005). Furthermore, injection of antisense oligonucleotides that blocked enJSRV envelope protein production, inhibited “trophoblast giant binucleate cell” differentiation as is normally observed on day 16, and led to pregnancy loss (Dunlap et al. 2006). However, there is so far no demonstration of cell–cell fusion mediated by enJSRV env and its exact role in placenta formation in sheep is unknown. Additionally, a fusiogenic and placenta-specific ERV envelope has also been identified in rabbits (Heidmann et al. 2009). The convergent evolution of fusiogenic ERV envelope genes in placental mammals strongly supports the notion that individual retroviral integrations can be domesticated independently of one another.

2.4.3 Structural Composition of HERV Envelope Proteins Functional and comparative studies show that the molecular cell-fusion mechanism of ERV envelope genes resembles the mechanism of viral fusion in exogenous viruses such as MLV. Many distinguishable motifs found in the TM part of the envelope protein are conserved among the different endogenous envelope proteins (Fig. 2.10). A peptide mimicking one of the heptad repeats in the TM of syncytin 1 functions as a fusion inhibitor (see Section 2.2.5) (Chang et al. 2004), indicating six-helix bundle formations during fusion of syncytin 1. Additionally, a structural

Fig. 2.10 Identification of motifs involved in regulation of the fusion of fusiogenic ERV envelope proteins, compared to the exogenous MLV. SP: Signal peptide; SPH: Histidine motif; FP: Fusion peptide; ISU: Immunosuppressive domain (black box); C(X)6 CC grey line; NHR CHR: Coiledcoil – heptad repeat structure (green); Transmembrane domain (gray box)

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prediction of the ectodomain of syncytin 2 resembles the structure of the corresponding domain of present-day exogenous retroviruses (Renard et al. 2005). All fusiogenic ERV envelope proteins identified contain cysteine motifs in TM and SU, similar to those involved in the formation of the disulfide bound between the two subunits in exogenous gamma-retroviruses (Chen et al. 2008, Pinter et al. 1997). Heterogeneity of the fusiogenic ERV envelope proteins is most pronounced for the SU subunit, which contains the regulatory and receptor-binding properties of the envelope proteins. However, they all feature the cysteine CXXC motif involved in disulfide bond isomerization during fusion (Wallin et al. 2004, 2005) (Fig. 2.10), and it may be speculated that this motif has the same function as in present-day exogenous retroviruses. Nevertheless, the localization of the CXXC motif separates the envelope proteins into two groups. The first group (syncytin 1, EnvPb1, syncytin Ory1) resembles the localization in exogenous retroviruses in the carboxy-terminal part of the SU, whereas the other group (syncytin 2, syncytin A, syncytin B) has an amino-terminal localization of the CXXC motif. Furthermore, syncytin 1 and syncytin 2 contain a motif reminiscent of the SPH motif found in the amino-terminal part of gammaretorviral SU, known to be involved in activation of the disulfide isomerization after receptor binding, as discussed earlier in Section 2.2.7. It has been shown that in the case of MLV this histidine residue can be substituted by a tyrosine (Qian and Albritton 2004), which is the amino acid residue found the SPY motif found in syncytin 1 and syncytin 2. However this substitution lowered the fusion activity of gammaretroviral envelope proteins. The other fusiogenic ERV envelope proteins lack the histidine motif, similar to the HTLV-1 envelope protein (Fig. 2.7). It remains to be determined whether this variable localization of CXXC and the tyrosine-substituted histidine motifs has an influence on the fusion regulation of syncytin 1 and syncytin 2, and how fusion is regulated in those ERV envelope proteins that lack a SPH motif. Nevertheless, the fusion-regulation process of the old gamma-retroviruses, represented by endogenous retroviruses, seems to be more divergent compared to present-day exogenous gammaretroviruses.

2.5 Conclusion Major advances over the past years have contributed to our detailed understanding of basic functions of the retroviral envelope protein, and a large number of specific cellular receptors for retroviral entry have been identified. Based upon this knowledge natural mechanisms of inhibition of cellular entry by retroviruses have been identified and specific types of synthetic inhibitors developed. The ability of retroviruses to form syncytia in cell culture has long been known for a number of exogenous retroviruses, while the possible role of cell–cell fusion in retroviral pathogenesis remains elusive. Some envelope proteins encoded by endogenous retroviruses of man and animals have been selectively conserved through evolution, and in some instances have retained their ability to drive cell fusion in a receptor-dependent manner possibly as a domestication process. Our knowledge of

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the physiological role played by such endogenous envelope proteins is still limited, but there is strong evidence that some envelope proteins are necessary for formation of the multinucleated layer of the placenta. Further studies of the normal and possible pathophysiological roles of such envelope proteins will benefit from our basic insight into the function of retroviral envelope proteins and our ability to interfere with this function.

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Chapter 3

Retroviral Membrane Fusions: Regulation by Proteolytic Processing and Cellular Factors Yoshinao Kubo

Abstract Retroviruses infect host cells by fusion between the viral envelope and host cell membranes mediated by retroviral envelope (Env) glycoproteins. Because of this membrane fusion activity, cells expressing the Env proteins often fuse with neighboring cells, resulting in syncytia. Retroviral membrane fusion is directly induced by the interaction between the Env proteins and cell surface receptors. In addition, many cellular factors could affect syncytium formation by the retroviral Env proteins. Because cell fusion events are also involved in the developmental processes of several tissues, studies on the regulation of the retroviral membrane fusion would contribute to the understanding of developmental biology as well as retrovirus infections. Precursor Env protein processing by furin or other cellular proteases into surface (SU) and transmembrane (TM) subunits is required for membrane fusion. Functions of the cytoplasmic tail of TM subunits to inhibit membrane fusion activity are conserved among many retroviruses. The mature SU proteins of several retroviruses are further cleaved by cathepsin proteases in acidic late endosomes to activate the membrane fusion. Fusion requires the formation of a “pre-fusion complex” at the viral entry site, and this complex includes many viral and cellular factors. Cholesterol- and sphingolipid-enriched raft microdomains of host cell and retroviral envelope membranes are both required for membrane fusion. Further, the cytoskeleton and its associated signaling factors, such as Rho, are also associated with Env-induced membrane fusion. Keywords Cathepsin · cell adhesion · cytoskeleton · endosome · envelope proteins · fusions · membrane fusions · microdomains · proteolysis · rafts · retroviruses Abbreviations AIDS ASCT

Acquired immuno deficiency syndrome Alanine, serine and cysteine selective transporters

Y. Kubo (B) Department of AIDS Research, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan e-mail: [email protected] L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_3, C Springer Science+Business Media B.V. 2011

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CAT1 CCR5 CXCR4 Env ERM FIV GLUT1 HERV HIV HTLV ICAM Lck LFA-1 MLV Pit ROCK SIV SU TM VCA VCAM VLA-4 WASP XMRV XPR

Y. Kubo

Cationic amino acid transporter 1 CC chemokine receptor 5 CXC chemokine receptor 4 Envelope Ezrin, radixin, moesin Feline immunodeficiency virus Glucose transporter 1 Human endogenous retrovirus Human immunodeficiency virus Human T-cell lymphotropic virus Inter-cellular adhesion molecule Leukocyte-specific protein tyrosine kinase Lymphocyte function-associated antigen 1 Murine leukemia virus Sodium-dependent phosphate symporter Rho-associated, coiled-coil containing protein kinase Simian immunodeficiency virus Surface Transmembrane Verprolin-homologous, cofilin-homologous, and acidic domain Vascular cell adhesion molecule Very late antigen-4 (Integrin alpha4beta1) Wiscott-Aldrich syndrome protein Xenotropic MLV-related virus Xenotropic/polytropic receptor

Contents 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Membrane Fusion by Retroviral Env Protein . . . . . . . . . . . 3.2.1 Cell Surface Receptor . . . . . . . . . . . . . . . . . . 3.2.2 Membrane Fusion Mechanism . . . . . . . . . . . . . . 3.3 Regulation of Retroviral Membrane Fusion by Proteolytic Processing 3.3.1 Processing of Precursor Env Polyprotein . . . . . . . . . . 3.3.2 R Peptide Cleavage . . . . . . . . . . . . . . . . . . . 3.3.3 Syncytium Formation in XC Cells by MLV . . . . . . . . . 3.3.4 Mechanism of R Peptide to Inhibit Membrane Fusion . . . . 3.3.5 Cleavage by Cathepsin Proteases . . . . . . . . . . . . . 3.4 Regulation of Retroviral Membrane Fusion by Cellular Factors . . . 3.4.1 Lipid Raft . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Cell Adhesion Molecules . . . . . . . . . . . . . . . . . 3.4.3 Cytoskeleton-Associated Molecules . . . . . . . . . . . . 3.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Retroviral Membrane Fusions

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3.1 Introduction Retroviruses including human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), human T-cell leukemia virus (HTLV), and murine leukemia virus (MLV) are enveloped with a lipid bi-layer derived from virus-producing host cells (Fig. 3.1). Retroviruses infect host cells by fusion between the viral envelope and host cell membranes after interaction with their cell surface receptors, similar to other enveloped viruses, and this reaction is induced by the envelope (Env) glycoproteins. Because of the membrane fusion activity of the retroviral Env proteins, cells expressing the Env proteins often fuse with neighboring susceptible cells to form multi-nucleated giant cells. This is known as syncytium-formation (Fig. 3.2). It

Fig. 3.1 Structure of a retrovirus particle. Retroviral particles have a lipid bi-layer envelope (envelope membrane) that is derived from the plasma membrane of virus-infected cells. The envelope proteins (Env) are expressed on the envelope membrane. The precursor Env proteins are proteolytically cleaved into surface (SU) and transmembrane (TM) subunits

Fig. 3.2 Morphology of syncytia induced by the R peptide-truncated ecotropic MLV Env protein in mouse NIH3T3 cells

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is generally thought that syncytium formation induced by the retroviral Env proteins reflects processes involved in viral entry into host cells. Syncytium formation induced by the HIV Env protein activates host cell apoptosis, ultimately resulting in cell death. This results in T-lymphocyte depletion, and may be associated with HIV pathogenicity (Ferri et al. 2000, Scheller and Jassoy 2001). Alternatively, such fusogenic retroviral Env proteins could also be used as a therapeutic for cancer treatment by inducing cell death of transformed cells through syncytium formation (Diaz et al. 2000, Galanis et al. 2001). Cell–cell fusions also play important roles for fertilization, and developments of syncytiotrophoblasts in placenta, osteoclasts in bone, and multi-nucleated skeletal muscle fibers. Interestingly, syncytiotrophoblast formation is induced by a human endogenous retrovirus (HERV) Env protein, named syncytin (Dupressoir et al. 2009, Mi et al. 2000). Thus, studies of how these cell fusion processes are regulated would contribute to understanding many biological phenomena, including viral infectivity, viral pathogenicity, cancer gene therapy, and tissue developments. The cell fusion process initiated by retroviral Env proteins is directly induced by the interaction of the Env proteins with their cognate cell surface receptors. Additionally, other viral and cellular factors influence Env-induced cell fusion. In this chapter, I will describe the regulation of retroviral Env-induced membrane fusion by proteolytic processing of the Env proteins and by cellular factors.

3.2 Membrane Fusion by Retroviral Env Protein 3.2.1 Cell Surface Receptor All retroviral Env glycoproteins are synthesized as a precursor polyprotein. The polyprotein is then cleaved into surface (SU) and transmembrane (TM) subunits by endoplasmic reticulum-associated cellular proteases. The SU and TM subunits bind each other via disulfide bonds (Wallin et al. 2004). Trimers of the SU-TM heterodimers are expressed on the surface of the viral particles and virus-infected cells (Sodroski 1999). The SU subunits interact with specific cell surface receptor proteins. The TM subunits anchor the Env trimer to membrane surface of viral particles and virus-infected cells, and induce membrane fusion resulting in syncytium formation and viral entry. The SU subunits of almost all HIV isolates utilize CD4 and either CXC chemokine receptor 4 (CXCR4) or CC chemokine receptor 5 (CCR5) as the cell surface receptors (Berger et al. 1999) (Table 3.1). However, HIV variants that can infect CD4-negative cells have been frequently isolated from patients with acquired immunodeficiency syndrome (AIDS) (Xiao et al. 2008, Zerhouni et al. 2004). Such CD4-independent HIV variants recognize CXCR4 or CCR5 as the sole receptor, and might be associated with hepatitis or nephropathy in AIDS patients by infection of CD4-negative liver or kidney cells (Marras et al. 2002, Xiao et al. 2008). CD4independent SIV variants are more frequently isolated from infected animals than

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Table 3.1 Cell surface receptors of retroviruses Retroviruses

Receptors

No. of transmembrane domains

HIV

CD4 CXCR4 or CCR5 CAT1 Pit2 XPR XPR GLUT1 ASCT2

single multiple multiple multiple multiple multiple multiple multiple

ecotropic MLV amphotropic MLV polytropic MLV xenotropic MLV HTLV HERV or syncytin

CD4-independent HIV variants (Edinger et al. 1999, Puffer et al. 2002). Almost all simple retroviruses recognize multi-membrane spanning proteins, like CXCR4 and CCR5, as the entry receptor (Overbaugh et al. 2001) (Table 3.1). Therefore, the CD4-independent HIV variants are thought to be prototypes of the CD4-dependent strains. MLVs are classified into four groups according to their host ranges; ecotropic, amphotropic, polytropic, and xenotropic MLVs. The ecotropic viruses can infect only rodent cells, mouse and rat. The amphotropic and polytropic viruses can infect many species of mammals including mouse, rat, mink, and human. The xenotropic virus can infect mink and human cells, but cannot mouse cells, although the virus has been isolated from mice. Interestingly, it has been recently reported that xenotropic MLV-related virus (XMRV) is isolated from humans with prostate cancer (Schlaberg et al. 2009, Urisman et al. 2006) or chronic fatigue syndrome (Lombardi et al. 2009). The different MLV classes recognize different multimembrane-spanning proteins as the cell surface receptors (Overbaugh et al. 2001); cationic amino acid transporter 1 (CAT1) for the ecotropic MLV (Albritton et al. 1989), and sodium-dependent phosphate symporter 2 (Pit2) for the amphotropic MLV (Kavanaugh et al. 1994). The polytropic and xenotropic MLVs recognize a same cell surface receptor protein, called the xenotropic/polytropic receptor (XPR), whose function remains unknown (Battini et al. 1999, Tailor et al. 1999). The polytropic Env can interact with the cell surface receptor of many types of mammals, and the xenotropic Env recognize the same receptor proteins of many mammals except for mouse. Other retroviruses of importance utilize other cell surface proteins. Glucose transporter protein 1 (GLUT1) has been identified as the HTLV cell surface receptor (Manel et al. 2003). The HERV type W Env protein or syncytin, which induces syncytiotrophoblast formation in placenta, interacts with a sodium-dependent transporter protein of neutral amino acids, alanine, serine, and cystein (ASCT2) (Lavillette et al. 2002). Almost all of the retroviral receptors are N-linked glycoproteins, and infections by many retroviruses are suppressed by sugar chains linked to their cognate receptors (Kubo et al. 2002, 2007b, Overbaugh et al. 2001, Tailor et al. 2003). Glycosylation-defective mutants of the ecotropic MLV receptor (CAT1) transport

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cationic amino acids into cells as efficiently as wild type CAT1 (Wang et al. 1996), indicating that the glycosylation is not required for the natural function. Whereas glycosylation of mouse CAT1 does not inhibit ecotropic MLV infection, glycosylation of the rat, hamster, and Mus dunni receptors do block infection (Kubo et al. 2002, 2004, Yoshii et al. 2008). These results suggest that glycosylation of these receptor proteins functions as innate immunity against retrovirus infections, and the host cell surface receptor proteins have evolved to suppress retrovirus infections. Many retroviruses interact with multi-membrane spanning transporter proteins as the entry receptors (Table 3.1). Because a single amino acid mutation in such multimembrane-spanning receptor proteins often abrogates their expression, and because transporter proteins have house-keeping functions to uptake essential nutrients into cells, the rate of evolution for the retroviral receptor genes would be expected to be relatively slow. If cellular proteins that have relatively higher evolution rates were used as retroviral entry receptors, the cellular proteins would easily evolve to become resistant to the viral infection, and such retroviruses would not survive. Due to the slow evolution rate, retroviruses recognizing multi-membrane spanning transporter proteins as the entry receptors continue to circulate (Yoshii et al. 2008).

3.2.2 Membrane Fusion Mechanism The SU-receptor interaction triggers a conformational change of the TM subunit (Fig. 3.3). The N-terminal hydrophobic fusion peptide of the TM subunit is exposed by this conformational change and inserts into the target cell membrane. The TM subunit adopts a hairpin-like structure through interaction of its N- and C-domains (Fig. 3.4). By this pathway of TM conformational changes, viral and cellular membranes are brought into close apposition and then are mixed. A fusion pore is then formed and expanded, allowing the retroviral core containing the viral genomes and enzymes required for replication to enter into the host cell cytoplasm. A peptide derived from the N- or C-domain of TM subunit inhibits retrovirus membrane fusion by suppressing the hairpin interaction of TM subunit N- and C-domains (Sodroski 1999).

Fig. 3.3 Mechanism of membrane fusion by the retroviral Env protein

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Fig. 3.4 Conformational change of TM subunit

Cell–cell fusion by the retroviral Env proteins should be induced by the same mechanism as virus entry, and therefore quantitative analysis of the Env proteininduced cell fusion is often used as an indicator of the retroviral entry into host cells. However, entry of many retroviruses into host cells is dependent on low pH, but syncytium formation by these retroviral Env proteins is not (Ragheb and Anderson 1994). Further, a non-infectious mutant of MLV Env can induce syncytium formation in a specific cell line (Kubo and Amanuma 2003). Therefore, the syncytium formation capability of the retroviral Env proteins does not always reflect viral entry into host cells, indicating that syncytium formation mechanisms of retroviral Env proteins are partially different from the membrane fusion mechanism that occurs during infection and entry.

3.3 Regulation of Retroviral Membrane Fusion by Proteolytic Processing 3.3.1 Processing of Precursor Env Polyprotein As mentioned above, the retroviral Env precursor polyprotein is proteolytically cleaved by cellular proteases in endoplasmic reticulum. Furin is one of the cellular proteases that process the HIV Env precursor protein into the SU and TM subunits (Hallenberger et al. 1992). However, the HIV Env precursor protein is also cleaved in furin-defective cells, indicating that other cellular proteases are capable of digesting the HIV Env precursor protein (Gu et al. 1995, Ohnishi et al. 1994). The other cellular proteases have not been identified yet. Mutant retroviral Env proteins that cannot be cleaved into SU and TM subunits cannot induce syncytium formation in susceptible cells (Dubay et al. 1995, Freed

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and Risser 1987, Goodman et al. 1993, Guo et al. 1990). These findings indicate that the proteolytic cleavage of retroviral Env precursor proteins is essential for membrane fusion activity.

3.3.2 R Peptide Cleavage The MLV TM subunit contains a 16-amino acid peptide at its C-terminus, called the R peptide, which is cleaved by the viral protease during virion maturation (Green et al. 1981, Henderson et al. 1984). Expression of an R peptide-truncated Env protein of ecotropic or amphotropic MLV in susceptible cells induces syncytia. In contrast, R peptide-containing Env cannot induce syncytia, indicating that the R peptide completely inhibits Env syncytium formation activity (Ragheb and Anderson 1994, Rein et al. 1994). R peptide cleavage-defective MLV Env mutant proteins have much lower infectivity than those with wild-type Env, even when the mutant Env proteins are incorporated into viral particles as efficiently as the wildtype Env (Kieman and Freed 1998, Kubo and Amanuma 2003, Kubo et al. 2007a). These results indicate that R peptide cleavage during virion maturation is required for virus entry into host cells. Expression of MLV Env proteins lacking the R peptide causes fusion of the virus-infected cells and neighboring cells, resulting in apoptotic cell death. As a consequence, production of progeny virus is reduced. These data suggest that the R peptide on Env is essential for progenitor virus production. The R peptide cleavage-defective mutants of polytropic and xenotropic MLV Env have much lower infectivity than the wild-type Env (Kubo et al. 2007a). This suggests that R peptide cleavage of polytropic and xenotropic MLV Env proteins activates the fusion activity, like ecotropic and amphotropic Env proteins. However, the R peptide truncated forms of polytropic and xenotropic Env proteins cannot induce syncytia in susceptible cells (Kubo et al. 2007a). Since the polytropic and xenotropic MLVs recognize the same cell surface receptor, the cell surface receptor could be the determinant for the inability of R peptide truncated polytropic and xenotropic Env proteins to induce syncytia. The C-terminal tails of Env proteins of spleen necrosis virus, gibbon ape leukemia virus, porcine endogenous retrovirus (Bovkova et al. 2002), and MasonPfizer monkey virus (Brody et al. 1994) are cleaved during virion maturation and inhibit membrane fusion activities of the Env proteins, like the MLV R peptide. Env proteins of feline leukemia virus and rat leukemia virus likely also have R peptide, because amino acid sequences of the cytoplasmic domains of these Env proteins are highly homologous to that of the MLV Env protein. C-terminal truncated forms of syncytin, involved in syncytiotrophoblast formation, show enhanced fusogenicity (Drewlo et al. 2006). These results suggest that the cytoplasmic tails of these retroviral Env proteins inhibit their membrane fusion activities. Cytoplasmic domains of HIV, SIV, HTLV, and feline immunodeficiency virus (FIV) Env proteins also inhibit the membrane fusion activity, though syncytium formation is not completely abolished (Hahn and Compans 1992, Kim et al. 2003,

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Lerner and Elder 2000, Ritter et al. 1993, Zingler and Littman 1993). Unlike the MLV R peptide, the Env cytoplasmic domain of these viruses is not cleaved during virion maturation. The membrane fusion inhibition by the cytoplasmic tail of HIV Env protein is abrogated by Gag protein processing by the viral protease (Murakami et al. 2004). Interaction between the Env cytoplasmic domain and Gag precursor protein could inhibit the membrane fusion activity of the HIV Env protein. Processing of the Gag precursor protein by the viral protease could release the Gag-Env interaction resulting in activation of membrane fusion. Together, these results indicate that the function of the Env cytoplasmic domains to inhibit membrane fusion activity is conserved among many retroviruses, and suggests that this inhibitory activity is important for retroviral proliferation, although the regulation mechanisms are different among different retroviruses.

3.3.3 Syncytium Formation in XC Cells by MLV The ecotropic MLV Env protein that contains the R peptide does not induce syncytia in almost all susceptible cells as mentioned above, but specifically can induce syncytia in rat XC and fu-1 cells (Jones and Risser 1993, Wong et al. 1977). By this property of XC cells, XC cells are widely used to titrate the ecotropic MLVs (Klement et al. 1969). Ecotropic virus-infected XC cells form syncytia and die resulting in plaques. The number of plaques correlates with the number of infectious viral particles. Because XC and fu-1 cells were both derived from muscle tissue (Svoboda 1960, Wong et al. 1977), cellular factors that regulate formation of multinucleated muscle fiber cells in myogenesis might be associated with the ability to form syncytia by the R peptide-containing Env protein. Tunicamycin, a N-linked glycosylation inhibitor, suppressed XC cell-specific syncytium formation, suggesting unknown glycosylated cellular factors are involved in the cell fusion (Kubo et al. 2003a). Syncytium formation in myoblast differentiation is also affected by glycosylation modulating agents (Spearman et al. 1987). The cell–cell fusion mechanism of R peptide-containing Env in XC cells is clearly different from the membrane fusion mechanism required for the viral entry into host cells, because virus bearing R peptide-containing Env protein cannot initiate infection, even in XC cells (Kubo and Amanuma 2003). Understanding the mechanism by which the R peptide-containing MLV Env induces syncytia in XC cells could contribute to resolving mechanisms involved in myogenesis.

3.3.4 Mechanism of R Peptide to Inhibit Membrane Fusion The disulfide bond within the SU-TM complex is isomerized during membrane fusion. It has been reported that isomerization of the SU-TM disulfide was suppressed in the R peptide-containing Env protein (Loving et al. 2008), and that a monoclonal antibody against the MLV SU protein more efficiently binds to the R

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peptide-truncated Env protein than to the R peptide-containing Env protein (Aguilar et al. 2003). These results suggest that the R peptide in the TM subunit controls the conformation of SU subunit through the disulfide bond. However, mutants of the R peptide-containing Env protein that have amino acid substitutions in the fusion peptide cannot induce syncytia in XC cells (Jones and Risser 1993), indicating that the fusion peptide of the R peptide-containing Env is exposed by the conformational change and inserted into cellular membrane of XC cells, like the R peptide-truncated Env (Fig. 3.3). In addition, the MLV R peptide inhibits the membrane fusion activity of SIV Env and influenza virus hemagglutinin proteins (Li et al. 2006, Yang and Compans 1996). It is unlikely that the MLV R peptide artificially fused to the heterologous viral spike proteins controls conformational changes of the chimeric proteins, as it does in the natural Env protein. Rather, unknown cellular factor(s) that interact with the R peptide might inhibit the membrane fusion activity of the Env protein. In XC cells, the inhibitory cellular factor might be attenuated by unknown glycosylated protein(s). Further studies are needed to understand the mechanisms by which the R peptide inhibits membrane fusion activity and the R peptide-containing Env protein induces syncytia in XC cells. The presence of cholesterol in the target cell membrane is required for HIV entry (see below). A cholesterol-binding agent, amphotericin B methyl ester, inhibits HIV infection by suppressing membrane fusion (Waheed et al. 2006). An amphotericinresistant HIV variant has been isolated, which has acquired the R peptide (Waheed et al. 2007). The Env cytoplasmic domain of this variant is cleaved by the viral protease during virion maturation, and the membrane fusion capability of the Env protein is activated. Thus, amphotericin B methyl ester could function to inhibit the conformational change of the HIV Env protein into the fusogenic form, and the cytoplasmic domain truncation of Env by the viral protease could rescue this conformational change in the presence of the cholesterol-binding agent.

3.3.5 Cleavage by Cathepsin Proteases Endosome acidification participates in infections by many retroviruses, such as ecotropic MLV (Katen et al. 2001, McClure et al.1990), avian leukosis virus (Mothes et al. 2000), mouse mammary tumor virus (Wang et al. 2008), Jaagsiekte sheep retrovirus (Bertrand et al. 2008), and equine infectious anemia virus (Brindly and Maury 2005, Jin et al. 2005). This suggests that entry of these retroviruses occurs through acidic endosomal compartments, and endosome acidification potentiates membrane fusion activity of Env (Fig. 3.5). Influenza virus infection also requires endosome acidification. Influenza virus cannot induce syncytia in susceptible cells at natural pH, but low pH treatment of influenza virus particles induces cell–cell fusion, indicating that the low pH treatment directly triggers a conformational change into a membrane fusion-inducing form. However, low pH treatment of retroviral particles does not induce cell fusion, suggesting that low pH is required but is not sufficient for the membrane fusion. Recently it has been reported that

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Fig. 3.5 Entry pathways of HIV and MLV

ecotropic MLV infection and syncytium formation are significantly suppressed in cathepsin B-knockout cells, and in cells treated with a cathepsin B inhibitor, CA074Me (Kumar et al. 2007). These data indicate that cathepsin B is required for membrane fusion activity by the ecotropic MLV Env protein. Cathepsin L is also required for the ecotropic MLV infection in cathepsin B-negative cells, suggesting that either cathepsin B or L is sufficient for the ecotropic virus infection (Yoshii et al. 2009). Activation of these cathepsin proteases requires low pH in acidic endosomes. These findings suggest that digestion of the Env protein by cathepsin B or L protease in acidic endosomes induces a full conformational change of the ecotropic Env protein to the membrane fusion active form. HIV infection appears to occur through the endosomal compartment, as with other retroviruses (Miyauchi et al. 2009). However, inhibitors of endosome acidification do not attenuate the HIV infection, but instead enhance it (Fredricksen et al. 2002, Wei et al. 2005). This result suggests that HIV entry into the host cell cytoplasm should occur at the plasma membrane or before the HIV particle-containing endosomes progress to acidic late endosomes through endocytosis (Fig. 3.5). If so, cathepsin proteases are not required for the membrane fusion by the HIV Env protein, because cathepsin proteases are activated by endosome acidification. Indeed, cathepsin inhibitors did not block HIV infection (unpublished data). However, multiple lines of evidence show that secreted cathepsins potentiate HIV infection and induce CD4-independent HIV infection of CD4-negative mammary epithelial cells (Ei Messaoudi et al. 2000, Moriuchi et al. 2000). These results suggest that cleavage of HIV Env by secreted cathepsin proteases triggers conformational changes to the fusion-active form without binding to CD4. Cell–cell fusion needed for multi-nucleated muscle fiber cell formation is impaired in cathepsin B-knockdown cells (Gogos et al. 1996) and by the CA-074Me

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cathepsin B inhibitor treatment (Jane et al. 2002). This result shows that cathepsin B is also required for the cell fusion in myogenesis, suggesting that the mechanism of syncytium formation in myogenesis is similar to fusion induced by the retroviral Env protein.

3.4 Regulation of Retroviral Membrane Fusion by Cellular Factors 3.4.1 Lipid Raft Cellular membranes contain many different types of lipid components and these lipids does not distribute uniformly within membranes. Cholesterol and sphingolipids are enriched in specific regions of the plasma membrane, called lipid rafts. Glycophosphatidylinositol-anchored proteins and signaling proteins are specifically localized to the raft domains, and lipid rafts play important roles for many biological events (Simons and Ikonen 1997). Lipid rafts could also be associated with the retroviral membrane fusion. Methyl-β-cyclodextrin extracts cholosterol from the plasma membrane, destroying raft structure. Treatment of target cells with methyl-β-cyclodextrin inhibits infection and cell fusion mediated by HIV and MLV Env proteins (Kamiyama et al. 2009, Lu et al. 2002, Manes et al. 2000, Popik et al. 2002, Viad et al. 2002). Treatment of target cells with a cholesterol-binding agent, amphotericin B methyl ester, also inhibits HIV infection and membrane fusion (Waheed et al. 2006, 2007). L-Cycloserine, an inhibitor of serine palmitoyltransferase that inhibits the first enzyme of the sphingolipid pathway, blocks HIV-induced syncytium formation and infection (Mizrachi et al. 1996). 1-Phenyl-2-palmitoylamino 3-morpholine 1-propanol and N-butyldeoxynojirimycin, which inhibit glucosyl transferase activity to ceramide, suppress HIV entry and membrane fusion (Fischer et al. 1995, Hug et al. 2000, Puri et al. 2004). These results reveal that raft microdomains in the host cell plasma membrane are required for membrane fusion by the HIV Env protein (Fig. 3.6). In addition, treatment of HIV particles with methyl-β-cyclodextrin inhibits their infectivity, indicating that raft microdomains of the HIV envelope membrane are also required for membrane fusion (Guyader et al. 2002). Further, treatment of host cells by a cholesterol synthesis inhibitor, statin, attenuates HIV entry (Del Real et al. 2004). Statin also inhibits protein prenylation, and the function of the signaling molecule Rho requires prenylation. Therefore, statin treatment also inhibits Rho signal transduction. It is thought that the inhibitory effect of statin on HIV entry is through suppression of Rho functions, but not by inhibiting choresterol synthesis. After the HIV Env proteins bind to host cells, the receptor proteins are gathered to the raft domains through a cytoskeleton-dependent process (Manes et al. 2000, Popik and Alce 2004, Viad et al. 2002). As a result, multiple Env-receptor interactions are induced and membrane fusion efficiently takes place in the raft domain. Therefore, the raft domains serve as sites where the membrane fusion occurs by the HIV Env proteins (Fig. 3.6).

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Fig. 3.6 Structure of HIV particle pre-fusion complex

CD4 is specifically localized to raft microdomains prior to HIV Env binding, but CXCR4 is not (Del Real et al. 2002, Manes et al. 2000, Nguyen and Taub 2002. It has been reported that HIV infection does not occur in target cells expressing a CD4 mutant that localizes outside raft microdomains (Del Real et al. 2002). These results suggest that raft localization of CD4 is required for HIV infection. However, another CD4 mutant protein that is not localized to raft microdomains has been reported to serve as the infection receptor as efficiently as wild-type (raft localized) CD4 (Percherancier et al. 2003, Popik and Alce 2004), suggesting that raft localization of CD4 is not necessarily required for HIV entry. However, methyl-β-cyclodextrin also inhibits the HIV infection in cells expressing the nonraft localized CD4 mutant, suggesting that HIV-cell fusion occurs through raft domains regardless of CD4 localization (Kamiyama et al. 2009, Popik and Alce 2004). Recently, it has been shown that methyl-β-cyclodextrin more significantly suppresses CD4-independent CXCR4-tropic HIV infection than CD4-dependent infection, suggesting that CXCR4 recruitment to the raft domain is primarily required for the HIV-1 infection (Kamiyama et al. 2009). Further studies are required to define the role of raft domains in HIV Env mediated-membrane fusion.

3.4.2 Cell Adhesion Molecules Because syncytium formation is induced between retroviral Env-expressing cells and neighbor susceptible cells, cell–cell contact enhanced by cell adhesion molecules could affect the cell fusion activity of retroviral Env proteins. A kind of integrin family cell adhesion molecule, LFA-1, facilitates HIV Env-mediated

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syncytium formation and viral entry (Fortin et al. 1999, Pantaleo et al. 1991). ICAM-1, a ligand of LFA-1, is incorporated into HIV particles (Beausejour and Tremblay 2004), and enhances the viral entry through the interaction with LFA-1 (Fortin et al. 1997, Paquette et al. 1998) (Fig. 3.6). The interaction between VLA4 and the cognate receptor VCAM-1 also enhances the HIV entry into host cells. Many other cell adhesion molecules, ICAM-2 (Butini et al. 1994), ICAM-3 (Biggins et al. 2007, Butini et al. 1994, Sommerfelt and Asjo 1995), LFA-3 (Kalter et al. 1991), CD44 (Rivadeneira et al. 1995) were reported to be associated with the HIV Env-mediated cell fusion or entry. All of these cell adhesion molecules enhance cell fusion activity, because the cell adhesion molecules strengthen the contact between Env- and receptor-expressing cells.

3.4.3 Cytoskeleton-Associated Molecules After a HIV particle binds to the host cells, the receptor proteins are gathered to the HIV-binding site. An actin polymerization inhibitor, cytochalasin D, attenuates the receptor clustering and the HIV entry, indicating that actin-dependent receptor clustering is required for HIV Env-mediated membrane fusion (Iyengar et al. 1998, Jolly et al. 2004, Kizhatil and Albritton 1997, Lehmann et al. 2005) (Fig. 3.6). The microtubule cytoskeleton also regulates HIV Env-mediated cell fusion and entry (Valenzuela-Fernandez et al. 2005). However, there is no evidence showing that the HIV receptor proteins directly interact with the cytoskeleton. Linker molecules between the HIV receptor and cytoskeleton are involved in the HIV Env-induced membrane fusion. Filamin A has been reported to function as the linker molecule between the HIV receptor proteins and cytoskeleton, and a dominant negative mutant of filamin A attenuated HIV Env-mediated cell fusion (Jimenez-Baranda et al. 2007) (Fig. 3.6). Ezrin, radixin, and moesin (ERM) proteins that function as linker molecules between certain membrane proteins and the cytoskeleton are also associated with the HIV Env-mediated membrane fusion and entry into host cells (Barrero-Villar et al. 2009, Kubo et al. 2008). ERM proteins have also been reported to inhibit nuclear import of the HIV core by stabilizing microtubule formation (Haedicke et al. 2008, Naghavi et al. 2007). The C-terminal verprolin-homologous, cofilin-homologous, and acidic (VCA) domain of the Wiscott-Aldrich syndrome protein (WASP) induces actin polymerization and branching, and has been shown to attenuate HIV infection (Komano et al. 2004). However, the WASP VCA peptide did not inhibit membrane fusion mediated by the HIV Env protein. It was speculated that actin networks induced by the WASP VCA peptide physically suppress nuclear import of the HIV core after virus entry into host cells. Additionally, it has been reported that actin depolymerization by cofilin is required for the nuclear import of the HIV core (Yoder et al. 2008) supporting the above speculation. These results suggest that the actin cytoskeleton positively and negatively controls membrane fusion by HIV Env and nuclear import of HIV core, respectively.

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Protein tyrosine kinase inhibitors, genistein and herbimycin A, suppress retroviral Env-mediated cell fusion and entry into host cells, suggesting that tyrosine kinases are involved in the membrane fusion activity (Cohen et al. 1992, Kubo et al. 2003b, Stantchev et al. 2007). A tyrosine kinase, Lck, enhances HIV Env-mediated syncytium formation (Briand et al. 1997, Yoshida et al. 1992), consistent with the above results. Because tyrosine kinase activity regulates cytoskeleton distribution and movement (Huveneers and Danen 2009), retrovirus Env-induced membrane fusion could be affected by tyrosine kinases through cytoskeleton rearrangement. Cytoskeleton rearrangement is also regulated by Rho signal transduction (Huveneers and Danen 2009). Therefore, cellular factors involved in Rho signaling have been shown to be associated with retroviral Env-mediated membrane fusion (Fig. 3.6). A dominant negative mutant of RhoA inhibits HIV entry into host cells (Jimenez-Baranda et al. 2007). Additionally, inhibition of Rho GTPase by statin attenuates HIV entry (Del Real et al. 2004). ROCK, a signaling molecule in the Rho pathway, stabilizes the actin cytoskeleton, and attenuates HIV infection (Jimenez-Baranda et al. 2007). HIV binding to target cells induces activation of Rac1, another signaling molecule in the Rho pathway (Pontow et al. 2004), and a Rac-1 dominant negative mutant or a Rac-1 inhibitor suppresses HIV Env-induced cell fusion (Pontow et al. 2004, 2007). These findings reveal that Rho signal transduction is associated with HIV Env-mediated membrane fusion through cytoskeleton rearrangement.

3.5 Conclusion Before the fusion between retroviral envelope and host cell membrane is induced by Env, many cellular factors as well as the viral proteins are gathered at lipid raft microdomain (Fig. 3.6). The pre-fusion complex contains cytoskeleton, and cytoskeleton-regulating Rho signal-associated molecules. Other critical cellular factors contained in the pre-fusion complex have yet to be discovered. The cellular factors involved in the pre-fusion complex could be also involved in other cell– cell fusion reactions, including the formation of muscle fibers, syncytiotrophoblasts, and osteoclasts. The detailed understanding of retrovirus-induced membrane fusion could contribute to the development of novel anti-HIV drugs and to the elucidation of the other cell fusion mechanisms involved in tissue developments.

References Aguilar HC, Anderson WF, Cannon PM (2003) Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the R peptide. J Virol 77:1281–1291 Albritton LM, Tseng L, Scadden D et al (1989) A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659–666

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Chapter 4

A Comparative Portrait of Retroviral Fusogens and Syncytins Philippe Pérot, Cécile Montgiraud, Dimitri Lavillette, and François Mallet

Abstract The strongest candidates for developmentally regulated cellular fusogens in mammals are Syncytins which contribute to cell–cell fusion leading to placental syncytiotrophoblast in higher primates, rodents, lagomorphs and sheeps. They consist of domesticated endogenous retroviral envelope glycoproteins (Env) whose fusion properties depend on the initial recognition of a specific receptor. In order to clearly understand Syncytins characteristics, we will first illustrate molecular details characterizing the maturation of class I fusion proteins by introducing envelopedriven fusion in an infectious context, i.e. virus cell fusion, exemplifying each step that lead to functional virions with the most relevant model such as HIV-1 lentivirus or MLV and type D interference group retroviruses. In a second part, we will comparatively present the current knowledge concerning Syncytins and the associated three levels of complexity. First, the placenta is probably more variable in structure than any of the mammalian organs. Second, Syncytins recognize specific and highly function-divergent/unrelated receptors. Third, some Syncytins were shown to exhibit other functions than fusion, such as proliferation, immunomodulation, receptor interference and anti-apoptotic properties. We will conclude by a brief overview of the consequences of Syncytin expression outside of its privileged tissue. Keywords Fusion · placenta · retrovirus · endogenous retrovirus · envelope · Syncytin · enJSRV · receptor · hASCT1 · hASCT2 · MFSD2 · HYAL2 Abbreviations ASCT ALV ASLV BaEV

Alanine, serine and cysteine selective transporters Avian leukosis virus Avian sarcoma leukosis virus Baboon endogenous retrovirus

F. Mallet (B) Laboratoire Commun de Recherche Hospices Civils de Lyon – bioMérieux, Cancer Biomarkers Research Group, Centre Hospitalier Lyon Sud, 69495 Pierre Bénite cedex, France e-mail: [email protected] Cécile Montgiraud and Dimitri Lavillette contributed equally to in this chapter L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_4, C Springer Science+Business Media B.V. 2011

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BLV CA CAT-1 CT cyt DRM EBV ECT en EnCa Env ER ERV ESCRT Exo FeLV FcEV FP GaLV GCM Gp GPI h HELLP HERV HIV HR HTDV HTLV HYAL2 IDO IFN Ig IL IP3 JSRV KoRV LLP LTR m M MA MAO MFSD2 MLV MMTV Mo-MLV

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Bovine leukemia virus Capsid Cationic amino acid transporter-1 Cytotrophoblast Cytoplasmic tail Rafts? Epstein-Barr virus Extravillous cytotrophoblasts Endogenous Endometrial carcinoma Envelope Endoplasmic reticulum Endogenous retrovirus Endosomal sorting complex required for transport Exogenous Feline leukemia virus Felis catus endogenous retrovirus Fusion peptide Gibbon ape leukemia virus Glial cell missing Glycoprotein Glycosylphosphatidylinositol Human Hemolysis, elevated liver enzymes and low platelets Human endogenous retrovirus Human immunodeficiency virus Heptad repeats Human teratocarcinoma-derived virus Human T-cell leukemia virus Hyaluronidase 2 Indoleamine 2,3-dioxygenase Interferon Immunoglobulin Interleukin Inositol-3-phosphate Jaagsiekte sheep retrovirus Koala retrovirus Lentivirus lytic peptides Long terminal repeat Mouse Mesenchyme Matrix Morpholino antisense oligonucleotide Major facilitator superfamily domain containing 2 Murine leukemia virus Mouse mammary tumor virus Moloney murine leukemia virus

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A Comparative Portrait of Retroviral Fusogens and Syncytins

M-PMV MS MSRV MuLV MVB MYA NC NO NWM OASIS ORF OWM PCR PcRV PDI PE Pit RBD RT SERV SIV SRV-1 SNARE SP SRP ST SU T TfR1 TGF TGN Th TM tm TNF TSE UA URE

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Mason-Pfizer monkey virus Multiple sclerosis Multiple sclerosis associated retrovirus Murine leukemia virus Multivesicular bodies Million years ago Nucleocapsid Nitric oxide New world monkeys Old astrocytes specifically induced substance Open reading frame Old world monkeys Polymerase chain reaction Papio cynocephalus retrovirus Protein disulfide isomerase Preeclampsia Sodium-dependent phosphate symporter Receptor-binding domain Reverse transcriptase Simian endogenous retrovirus Simian immunodeficiency virus Simian retrovirus-1 Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor Signal peptide Signal recognition particle Syncytiotrophoblast Surface unit Trophoblast Transferrin receptor 1 Transforming growth factor Trans-Golgi network T helper cells Transmembrane unit Transmembrane domain Tumor necrosis factor Trophoblast specific enhancer Uterine arteries Upstream regulatory element

Contents 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Contribution of the Envelope to the Retroviral Life Cycle . . . . 4.2.1 Synthesis of Env Glycoprotein and Viral Assembly . . . . 4.2.2 Virus-Host Cell Membrane Fusion: A Multistep Mechanism 4.2.3 Rous Meets Mendel . . . . . . . . . . . . . . . . . .

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66 4.3 Syncytins and Cell–Cell Fusion . . . . . . . . . . . . . . . . . . 4.3.1 Integration, Domestication Steps and Biological Functions of Endogenous Viral Glycoproteins . . . . . . . . . . . . . 4.3.2 Fusion Mechanism and Receptor Recognition . . . . . . . . . 4.3.3 Retroviral Envelopes Are Involved in the Placenta Development 4.3.4 Syncytin-1 Expression Outside of Its Privileged Tissue . . . . 4.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4.1 Introduction Our life begins with fusion of our mother’s oocyte with one of our father’s spermatozoids. A consequent successful pregnancy depends on the 10 m2 placental syncytiotrophoblast resulting from fusion of abundant cytotrophoblastic cells. As the embryo develops, skeletal muscle differentiation depends on the fusion of mononucleated myoblasts to form multinucleated muscle fibers. In the adult body, macrophages can fuse to form either multinucleated osteoclasts that control the maintenance of the bones or multinucleated giant cells that are important for the immune response. Last but not least, fusion as a driver of embryonic stem cell differentiation suggests a new role of cell fusion in mammalian development. Overall, cell fusion is a process in which two or more cells become one by merging their plasma membranes. Fusion, with the exception of gametes and stem cells, produces only terminally differentiated, non-proliferating tissue, and is thus mainly involved in tissue maintenance or regeneration. The fused cells (syncytia) that contain several nuclei within a single cytoplasm may be homokaryons (homotypic fusion) or heterokaryons (heterotypic fusion) as derived from the fusion of similar or different origin cell types, respectively. In any case, fusion of two separate lipid bilayers in non aqueous environment first requires that they come in close contact. Second, an intermediate stage is characterized by the merger of only the closest contacting monolayer, a process called hemifusion. Third, the fully completed fusion results in whole bilayer merging following by the opening of the pore. It remains questionable whether cell–cell fusion involves the same type of mechanisms than in other membrane fusion events, such as intracellular vesicle fusion mainly based on SNAREs proteins (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and virus-cell fusion achieved by transmembrane viral fusion proteins (Chen and Olson 2005). However, we might expect that longconserved syncytial structures, such as skeletal muscle that have become integral to the body plans of multiple phyla, may be formed by mechanisms that have been mostly conserved during evolution (Mohler 2009). It is thus striking to notice that little is known about the molecular actors that are involved in regulating and completing cell–cell fusion, and of what is known there is little conservation between species, suggesting that these mechanisms might have evolved independently (Chen et al. 2007). The multiple transmembrane-domain tetraspanin protein CD9 on the egg surface and the single transmembrane-domain protein IZUMO with an extracellular

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immunoglobulin (Ig)-like domain on the spermatozoid surface contribute to the fusion of the mice gametes, although it is still a matter of conjecture whether the two molecules interact directly in trans to achieve the membrane fusion reaction (Inoue et al. 2005). Transmembrane-domain proteins with extracellular Ig-like domains have been implicated in homotypic macrophage fusion in rats (Han et al. 2000) and in cell–cell tethering prior myoblast fusion in drosophila melanogaster (Bour et al. 2000). Interestingly, the strongest candidates for developmentally regulated cellular fusogens in mammals are Syncytins, a family of single-pass transmembrane proteins, which contribute to cell–cell fusion leading to placental syncytiotrophoblast in higher primates, rodents, lagomorphs and sheeps. They consist of domesticated endogenous retroviral envelope glycoproteins whose fusion properties depend on the initial recognition of a specific receptor. With the exception of retrovirus-derived Syncytins, none of the cell surface proteins identified in the various cell–cell fusion processes resemble SNAREs or class I fusion protein, (i.e. fusion does not appear to be mediated by an α-helical bundle). Though, fusogenic proteins contribute to decrease the kinetic barrier to allow the fusion of the two bilayer membranes. Viral fusion proteins do so by using the force energy released during a protein conformational change to draw together the membranes. The understanding of the Syncytins dependant cell–cell fusions will likely parallel the mechanism of at least retroviral infection. Indeed, Syncytins are host domesticated genes derived from ancient retroviruses infections of the host germ line. Syncytins appear to group relatively distinct actors that may exhibit common principles leading to membrane fusion and hence are good examples of the various scheme of evolution to establish similar but different structures (microscopic and macroscopic) with similar roles. Such a dichotomy between distinct players with common principles was indeed proposed for all fusion processes by Martens and McMahon (2008). Three classes of viral fusogens have been described. The class I and II fusion proteins are characterized by trimers of hairpins containing a central α-helical coiled-coil or β-sheets structure, respectively, while the class III fusion proteins have a mixed secondary structure (Weissenhorn et al. 2007). We will first introduce envelope-driven fusion in an infectious context, i.e. virus-cell fusion, by illustrating each step leading to functional virions with the most relevant model such as HIV-1 lentivirus or MLV and type D interference group retroviruses. The purpose is to illustrate molecular details characterizing the maturation of class I fusion proteins, defined by the following four characteristics: the cleavage of an envelope protein precursor leading to surface and transmembrane subunits, a fusion peptide located just next to the cleavage site (except avian ASLV), a trimeric complex association, and the ability to form a hairpin structure, also called a coiled-coil structure, in its active fusion conformation. The progression of these structural rearrangements slows down the kinetic barrier between hemifusion and fusion-pore formation. Intriguingly, without unequivocal evidence of infectious agent, retroviral particles were observed in physiological situation (Lyden et al. 1994) but also in pathological ones (Perron et al. 1989; Boller et al. 1993) in man. They could derived from endogenous retroviral sequences, as the human genome (Lander et al. 2001) but also the mouse genome (Waterston et al. 2002) contain a huge amount of

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endogenous retroviruses, reaching 8.5 and 9% of these genomes sizes, respectively. Although human and mouse species contain essentially different retroviral families, some of their coding sequences are still intact, e.g. 18 envelopes ORFs were identified in the human genome (Blaise et al. 2003; Blaise et al. 2005) including the two human Syncytins. This huge repertoire awaiting the identification of functions will be illustrated, as it may represent a third condition beside infectious viruses and domesticated envelope Syncytins. In a second part, we will comparatively present the current knowledge on Syncytins. Outstandingly, the situation comprises at least three levels of complexity. First, the placenta is probably the more variable in structure than any of the mammalian organ. Placentas are variously classified, as regard to (i) their form, being discoid (primates, lagomorphs and rodents) or cotyledonary (ruminant), (ii) the type of layer between fetal trophoblast and maternal endometrial surface, hemochorial (primates, lagomorphs and rodents) or epithelio- and syndesmochorial (ruminants), and (iii) the structure of the maternal-fetal interdigitation, villous type or villi (primates and sheep) or labyrinth (lagomorphs and rodents) (Bernirschke K, Comparative placentation at http://placentation.ucsd.edu). The latter, a continuous syncytiotrophoblast layer that covers the entire surface of the human placental villi which floats in maternal blood, is responsible of ion and nutrient exchanges and synthesizes steroid and peptide hormones such as progesterone and human chorionic gonadotropin (hCG) required for human gestation. Second, Syncytins recognize specific and highly function-divergent/unrelated receptors. In human, Syncytin-1 recognizes hASCT1 (Blond et al. 2000) and hASCT2 (Lavillette et al. 2002) receptors while Syncytin-2 binds to MFSD2 receptor (Esnault et al. 2008). In rodents, Syncytin-A and Syncytin-B possess unidentified but distinct receptors (Dupressoir et al. 2005), and in lagomorphs Syncytin-Ory1 functionally recognizes hASCT2 (Heidmann et al. 2009). In sheep, enJSRV envelope(s) interacts with HYAL2 (Dunlap et al. 2006). This illustrates that proteins involved in cell–cell fusion, such as Syncytin partner receptors, are likely to play pleiotropic roles in other cellular processes, e.g. transport of small molecules, but also modulation of membrane structures, with specificity being achieved through the coupling of these proteins to different upstream and downstream effectors. Third, Syncytins were shown to exhibit other functions than fusion, such as proliferation (Strick et al. 2007; Larsen et al. 2009), immunomodulation (Mangeney et al. 2007), receptor interference (Blond et al. 2000; Ponferrada et al. 2003) and anti-apoptotic properties (Strick et al. 2007; Knerr et al. 2007), these functions being not shared by all these proteins. We will conclude by a brief overview of the consequences of Syncytin-1 expression outside of its privileged tissue.

4.2 Contribution of the Envelope to the Retroviral Life Cycle Retroviral classification was initially based on virion morphology observed with electronic microscopy during maturation and assembly of particles (Coffin 1992). Accordingly, retroviruses are designated A-, B-, C- and D-type. The

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International Comity of Taxonomy has now established seven genera of Retroviridae based on sequence homologies: Alpharetoviruses correspond to avian type C (Avian leukosis virus, ALV) retroviruses, Betaretroviruses to type B (Mouse Mammary Tumor Virus, MMTV) and D (Simian retrovirus-1, SRV-1) retroviruses, Gammaretroviruses to mammalian type C retroviruses (Murine Leukemia Virus, MLV), Deltaretroviruses to the ancient group of HTLV-BLV (Human T-cell Leukemia Virus/Bovine Leukemia Virus), Epsilonretroviruses (Waileye Dermal Sarcoma Viruses family), Lentiviruses group which contains HIV and SIV (Human and Simian Immunodeficiency Viruses) and Spumavirus including Human Foamy Viruses (van Regenmortel et al. 2000). Retroviruses are RNA enveloped viruses. They infect cells via a cellular receptor recognition followed by the fusion of virus and cell membranes. Upon entry, the next step of the retroviral life cycle consists of a retrotranscription stage mediated by the viral reverse transcriptase protein that converts the viral genomic RNA in double strand DNA. Subsequently, the viral genetic material is targeted to the nucleus and stably integrated in the host cell genome by the viral integrase. The integrated viral DNA is named provirus and is flanked by two Long Terminal Repeats (LTR) that act as transcriptional regulatory elements. The 5 LTR contains the promoter and enhancer signals while the 3 LTR contains the polyadenylation signal terminating the transcription. All the replication competent retroviruses contain at least three genes coding for the structural proteins (gag), the enzymatic proteins (pro-pol) and the envelope glycoprotein (env). During its life cycle the virus uses the gene replication machinery of the host cell. Herein, we will focus on the characteristics of the envelope (Env) protein that is composed of one surface unit (SU) and one transmembrane unit (TM) which is itself subdivided into three domains, an ectodomain, a strict transmembrane domain (tm) and a cytoplasmic tail (cyt) (Fig. 4.1a). Env glycoprotein will undergo several modifications to generate a mature and functional glycoprotein addressed to the plasma membrane in order to contribute to the virus infection-competency. Functionally, the SU domain is involved in receptor recognition and the TM subunit contains both the fusion peptide and the heptad repeat domains involved in fusion.

4.2.1 Synthesis of Env Glycoprotein and Viral Assembly During virus production, the host cell is basically preserved since the expression of fusogenic competent glycoproteins is highly controlled. Sequentially, the Env protein synthesis is initiated by the free-ribosomes, next modifications take place in the endoplasmic reticulum and then an oligomerized precursor is transported by vesicles to the golgi apparatus. Abundant glycoprotein at the surface of the cell could induce cellular death by syncytia formation, toxicity via receptor interaction, or immune recognition. That’s why the localization and the amount of the oligomerized retroviral envelope glycoprotein at the host cellular surface are highly modulated by fine trafficking and sequestration mechanisms. The receptor interference mechanism

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Fig. 4.1 Structure and synthesis of retroviral envelope. (a) Schematic portrait of an envelope prototype. SP, signal peptide (grey). SU, surface unit (yellow): contains RBD, receptor binding domain (gold yellow) and C, C-terminal domain (light yellow) with CXXC motif (generally CC with = L,I,V,F,M or W). (K/R)X(K/R)R, furin cleavage site. TM, transmembrane unit (hatched boxes): contains FP, fusion peptide (red); leucine zipper motif with HR1 (blue) and HR2 (green) heptad repeats followed by the CX6 CC motif; tm, trans-membrane anchorage domain (red, hatched); cyt, cytoplasmic tail with C-terminal R peptide (blue) containing YXX motif. Note that the ectodomain part of the TM contains a so-called immunosuppressive domain QNRX2 LDXLX5 GXC joining the CX6 CC motif (not illustrated). (b) Schematic maturation process of the envelope glycoprotein. The successive immature forms of the envelope glycoprotein are illustrated (petrol blue). Initial glycosylation sites (branch trees with open circles), disulfide bonds (C–C), palmitoylation sites (broken lines) and final glycosylation sites (branch trees with dark circles) are indicated. Color codes and abbreviations used in the final trimeric structure expressed at the plasma cell membrane are as given in a; post translational modifications and disulfide bonds are omitted

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can also limit the amount of receptors available for fusion between infected cells. Finally, Env fusion competency may be a late event that occurs during virus budding as described for MLV. 4.2.1.1 Synthesis and Maturation of Env Glycoprotein As common cellular proteins, retroviral Env translation is initiated by free ribosomes in the cytosol. The signal peptide (SP) located in the N-terminus of the Env glycoprotein is the first synthesized segment. This initial step and the following ones are illustrated in Fig. 4.1b. SP length varies depending on the retrovirus family but its composition is conserved with an hydrophobic signal, recognized by the SRP (Signal Recognition Particle), that allows the anchorage of the nascent chain to the endoplasmic reticulum (ER). The nascent protein is translocated through the membrane of ER. At the end of the synthesis, the extracellular part of Env is folded in the lumen of the ER, as for cellular membrane proteins. The release of the protein in the lumen is impaired by a stop transfer region composed of a hydrophobic sequence followed by aromatic and charged amino acid which will delimitate the membrane anchored domain or transmembrane domain (tm) (Hunter and Swanstrom 1990). This tm domain is an α–helix constituted by at least 23 amino acids (for HIV-1) and a maximum of 36 amino acids (for MMTV) but it contains unexpected residus for alpha helix structure in the context of a membrane (helix breaker amino acids like glycine and proline or positively charged tryptophane or cysteine). The Env N-extremity (ectodomain in the future virion) is then located in the lumen of ER while the C-terminal part (cyt) of the protein remains in the cytosol. ER is then the site of co- and post-translational modifications such as N-glycosylations, protein folding, disulfide bonds formation and oligomerization (Ratner 1992). After the SP cleavage, the precursor is modified by N-glycosylations. Depending on the retrovirus, the number and the location of glycosylation sites are variable. For HIV-1, the protein presents an unusual highly glycosylation with 24–32 sites and sugars account for half of the molecular weight of the Env protein (Mizuochi et al. 1990). For the other retroviruses, the number of glycosylation is around 8. Almost all glycosylations are in the SU, and except for gammaretroviruses for which there is no glycosylation in the TM, others have 1 (Betaretroviruses like BaEV) to 7 (HIV-1) glycosylations in TM reflecting the weak exposition of this subunit hidden by SU. In all cases, glycosylations are essential for the folding, the trafficking, the cleavage and the recognition of the receptor by the Env protein (Polonoff et al. 1982). For example, N-linked glycans are critical determinants for the efficient recognition of CD4 T cells by HIV-1 gp120, since mutant protein lacking one N-glycan did not effectively stimulate CD4+ T cells (Li et al. 2008a). For MLV, due to the fewer number of glycosylations than for HIV-1 Env, their roles have been more characterized and if some glycosylations are not crucial for incorporation, they are involved in the stability of the postcleavage envelope complex (Li et al. 1997). Following glycosylation events, intramolecular disulfide bonds are formed in SU and TM subunits to generate loops in the secondary structure of the envelope protein. The cysteines of the SU involved in these bonds are well conserved in a

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subgroup of retroviruses suggesting a similar domain organization. These cysteines are crucial for the folding and the transport of the envelope to different cellular compartments. The substitution of some cysteines in SU and TM of HIV-1 (Bolmstedt et al. 1991; Dedera et al. 1992) or other retroviruses like MuLV (Thomas and Roth 1995; Freed and Risser 1987) leads to a non cleaved envelope glycoprotein precursor retains into the cells. Finally, the loops generated by the disulfide bonds are essential for cellular receptor recognition (MacKrell et al. 1996). In addition, the TM of γ– and some β–retroviruses (but not for lentiviruses) contains specific cysteines that are important for SU-TM intermolecular association as it will be developed later. The disulfide bond formation is part of a more global control of modifications of the retroviral Env by the cell (Braakman and van Anken 2000). This control involved protein disulfide isomerase (PDI) (Fenouillet et al. 2007), and also chaperone molecules like GRB78 Bip (Earl et al. 1991), calnexin (Li et al. 1996) and calreticulin (Otteken and Moss 1996) as described for HIV-1. The failure to pass the quality control leads to a non cleaved envelope glycoprotein precursor that is kept either in endoplasmic reticulum or in the golgi apparatus. Retroviral Env glycoprotein leaves the ER in a trimeric form to reach the Golgi where N-glycans are matured, O-glycans added (Pinter and Honnen 1988; Bernstein et al. 1994) and the cysteines at the hedge of the tm domain are palmitoylated. (Yang et al. 1995) (Fig. 4.1b). The appropriate trimeric conformation (with glycosylation and disulfide bonds) being obtained, the precursor is then cleaved at a highly conserved site (Fig. 4.1a) among retroviruses by the furin-convertase protein into its two subunits e.g. gp120 (SU) and gp41 (TM) for HIV-1. The Env complex of HIV1, as described for most retroviral Env glycoproteins, is trimeric with six individual subunits (three gp120 and three gp41 subunits). It is the TM subunit that triggers the oligomerization, as the TM (associated or not to the SU) is always detected in the oligomerized forms (Einfeld and Hunter 1988; Earl et al. 1990). The main determinant of this trimerization is a region in heptad repeat (Gallaher et al. 1989; Poumbourios et al. 1997) with high homology with leucine zipper domain (Fass et al. 1996; Weissenhorn et al. 1997; Owens and Compans 1990). Interestingly, the SU is also a trimer when it is shedded (Tucker 1991; Owens and Compans 1990) whereas soluble SU expressed alone is usually a monomer (Poumbourios et al. 1997). Hence, TM initiates the trimerization and, after that, SU can stay as a noncovalently linked fragile trimer. The trimerization gives the required environment for the fusion by masking the fusion peptide that will be later freed following receptor binding and also confers the fusogenic potential to the glycoprotein. The two SU and TM subunits are either linked in a covalent or non-covalent way. For HIV-1, the existence of the soluble gp120 protein indicates a non-covalent link between SU and TM (Kowalski et al. 1987). The regions implicated in this interaction are principally the C1 and C5 region of the SU and the leucine zipper domain and the CX5 C region of the TM (Lopalco et al. 1993; Schulz et al. 1992). For most others retroviruses a covalent link was described at one point. In all the case, except MMTV and JSRV, a disulfide bond between the SU and the TM is formed between the highly conserved CX6 CC motif of the TM and the CXXC of the SU (Sitbon et al. 1991; Schulz et al. 1992; Pinter et al. 1997). This CXXC motif is extremely rare in cellular proteins

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and is similar to a motif found in the catalytic site of enzyme involved in thiol isomerisation like PDI or thioredoxin (Sanders 2000; Pinter et al. 1997). This motif in the SU has been shown to be part of an autocatalytic isomerisation function of SU to destroy the initial bond between SU and TM that was established during Env synthesis and to create an intra-SU bond inside the CXXC motif (Wallin et al. 2004; Li et al. 2008b). This disulfide bond isomerisation is crutial for the fusogenicity of gammaretrovirus (MLV) (Fenouillet et al. 2008). It can be interesting to mention that HIV-1 bond can be reconstituted after recreating the motif in SU and TM (Binley et al. 2000). 4.2.1.2 Cellular Localization of Env Glycoprotein and Viral Assembly Complex multilevel interactions have been described between Env, Gag and sorting proteins involved in traffic of molecules or vesicles inside the cells. These proteins are involved in both Env trafficking and virus budding. To limit the quantity of Env at the cell surface, the Env undergoes endocytosis and is trafficked in endosomal pathways. These cell localizations are driven by traffic peptidic motifs, like for cell proteins, that have been characterized to direct cellular transmembrane proteins into different endosomal compartment (Bonifacino and Traub 2003). Lentiviruses, including HIV-1, are unusual in having transmembrane glycoproteins with much longer cyt intracytoplasmic tail (150 amino acids) than most other retroviruses (20–50 amino acids) (Hunter and Swanstrom 1990), suggesting that this domain has one or more functions specific to lentivirus replication or persistence. Two groups of motifs have been identified in HIV-1 cyt. The first group consists of three structurally conserved amphipathic alpha-helical domains, designated as lentivirus lytic peptides 1, 2, and 3 (LLP-1, LLP-2, and LLP-3) (Xu et al. 2006). LLP domains have been implicated in various functions, including Env cell surface expression, Env fusogenicity, and Env incorporation into virus particles (Piller et al. 2000). Several studies have suggested that Env is incorporated into virions via interactions between LLP and the matrix region of Gag. The second group of motifs regulates the intracellular trafficking of Env. At steady state, Env is predominantly located in the trans-Golgi network (TGN) (Takeda et al. 2003). This intracellular distribution results from dynamic cycling of Env between the cell surface, the endosomal compartment, and the TGN. Newly synthesized Env proteins undergo endocytosis after their arrival at the cell surface. Env internalization is mediated by the interaction of Y712SPL (YXX on prototype, Fig. 4.1a), a membrane-proximal tyrosine-based signal in the gp41 cyt tail, with the adaptor protein (AP) complexes of the cellular sorting machinery, involving the clathrin adaptor AP-2 in particular (Berlioz-Torrent et al. 1999). The cytoplasmic tail of many other retroviruses also contains a YXX motif, including gammaretrovirus like MLV (Song and Hunter 2003), RD114 (Sandrin and Cosset 2006), HTLV-1 (Berlioz-Torrent et al. 1999), M-PMV (Song and Hunter 2003; Song et al. 2005) and BLV (Inabe et al. 1999; Novakovic et al. 2004). The gp41 cyt also interacts with the TGN- and endosomebased clathrin-associated adaptor AP-1 via a dileucine motif (Berlioz-Torrent et al. 1999; Wyss et al. 2001) which induces its cellular retention. Some dileucine motifs

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are also present in the cytoplasmique tail of MLV, RD114, M-PMV or BLV (Sandrin and Cosset 2006; Grange et al. 2000). Finally, Y802W803, a diaromatic motif involved in the retrograde transport of Env to the TGN, was also identified in the gp41 cyt (Takeda et al. 2003) and interacts with TIP47, a protein required for the retrograde transport from cell surface to the TGN in link with matrix (MA) interaction (Lopez-Verges et al. 2006). A retrograde acid cluster motif has also been identified in RD114 Env (Bouard et al. 2007) that induces a retrograde transport of the Env from the late endosome via interaction with PACS1 complexes. It should be note that the palmitoylated cysteines located in the tm are also involved in cell distribution of Env by contributing to the association with lipid rafts (Bhattacharya et al. 2004). These rafts seem to serve as platforms for virus assembly and budding (Suomalainen 2002). For example, membranes of HIV-1 virions have a higher cholesterol rate than the original infected cell, and HIV-1 virions produced in cells with synthesis defects in sphingolipid and cholesterol are less infectious (Brugger et al. 2006). If the cellular distribution of Env is linked to interaction with trafficking cell molecules, there is evidence indicating that an interaction between the TM cytoplasmic tail and the MA domain of the viral Gag polyprotein mediates Env packaging into particles. Gag structural HIV-1 polyprotein precursor consists of MA, CA (capsid), NC (nucleocapsid), and p6 proteins. The budding ability of retroviruses requires only Gag proteins. Indeed, Gag expression in the absence of other retroviral proteins is sufficient to the liberation of Env-free virions, but these pseudo virions are released independently of the cellular poles. However, when Env is coexpressed, the budding is restricted to the basolateral membrane and mutations of the cyt tyrosine in the membrane-proximal tyrosine-based signal Y712SPL disturb the polarized release of HIV-1 in polarized epithelial cellular models (Lodge et al. 1997; Owens et al. 1991). It is unclear how viral RNA, Gag and Env proteins reach the same site of the cellular plasmic membrane for perfect assembling and budding of the virion. It should be mention that HIV-1 and SIV budding are not only polarized in epithelial cells. In lymphocytes, the release of viruses is restricted to domain of contact between two cells or even between the cell and the culture plate (Bugelski et al. 1995; Pearce-Pratt et al. 1994). This is a budding in a virologic synapse (Morita and Sundquist 2004) and it is also driven by Env cyt motif. This polarisation of budding might have a physiological importance for the cell–cell transmission of the virus. In the case of MLV virus, a model has been developed that allows a better comprehension of Gag and viral RNA trafficking. It was shown that recruitment of glycoproteins by the gammaretroviral core proteins takes place in the intracellular compartments and not at the cell surface. Moreover, gammaretroviral core proteins could relocalize Env glycoproteins in late endosomes and could allow incorporation on viral particles (Sandrin et al. 2004; Bouard et al. 2007). Finally, it was proposed that the retrovirus budding depends on the cell types but might depend of the status of the infection and condition of the cells. In T-lymphocytes, it was initially described that the assembly and budding takes places at the plasma membrane (Barre-Sinoussi et al. 1983; Gelderblom et al. 1987), but recent reports indicated also an assembly in intracellular vesicle containing virus (Grigorov et al. 2006; Joshi et al. 2009). Similarly, in macrophages, HIV-1 assemble and bud in MVB

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(multivesicular bodies) (Nydegger et al. 2003; Sherer et al. 2003; von Schwedler et al. 2003) and the viruses are then released outside the cell by fusion of the MVB with plasma membrane (Trojan Horse hypothesis). This is in agreement with the suggestion that retroviruses exploit a cell-encoded pathway of intercellular vesicle traffic, exosome exchange, for both the biogenesis of retroviral particles and a lowefficiency mode of infection (Gould et al. 2003; Fang et al. 2007). However, it was recently proposed that the vesicle containing viruses might have different genesis with some vesicles coming from the plasma membrane invaginations (Welsch et al. 2007). The reason for this discrepancies are not clear but involved Gag interaction with membrane, ESCRT (endosomal sorting complex required for transport) localizations, interferon induced proteins and lipidic composition of microdomain (Ono et al. 2004). 4.2.1.3 Fusion Competency Gammaretrovirus virions assemble and bud from the infected cells as immature particles that must undergo an additional proteolytic maturation to become infectious (Brody et al. 1992; Christodoulopoulos and Cannon 2001; Green et al. 1981; Rein et al. 1994). This maturation concerns the viral protease dependent cleavage of the so-called R peptide at the C-terminus of the cytoplasmic tail (Green et al. 1981; Rein et al. 1994) (see location on Fig. 4.1a). The R peptide inhibits the fusion, and different hypotheses have been proposed. Firstly, the R peptide contains the YXXφ internalization motif and the removal of this motif following the cleavage of the R peptide might induce higher amount of envelope at the surface membrane and consequently more fusion (Song and Hunter 2003). Secondly, another explanation is that following the R peptide cleavage, the remaining cyt tail forms a membraneembedded amphiphilic alpha-helix domain destabilizing the membrane (Zhao et al. 1998; Rozenberg-Adler et al. 2008). Thirdly, it was proposed that, as the R peptide contains a palmitoylation site, its removal induces the close trimerization of the cyt tail and drastic conformational changes in the ectodomain of Env (Aguilar et al. 2003) which might influence Env fusogenicity by destabilizing the SU-TM complexes. These conformational changes are necessary for the isomerisation of the SU-TM disulfide MLV Env (Loving et al. 2008). The R peptide cleavage is the last step leading to a fusion competent infectious MLV retrovirus but this final modification does not exist in lentiviruses which harbour a long cytoplasmic tail. However, studies indicate that artificial (HTLV, HIV or SIV) or natural (SIV) shortening of the cytoplasmic tails change the conformation of ectodomain (Edwards et al. 2002; Spies et al. 1994) and increase the fusogenicity of the Env in cell–cell fusion (Kim et al. 2003; Edwards et al. 2002; Spies et al. 1994).

4.2.2 Virus-Host Cell Membrane Fusion: A Multistep Mechanism Glycoproteins from enveloped viruses evolved to combine two main features. They have the capacity to bind with a specific cellular receptor and they harbour a fusion domain (peptide fusion and transmembrane domain) that can be activated to mediate the merging (fusion) of viral and cellular membranes.

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Fig. 4.2 Virus-host cell membrane fusion. a Schematic representation of six prototype stages beginning with the fusion competency acquisition of the envelope glycoprotein (1) based on R peptide release by viral protease and ending with the gathering of viral and cellular membranes (6) induced by the anchorage of the fusion peptide into the cell membrane. Red arrow symbolizes the R peptide cleavage. b Schematic drawing of the successive steps leading to lipidic pore formation. (1) proximal leaflets of viral (green) and cell (black) membranes coming into immediate contact,

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Three different classes of viral fusion proteins have been identified to date based on key structural features at pre- and post-fusion stages. Many studies mentioned that the structural transition from a pre- to a post-fusion conformation leads to a stable hairpin conformation. This concerns the class I fusion proteins, characterized by trimers of hairpins containing a central α-helical coiled-coil structure, and the class II fusion proteins characterized by trimers of hairpins composed of beta sheets structures. A third class of fusion proteins has been described recently, that also forms trimers of hairpins by combining the two structural elements alpha-helix and beta-sheet structures (Weissenhorn et al. 2007). Three main steps are described for achieving the pre- to post conformational changes. The first one, after Env activation upon receptor binding or acidification of the endosomal compartment, exposes the fusion peptide that is projected toward the top of the glycoprotein, allowing the initial interaction with the target membrane (Fig. 4.2a, drawings 1–4). The second one is the folding back of the C-terminal region onto a trimeric N-terminal region (Fig. 4.2a, drawings 5–6) that leads to the formation of a post-fusion protein structure with the outer regions zipped up against the inner trimeric core in an antiparallel coiled coil structure. The final and third step also requires further refolding of the membrane proximal and transmembrane regions in order to obtain a full-length post-fusion structure where both membrane anchors are present in the same membrane (Fig. 4.2b). 4.2.2.1 Receptor Binding and Peptide Fusion Liberation HIV-1 fusion is mediated by specific interaction of the viral envelope glycoprotein with the cell surface CD4 molecule that serves as the primary receptor, and additionally a chemokine receptor CCR5 or CXCR4 as HIV-1 co-receptors. Both receptors and the co-receptor binding sites are on gp120 although the membrane fusion is triggered by conformational changes in the transmembrane protein gp41. The viral entry can be blocked by three categories of agents (Qian et al. 2009) (i) attachment inhibitors/antagonists targeting CD4, CCR5 and CXCR4 (ii) inhibition of the post-binding conformational changes, (iii) fusion inhibition. During fusion process, heptad repeats HR1 and HR2 form a six helix bundle structure. Synthetic peptides based on the HR1 and HR2 sequences of gp41 have anti-HIV-1 properties; this is up to date the most successful HIV-1 entry inhibitors class. Receptors of type C and D retroviruses are cell membrane anchored proteins that transport small molecules. The receptor of ectopic MLV type C retroviruses is CAT-1, a cationic amino acid (like lysine or arginine) transporter (Kavanaugh et al. 1994). The receptor of amphotrophic MLV is Pit-2 and the receptor of MLV10A1 is Pit-1. Pits-1 is also the receptor for GALV and FeLV. Pit-1 and Pit-2 are two inorganic phosphate transporters. Nevertheless, in some cases, these viruses are able

Fig. 4.2 (continued) (2) hemifusion stalk with proximal leaflets fused and unfused viral (blue) and cell (red) distal leaflets, (3) unfused stalk expansion leading to the hemifusion diaphragm, (4) fusion pore forming the hemifusion diaphragm bilayer, (5) core release into the cell

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to recognize the common parts of Pit-1 and Pit-2 receptors and to infect human cells by using one or the other indifferently (Tailor et al. 2000). Two receptors have been identified for the type D retroviruses interference group: ASCT1 and ASCT2, two neutral amino acid transporters and receptors for BaEV, SRV and RD114 viruses. Retroviruses of the RD114 family recognize an hypervariable region in the second bundle of ASCT2 receptor. Most of the time the viruses use the part of the transporter that is involved in the transport function of this molecule: this region is under strong selection pressure to keep the function and therefore the polymorphism is limited and the infectivity, i.e. the recognition of the receptor by the viruses, is conserved. Furthermore these type D retroviruses can use either ASCT1 or ASCT2 by recognizing the conserved domain. Two types of fusion mechanisms can occur, namely pH independent and pHdependent. In the first case, the recognition between virus and receptor directly triggers conformational changes in the glycoprotein that leads to the direct fusion between the two membranes (viral and plasma) and to the liberation of the viral genetic material. The activation of Env at neutral pH allows the fusion in vitro and in vivo of Env-expressing cells co-cultured with receptor-expressing cells. The fusion leads to the merging of cytoplasms and to the generation of multinucleated cells named syncytia. In the second case, for pH-dependent fusion, the interaction between the Env and the receptor is followed by an endocytosis of the virus-receptor complex before the acidification of the endosome triggers conformational changes in the glycoprotein. For the pH dependent virus, such a fusion can be reproduced in vitro in cell culture or in liposome-virus fusion assay after decreasing the pH in the test tube, but cannot occur in vivo. Most retroviruses use a pH-independent fusion mechanism, with a few exceptions for MMTV, JSRV and ASLV. The proposed mechanism for ASLV virions is an intermediate since entry occurs in two steps, beginning with a receptor-priming that in turn induces Env conformational changes allowing the Env to become sensitive to the low pH. This hybrid mechanism does not lead to cell–cell fusion in vivo. JSRV also uses a receptor-priming for fusion activation of Env at low pH but the mechanism is slightly different that for ASLV (Cote et al. 2009) and requires dynamin-associated endocytosis (Bertrand et al. 2008). MMTV is so far considered as a classical pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) and trafficking to a low pH compartment (Wang et al. 2008). Finally, it should be note that viruses that use a pH independent mechanism of activation of Env may still enter the cell by endocytosis without any requirement for acidification activation of Env into the endosomes. So far, there are many different endocytosis pathways that have been described (Marsh and Helenius 2006; Mercer and Helenius 2009) as being used by both pH-dependant and pH-independent viruses. However, re-investigations of the entry pathways are clearly needed for many pH-independent viruses that were thought not to rely on endocytosis. For example, Nipah paramyxoviruses that can fuse cells at neutral pH seem to use macropinocytosis for entry (Pernet et al. 2009). Macropinocytosis and phagocytosis have also been proposed for HIV-1 entry even if it is unsure that this entry can lead to productive infection (Marechal et al. 2001; Trujillo et al. 2007).

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Let’s describe the several critical domains which are directly involved in the fusion process. For the most part, in retrovirus-cell fusion, a fusion unit typically contains a unique transmembrane domain and a fusion peptide – a sequence of 10–30 residues that forms an amphiphilic domain usually at the N-terminus of the TM (Chernomordik and Kozlov 2003). The hydrophobic fusion peptide domain is sequestered in all previously described Env biosynthesis steps. The final acquisition of the fusion state competency is triggered by the receptor binding alone and/or a low pH surrounding the endosomes and globular head domains dissociation. This movement allows a loop-to-helix transition of a polypeptide segment of TM that was previously buried underneath the SU heads, projecting the fusion peptide ∼100 Å towards the target membrane, where it inserts irreversibly. In the case of class I fusion proteins like retroviruses, this occurs by a “spring-loaded” mechanism. This initial change is proposed to result in a “pre-hairpin intermediate”, an extended structure that is anchored both in the target membrane by the fusion peptide and in the virus membrane by the tm segment (Fig. 4.2a drawings 5 and 6). The HR2 Cterminal end of the long TM α-helix jackknifes back, reversing the direction of the viral-membrane-proximal segment of TM, which then interacts in an anti-parallel fashion with the groove formed by the N-terminal HR1 trimeric coiled coil. The final post-fusion conformation of TM is therefore a highly stable rod with the TM and fusion-peptide segments together at the same end of the molecule, a structure termed a “trimer of hairpins”. The hairpin structure brings the two membranes proximal and provides free energy to overcome the barrier of membrane merging (Melikyan 2008). Membrane fusion occurs, which leads to pore formation and release of the viral genome into the cytoplasm. In addition, compare to cellular glycoproteins, the retroviral TM ectodomain also contains a hydrophobic domain abnormally enriched in tryptophane in the juxtamembrane domain (Salzwedel et al. 1999; Suarez et al. 2000). This domain contributes to the conformational change and membrane destabilization during the fusion process of HIV-1 (Munoz-Barroso et al. 1999), and antibodies (Lorizate et al. 2006; Purtscher et al. 1994) or peptides (Moreno et al. 2006) directed against this domain inhibit the entry. This juxtamembrane domain is also critical for fusion of many envelope viruses beside retroviruses, including paramyxoviruses and coranaviruses. 4.2.2.2 Pore Formation and Fusion of the Target Membranes The hypothesis of the pore model in viral membrane fusion mechanism (Fig. 4.2b) is supported by experimental results. The first evidence for a hemifusion intermediate was achieved by studying influenza virus entry that occurs after the hemagglutinin glycoprotein binding to the host cell. The substitution of the hemagglutinin transmembrane domain by a glycosylphosphatidylinositol (GPI) revealed the importance of the transmembrane region for the fusion pore opening and expansion. Hemifusion structures are connections between outer leaflets of apposed membranes, whereas the inner leaflets remain distinct. This is a transient structure that either dissociates or gives rise to the fusion pore (Chernomordik and Kozlov 2008). Interestingly, the

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helix breaker residues within the tm domain are critical for the hemifusion and pore opening step of the fusion process mediated by different retroviral Env, like HIV-1 (Owens et al. 1994) and Mo-MLV (Taylor and Sanders 1999). In addition, a hemifusion intermediate has been detected in the case of HIV-1 Env-mediated fusion (Munoz-Barroso et al. 1998) by using peptide inhibitors that target a pre-fusion or prehairpin structure such as HIV-1 gp41 T-20. Then, the pore is formed and it allows a connection between two compartments initially separated by the apposed membranes. The membrane ability to hemifuse and develop fusion pore has been found to depend on the lipid microdomain composition, e.g. cholesterol (Chernomordik and Kozlov 2003). A potential lipid dependence of virus entry processes has been first deducted from experiments on influenza virus suggesting the implication of lipid rafts (Takeda et al. 2003). For retroviruses, the tm palmitoylations which contribute to the Env localization in raft domains (Li et al. 2002) influence indirectly the fusion process (Gebhardt et al. 1984; Ochsenbauer-Jambor et al. 2001). As an alternative to lipidic pore hypothesis, a direct fusion has also been proposed. The fusion pore is a full proteic channel-like structure dependent only on the transmembrane domains of the glycoproteins. In this model, the pore is opened by the joining of two hemipores located on each membrane (Chernomordik and Kozlov 2008; Chernomordik and Kozlov 2005). After fusion pore opening and enlargement (Melikyan et al. 2005), the genetic material enters the cytoplasm of the cell and enters the nucleus.

4.2.3 Rous Meets Mendel In humans, virus-like particles without trivial evidence of inter-individual transmissibility were identified in disparate contexts such as placenta, autoimmune diseases, e.g. body fluids of multiple sclerosis patients, and cancers, e.g. seminomas, lymphomas or plasma of breast cancer patients. In the seventies, numerous electron microscopic studies have described the presence of virus related particles in placental chorionic villous tissues of humans (and primates). Further studies then revealed some retroviral characteristics of these particles such as ultrastructural features and RT activity (Lyden et al. 1994). In addition, retroviral envelopes were detected in placenta sections by immunohistochemical techniques in human (Venables et al. 1995) and in baboon (Langat et al. 1999). Retrovirus-like particles associated with reverse transcriptase (RT) activity have been described by several groups in cell cultures from patients with multiple sclerosis (MS) (Perron et al. 1989; Haahr et al. 1994). Infectious properties of these particles are at least not trivial to ascertain if not doubtful. However, using PCR techniques, a reconstructed retroviral genome was defined as Multiple Sclerosis associated RetroVirus (MSRV) (Perron et al. 1997; Komurian-Pradel et al. 1999). MSRV is closely related to the HERV-W (Human Endogenous RetroVirus) family and particularly the ERVWE1/Syncytin-1 locus (Blond et al. 1999). Though, no full length replication competent virus has been experimentally isolated (Voisset et al. 1999). Nevertheless, it has been demonstrated that MSRV particles cause

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T lymphocyte-dependent death with brain haemorrhage in humanized SCID mice (Firouzi et al. 2003). MSRV envelope protein has been proposed to exert various immune properties, e.g. as triggering a superantigen effect (Perron et al. 2001) and activating innate immunity (Rolland et al. 2006). Note that HERV-H related elements were also associated with particles observed in MS (Christensen et al. 1998), dually found with viruses from the herpes group, most likely Epstein Barr Virus (EBV). Cancers of the male reproductive system appears to be a favourable context for virus-like particles detection. Thus, HTDV (Human Teratocarcinoma-Derived Virus) is only expressed in the male germ line tumor context (Boller et al. 1993), and similar particles were observed in testicular germ cell tumors (or seminomas) (Herbst et al. 1999). In both situations, HERV-K transcripts could be associated with the particles. By electron microscopy and immunogold staining, HERV-K like particles were also visualized in the plasma of individuals with lymphoma, but these particles seem to be defective, as surface spikes and free mature virus particles were never observed (Contreras-Galindo et al. 2008). In all the situations exemplified above, although nucleic acid material could be associated with the particles, it remains unclear whether such particles could result from expression of a single retroviral loci, a trans complementation process or even more complex phenomenon involving genetic material recombination. As a corollary, infectivity of these particles has not been demonstrated. A clearer view was expected from the publication of several mammalian genomes, including human (Lander et al. 2001) and mouse (Waterston et al. 2002) genomes. Genomes of mammalian species harbor a large amount of retrovirus-like sequences. These endogenous retroviruses (ERVs) are remnants of ancient retroviral infections that initially occurred in the host germline. Throughout evolutionary time, these initially stably-integrated sequences have derived into gene families by retrotransposition events, and have accumulated genetic defects as a consequence of the host domestication. This general drift basically resulted in gene silencing. Generally, the retro-elements are free-Env and are not able to dissemination between cells. Intriguingly, the human genome but also the mouse genome contains a huge amount of endogenous retroviruses, reaching 8.5 and 9% of these genomes sizes, respectively. Deciphering the human genome showed that the HERV-K family contains tens of almost complete but mutated proviruses that allow the expression of viral proteins which appears able to form retroviral particles. However, no complete proviruses able to produce replication competent and infectious viral particles have been detected. The HERV-K113 locus though to be the more recent element of the family and that contains intact ORFs for all the viral proteins does not produce any particles (Lee and Bieniasz 2007). Trans-complementation between different HERV-K(HML2) proviruses could theoretically produce infectious particles, although not demonstrated to date. Interestingly, the infectious potential of HERV-K particles was artificially restored by generating a consensus HERV-K (HML-2) provirus named Phoenix supposed to be the HERV-K family progenitor (Dewannieux et al. 2006); this consensus contained at least 20 amino acid changes

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on the overall sequence as compared to individual proximal HERV-K loci. By electronic microscopy, this resurrected HERV-K forms viral particles in transfected cells. The budding of its particles is similar to γ-, δ-retroviruses or lentiviruses with no particles preassembling into the cytoplasm. As cited earlier, MSRV is closely related to the HERV-W family including the Syncytin-1 encoding ERVWE1 locus which is the only W-locus bearing a fulllength envelope. The sequencing of ERVWE1 envelope confirmed that the MSRV envelope was not encoded by the ERVWE1 locus (Mallet et al. 2004). It was thus proposed that MSRV particles (if not derived from an as yet uncharacterised exogenous retrovirus) may result from transcomplementation of dispersed HERV-W copies simultaneously activated (Dolei 2005), what appears poorly probable as regards to the HERV-W elements identified in the human genome. However, it could not be formally excluded that MSRV/HERV-W genome (associated with particles) may result from very complex recombination events involving several loci on distinct chromosomes (Laufer et al. 2009). Although complete genomes analyses did not clearly explained the mechanisms leading to the formation of endogenous retroviral particle, they uncovered the extreme plasticity of these retroviral elements. Koala retrovirus (KoRV) provides a unique opportunity to study the process of ongoing endogenisation as it still appears to be spreading through the koala population. Interestingly, infectious viral particles are produced by the endogenous form of KoRV and high levels of viraemia have been linked to neoplasia and immunosuppression (Tarlinton et al. 2008). It remains unclear how the host can react when exogenous and endogenous forms of a virus are coexisting within the genome and his environment. Studies on Koala might answer this question. Interplay between the primitive virus world and the evaluated eukaryotic one could be observed at the env level. Thus, infectious retroviruses appear to have burst from our far ancestors genome by transcomplementation of cellular retrotransposons with viral envelopes genes (Malik et al. 2000). Another type of capture exists between retroviruses of distant species, consisting in the swapping of envelopes observed for species in the same environment or linked by the food chain. For example, the RD114 virus comes from two genetic recombinations resulting in two env-captures. First, the SERV (simian endogenous retrovirus) env was captured by the PcRV (Papio cynocephalus retrovirus) leading to the BaEV (baboon endogenous retrovirus). Second, the acquisition of BaEV env by FcEV (felis catus endogenous retrovirus) led to the emergence of RD114 virus (Kim et al. 2004). Last, endogenous retroviruses as remnants of ancient retroviral infections that initially occurred in the host germline represent an intriguing heritage. More precisely, as a consequence of at least 30 distinct chapters of retroviral infection during the past 90 million years, the current human genome contains 18 coding envelope genes (de Parseval et al. 2003; Blaise et al. 2005) (Table 4.1). The most represented family is the HERV-K(HML2) family which contains six coding env genes lacking fusogenic activity. Three Env proteins belonging to HERV-W, HERV-FRD and HERV-P families, namely Syncytin-1, Syncytin-2 and EnvP(b) respectively, have fusogenic properties.

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Table 4.1 Human viral heritage of envelopes open reading frames containing canonical retroviral motives Name

RNA expression

Protein expression

Fusogenity

References

envH1 envH2 envH3 envK1 envK2 envK3 envK4 envK5 envK6 envT envWa envFRDb envR envR(b) envF(c)2 envF(c)1 envV envP(b)

NEc NEc NEc NEc NEc NEc NEc NEc NEc NEc Placenta Placenta All tissue NEc NEc NEc Placenta All tissue

NEc NEc NEc NEc NEc NEc NEc NEc NEc NEc Placenta Placenta NDd NEc NEc NEc NDd NDd

NDd NDd NDd NDd NDd NDd NDd NDd NDd NDd Yese Yesf NDd NDd NDd NDd Noe Yese

Lindeskog et al. (1999) de Parseval et al. (2001) de Parseval et al. (2001) de Parseval et al. (2003) Barbulescu et al. (1999) Donner et al. (1999) Barbulescu et al. (1999) Turner et al. (2001) Turner et al. (2001) de Parseval et al. (2003) Blond et al. (1999) de Parseval et al. (2003) Cohen et al. (1985) de Parseval et al. (2003) de Parseval et al. (2003) de Parseval et al. (2003) Blaise et al. (2005) Blaise et al. (2005)

a Syncytin-1. b Syncytin-2. c No

expression. determined. e In vitro. f In vivo. d No

4.3 Syncytins and Cell–Cell Fusion In spite of ERVs have been thought to be a non-functional part of the genome for a while, the past 10 years identified open reading frames of envelope genes in human, mouse, rabbit and sheep genomes (Fig. 4.3a), and associated with transcription activity and fusogenic glycoproteins synthesis (Fig. 4.3b) likely involved in biological functions. This is the case for the two human envelopes genes ERVWE1/Syncytin-1 and ERVFRDE1/Syncytin-2, located on chromosome 7q21.2 and 6p24.1, respectively, as well as for the two Syncytins-related A and B in mice, both pairs associated with fusion steps occurring in placental development process. Recently the novel Syncytin-Ory1 was identified in rabbit given a new example of syncytin gene within a third order of mammals (Heidmann et al. 2009). The ovine species also provide a quite interesting model of endogenisation process since the exogenous and pathogenic Jaagsiekte Sheep Retrovirus (JSRV) coexists with at least 27 highly related endogenous counterparts (enJSRVs), accounting for envelope genes in the ovine genomic DNA with evidences for open reading frames (Arnaud et al. 2007). enJSRVs play a crucial role in the sheep placental morphogenesis and

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Fig. 4.3 Structure, phylogeny and fusion capacities of Syncytins. a Envelopes structures of Syncytins and schematic representation of their cognate receptors. FP: fusion peptide; tm: transmembrane domain; cyt: cytoplasmic tail. Black dots indicate the predicted N-glycosylation sites. SDGGGX2DX2R, consensus motif conserved in type D retroviral interference group, is indicated in human Syncytin-1 and rabbit Syncytin-Ory1. b Demonstration of Syncytin-1 cell–cell fusion property. TELacZ cells (dark blue nucleus) expressing Syncytin-1 envelope glycoprotein

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their envelope expression in the reproductive tract is mandatory for a successful pregnancy (Dunlap et al. 2006). The focus point in this chapter will be now to discuss if these endogenous viral proteins of the genome still remain fusogenic in the same way an exogenous retrovirus envelope glycoprotein is (including transcription strategy, maturation steps, receptor recognition and fusion process), and to address the question of the domestication by the host and adaptive response though integrative and evolutionary points of view.

4.3.1 Integration, Domestication Steps and Biological Functions of Endogenous Viral Glycoproteins 4.3.1.1 Integration Dating and Orthologues One starting point in the discussion about the endogenous envelopes found in genomes could be the estimation of the age of the proviruses. This can basically be done by two approaches, bringing additional informations. One way is to assess a phylogenic lineage by tracing the presence of a similar DNA sequence at the same genomic loci in the genome of different species, and to conclude by a unique hypothetical integration event into the germline of a common ancestor. Another way is to consider the divergence between the 5 and 3 LTR and assuming a molecular clock is acting randomly through the genome, to generate variations over time between two originally and identical provirus sequences (the 5 LTR and 3 LTR being identical at the time of integration in the host genome). The first striking point are the unshared properties of both families and integration times within the humans, mice, rabbits and sheeps endogenous envelopes (Fig. 4.3c). ERV-W elements have been identified in hominoidae (human, chimpanzees, gorillas, orangutans and gibbons) and Cercopithecidae (old world monkeys) (Kim et al. 1999; Voisset et al. 1999) indicating that what we call today the human ERV-W family, HERV-W, derived from an ancestral virus which entered the genome after the divergence between Catarrhini and Platyrrhini, i.e. less that 40 million years ago (MYA). The ERVWE1/Syncytin-1 locus results from a complete proviral retrotransposition event into the germ line of an ancestor before Hominoidae and Cercopithecidae divergence more than 19–25 MYA (Caceres and Thomas 2006; Bonnaud et al. 2005). A full length envelope ORF corresponding to functional envelope glycoprotein was preserved in Hominoidae but genetic drift led to truncated envelope genes in old world monkeys. In contrast, the FRD family containing the HERV-FRD envelope Syncytin-2 is found in all simians, from New

Fig. 4.3 (continued) co-cultured with indicator XC cells (light blue nucleus) expressing hASCT2 receptor generates multinucleated large syncytia (left part). TELacZ-Syncytin-1 cells co-cultured with XC cells lacking hASCT2 do not fuse (right part). c Phylogenetic tree depicting the conservation among species of the six envelope-open reading frames harbouring retroviral canonical motifs (branches of the tree are only illustrative). NWM: new world monkeys; OWN: new world monkeys

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World monkeys to human, suggesting a divergence split at least 40 MYA (Blaise et al. 2003). Moreover, the human, mouse and rabbit genes are not orthologs since Syncytin-A and -B entered into the rodent lineage before speciation of Muridae about 20 MYA (Dupressoir et al. 2005), i.e. after the speciation of rodents and primates while the Syncytin-Ory1 integration took place before the divergence between Lepus and Oryctolagus/Sylvilagus around 12 MYA (Heidmann et al. 2009). The situation is also different in domesticated sheep (Ovis aries) and other species within the Caprinae subfamily, where the endogenous retroviruses JSRV (enJSRVs) start to invade the genome at least 5–7 MYA and are likely still colonizing it today as given evidences by the restricted presence of recent enJSRVs loci into the genome of only some breeds or even some animals of the same breed of domesticated sheep (Arnaud et al. 2007). In order to better understand which mechanisms may have led to a positive selection of organisms harboring embarked viral genes, many arguments in favor of a domestication scenario have been deployed, especially about the ERVWE1/Syncytin-1 locus. Indeed, the proposed evolutionary pathway occurring in Hominoidae is opposed to the genetic drift in Cercopithecus. A ∼4.3 kb region, comprising the HERV-W 5 LTR-gag-pol fraction, was deleted in Cercopithecus and was followed by a genetic drift of the Env/Syncytin-1 ORF (Bonnaud et al. 2005; Caceres and Thomas 2006). Remarkably, the Syncytin-1 ORF has been conserved in all Hominoidae, while gag and pol regions have accumulated numerous stop and frameshift mutations (Mallet et al. 2004), supporting the idea of a specific preservation. Meanwhile, the analysis of 155 individuals including Caucasians, Asians, Africans, Metis and Ashkenazi people has revealed a positional conservation of the Syncytin-1 locus and the preservation of the envelope ORF (Bonnaud et al. 2004; Mallet et al. 2004), while a close examination of 24 ERVWE1 provirus sequences has showed an unusually low polymorphism in the 5 LTR (1 base per 18 kb as compared to 1 base per 2 kb for coding sequences) (Mallet et al. 2004). An additional specificity of the ERVWE1 provirus is the MaLR-LTR trophoblast specific enhancer (TSE) located upstream the ERVWE1 provirus that is highly conserved with no polymorphism observed in the 48 human sequences analyzed. Although the envelope gene may be considered under selective pressure depending on the part of the gene we focus on, the striking feature for the ERVWE1 locus is a 12 bp deletion observed in the Syncytin-1 intracytoplasmic tail gene region and that constitutes a specific signature of this locus. This deletion is unique among all ERV-W copies in available human and chimpanzee genomes (Bonnaud et al. 2005), and is crucial for the envelope fusogenicity (Bonnaud et al. 2004; Cheynet et al. 2005). Interestingly, the comparison of the FRD/Syncytin-2 envelopes among simians has also revealed a limited number of mutations, and pseudotypes experiments demonstrated that only one mutation occurring in the transmembrane subunit of the protein can be responsible for the loss of infectivity (Blaise et al. 2004). Notably, the alignment of endogenous and exogenous JSRV envelopes reveals similar deletions in the cytoplasmic tail of enJSRVs env as compared to the exogenous one (Palmarini et al. 2001). Altogether, these elements may infer the hypothesis of a positive selection and domestication of retroviral envelopes.

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4.3.1.2 Endogenous Retrovirus Envelopes Are Expressed in the Placenta and in the Testis Suggesting a Direct Involvement in Developmental Process The HERV-W family was molecularly characterized following the isolation of cDNA clones in the placenta that revealed viral sequences genes expression, especially with similarities to the avian retrovirus primer binding site (Blond et al. 1999). In 2000, protein truncation tests within this endogenous family revealed only one open reading frame (ORF) coding for a putative envelope gene associated with a functional U3 promoter (Voisset et al. 2000). One year later, Blond and Mi concomitantly associated an HERV-W envelope protein with fusion events in TE671 and BeWo cells, and the name Syncytin was proposed by Mi (Blond et al. 2000; Mi et al. 2000). Heidmann and colleagues then conducted a genome wide screening that identified a second envelope protein, belonging to the HERV-FRD family, and expressed exclusively in the human placenta. They named Syncytin-2 this putative new fusogenic Env-FRD protein (Blaise et al. 2003). A similar in silico approach was done in the murine genome, identifying the two coding envelopes genes present as unique copies and with a placenta specific expression: Syncytin-A and Syncytin-B (Dupressoir et al. 2005), and recently in the rabbit genome, identifying the Syncytin-Ory1 gene (Heidmann et al. 2009). If the situation is much more different in the ovine genome, where approximately 27 copies of endogenous betaretrovirus (enJSRVs) were detected, RT-PCR and in situ hybridization clearly indicate a conceptus (embryo/fetus and extra embryonic membranes) localization of enJSRVs env transcripts during gestation (Dunlap et al. 2006). Although human Syncytins were abundantly described in the placental tissues, initial works also mentioned a weaker but significant transcription in the testis without any protein evidence (Mi et al. 2000). Envelope-specific RT-PCR established expression in the human testis of both Syncytin-1 and Syncytin-2 (de Parseval et al. 2003), and a multiplex degenerated PCR screening for a consensus pol region has revealed a general expression of the HERV-W family in testis (Pichon et al. 2006) and epididymis (Crowell and Kiessling 2007). This is consistent with old studies that identified the epididymal epithelium as a principle reservoir for retrovirus expression in the mouse (Kiessling et al. 1989). This knowledge points out that endogenous envelopes expression are usually associated with developmental tissues, and so far raise the question of whether or not Syncytins play a direct role in the mammalian placentation.

4.3.1.3 Biological Function of ERVs Envelopes The keen interest in ERVs envelopes expressed in placentas is fed by in vivo or ex vivo demonstrations that directly link Syncytins with fusion events during placental development. Although the role of Syncytin-1 in human placentation awaits a definitive demonstration (e.g. infertility associated mutation), recent knock-out gene experiments in mice clearly achieved this goal in rodent model and demonstrated for the first

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time the critical role of Syncytin-A in placenta morphogenesis. Using a homologous recombination strategy, Syncytin-A null mouse embryos exhibited growth retardation with an altered placenta labyrinth architecture and died in utero (Heidmann et al. 2009). This is consistent with previous in vitro works that used specific antibodies and antisense oligonucleotides to show a decrease in syncytia cell formation after Syncytin-A inhibition (Gong et al. 2007). In addition, the endogenous retroviruses of sheep, enJSRVs, play a fundamental role in sheep conceptus growth and trophectoderm differentiation via their envelope glycoproteins. Indeed, in vivo experiments using an enJSRV envelopes specific morpholino injection trigger the lost of pregnancy by day 20 after injection (Dunlap et al. 2006). These kind of in vivo experiments obviously cannot be performed in human. Yet, primary cultures of human villous cytotrophoblasts cells give a unique opportunity

Fig. 4.4 Involvement of Syncytins in placenta development. a Assays reporting the biological effect of Syncytins. b Ex vivo or in vivo specific inhibition of Syncytins expressions. From left to right: Syncytin-1-induced human primary trophoblasts fusion and differentiation results in syncytia formation ex vivo (a). Inhibition by specific antisense oligonucleotide largely reduces syncytia formation (b). Electron micrograph of Syncytin-A+/+ mouse placenta shows tight apposition of the syncytiotrophoblast I and II layers (ST-I; ST-II); stgc: sinusoidal trophoblast giant cells (a). Syncytin-A–/– null mouse embryo interhemal domains shows unfused trophoblast I cells (T-I) (b). Micrograph of the normal development of a sheep conceptus (a). Retarded growth of a sheep conceptus recovered after an envelope enJSRV morpholino antisense oligonucleotide (MAO-env) injection (b)

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to study placenta cells as closely related as possible to tissue environment. Thus, by using specific antisense oligonucleotides and siRNA strategies, expression of Syncytin-1 mRNA and protein as well as the syncytium formation by cell fusion events were dramatically reduced (Frendo et al. 2003b; Vargas et al. 2009). In addition to that, Vargas and colleagues recently compared these results using the same targeting strategy against Syncytin-2, and interestingly showed that Syncytin-2 inhibition in primary cells culture also leads to a decrease in fusion index that is more important than for Syncytin-1 (Vargas et al. 2009). The conclusion is that Syncytin2 could also be a major determinant of trophoblast cell fusion, and in a coherent vision this underlines there should be more than one ERV envelopes proteins acting upon trophoblast cell fusion in human. Parallel procedures demonstrating the involvement of human, mouse and sheep Syncytins in placenta development are illustrated in Fig. 4.4. Note that early works identified ERV-3 (HERV-R) envelope as the first candidate for placental functions. The ERV-3 envelope protein is detected specifically in the multinucleated syncytiotrophoblast in vivo (Venables et al. 1995) and ERV-3 Env expression affects proliferation and differentiation of BeWo cells in vitro (Lin et al. 1999; Lin et al. 2000). However, the observation that approximately 1% of the Caucasian population has a mutation in ERV-3 env inducing a stop codon, and consequently resulting in a truncated envelope lacking both the fusion peptide and the immunosuppressive domain (de Parseval and Heidmann 1998) has drastically lowered the scientific efforts regarding involvement of ERV-3 in placental development. Indeed, a second hypothetical function of Syncytins is related to their putative immunosuppressive activity (see below) due to the presence of a putative immunosuppressive region conserved among murine, feline, and human retroviruses (Cianciolo et al. 1985). So far we saw that Syncytins are involved in developmental fusion process. In the next part of this chapter we aim to focus on the mechanistic comparison between exogenous and endogenous envelope glycoproteins at the synthesis and maturation steps.

4.3.2 Fusion Mechanism and Receptor Recognition 4.3.2.1 Maturation The different steps leading from a brand-new translational product in the cytosol to a functional membrane-anchored envelope glycoprotein has been discussed previously. Basically, endogenous envelopes still remain glycoproteins, engaged in the classical reticulum-golgi apparatus where post translational events occur, before to be address to the plasma membrane and to become functional. Thus, precursor synthesis and glycosylation, disulfide bonds, trimerization, peptide cleavage and the importance of the cytoplasmic tail will be illustrated here introducing specific Syncytins knowledge, in order to support our previous descriptions as well as to focus on endogenous envelope specific characteristics.

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Precursor, Furin Cleavage and Glycosylation Studies in BeWo cells models have described in a fine way the maturation of Syncytin-1. Syncytin-1 is first synthesized as a 73-kDa precursor (gPr73) before to be cleaved at a conserved RNKR furin cleavage site into two subunits, SU (gp50) and TM (gp24) (Cheynet et al. 2006). Although polypeptides size of Syncytin-1 and -2 is the same (538 amino acids), sizes of SU and TM are different after processing (Chen et al. 2008). PNGase F digestion and tunicamicyn treatment predicted and confirmed seven N-glycosylation sites for the Syncytin-1. These results indicate that Syncytin-1 is a moderately glycosylated protein, with one glycosylation site in the TM subunit which is essential for correct envelope protein folding, and with highmannose N-glycans on the six glycosylation sites of the carboxy-terminal domain of the SU (Cheynet et al. 2006). Furin inhibition experiments conducted on Syncytin-2 have also established the furin to play a major role in the proteolytic cleavage of the HERV-FRD envelope proprotein (Chen et al. 2008), where the cleavage consensus sequence is also found. Interestingly, using knock down experiment, furin has been proposed to have a possible role in promoting trophoblast cell migration and invasion in human placenta (Zhou et al. 2009). Bioinformatics analyses and sequence alignments suggest that Syncytin-A and -B exhibit most features of membrane fusion proteins, including the conserved cleavage site RNKR, which separate the SU and TM subunits (Dupressoir et al. 2005; Peng et al. 2007). Finally, the same feature is observed for the Syncytin-Ory1 sequence that exhibits a RQKR site (Heidmann et al. 2009) and for the enJSRV sequences that harbour the cleavage furin motif site. Disulfide Bonds and Trimerization Considering the Syncytin-1 TM gp24 sequence, it appeared that a leucin zipper-like LX6 LX6 NX6 LX6 L and a CX6 CC motifs are present, suggesting that SU and TM may covalently link together and form homotrimers (Cheynet et al. 2006). Indeed, Syncytin-1 sequence contains a typical disulfure isomerase motif in the SU domain (CC). As previously mentioned for MLV, the first two cysteines of the CX6 CC motif can form a stable disulfide bond, leaving the third cystein free to form a disulfide bond with the CC motif (Fass et al. 1997). Mutational experiments using neutral substitution in the CX6 CC motif did not affect the protein precursor expression level, but impaired syncytia formation, suggesting that disulfide bonds likely contribute to the correct folding of the envelope. In accordance to that, Chen and colleagues demonstrated that the disulfide bridge-forming CX7 C motif of the Syncytin-2 was essential for the fusogenic activity (Chen et al. 2008). Cytoplasmic Tail and R Peptide The cytoplasmic tail region of numerous retroviral envelopes plays a critical role in the fusion triggering. In the retrovirus life cycle, the presence of an R peptide basically prevents the fusion to occur, notably because of the presence of the YXX

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motif described above. During viral packaging, the R peptide is proteolytically cleaved and this event enables envelopes to cause membrane fusion, as described by mutagenesis experiments (Yang and Compans 1996). We illustrate how Syncytins used various strategies that diverge from envelopes of infectious retroviruses to adapt to their physiological functions. Surprisingly, sequences comparison of the Syncytin-1 locus with all other HERVW envelope elements revealed a 12-bp (corresponding to four LQMV amino acids) deletion in its cytoplasmic tail (Bonnaud et al. 2004). Moreover, insertion of these four amino acids into Syncytin-1 tail completely abolished the fusogenic potential (Bonnaud et al. 2004). This result argues that Syncytin-1 is constitutively fusion competent, as opposed to exogenous retroviruses envelopes, and is coherent with a domestication point of view since no viral protease open reading frames exist anymore in the human genome (Voisset et al. 2000) (Fig. 4.5). Furthermore, the role of the cytoplasmic domain of Syncytin-1 has been systematically investigated by producing a series of C-terminal truncated variants, leading to the conclusion that residues adjacent to the membrane domain are required for optimal fusion probably by forming a helical structure, while final C-terminal residues more likely act as a fusion inhibitor domain (Drewlo et al. 2006; Cheynet et al. 2005). Remarkably, a truncation mutant which shortens the cytoplasmic tail precisely at the site of the LQMV-deletion motif exhibits higher fusogenic properties than the wild-type protein (Cheynet et al. 2005). Even if no work on Syncytin-2 has been done in such a fine way to assess the fusogenic properties modulation its cytoplasmic tail, we

Fig. 4.5 Comparative evolution of Syncytins cytoplasmic tails: from viruses to genes. The first five amino acids correspond to the transmembrane domain. Experimentally determined (GaLV, MLV, exoJSRV) and putative (W Rep. and FRD Rep.) protease cleavage site (black line) and YXX signaling motif are indicated in lowercase. Comparison of the Syncytin-1 protein (Syn-1) with the HERV-W family consensus sequence obtained from Repbase (W Rep.) shows a four amino acids deletion (LQMV) in the domesticated fusogenic protein, overlapping the ancestral viral protease cleavage site. The underlined leucine indicates a C-terminal truncation mutant exhibiting hyperfusogenic activity and significant pseudotyping capacity. Comparison of the Syncytin-2 protein (Syn-2) with the Repbase FRD consensus sequence (FRD Rep.) shows a stop codon that shortens the Syncytin-2 cytoplasmic tail and no evidence of viral protease cleavage site. Alignment of enJSRV and exoJSRV shows the placenta-expressed enJSRV has accumulated mutations surrounding the protease cleavage site and lacks downstream tyrosine (Y) residue. Genebank accession numbers: MLV: M14702; GaLV: AF055060, Syncytin-1: GQ919057, Syncytin-2: HEU27240, enJSRV: enJS56A1 and exoJSRV: AF105220

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identified a stop codon in the cyt of Syncytin-2, as opposed to the RepBase prototype, resulting in a shortening of the tail (Fig. 4.5). Moreover, the protease cleavage site appears absent as regard to the FRD family consensus genome. Unlike the endogenous retrovirus enJSRV, the exogenous JSRV is pathogenic for sheep and is responsible for a transmissible lung cancer in sheep via its envelope glycoprotein acting as a dominant oncoprotein (Palmarini et al. 1999). Studies on the cytoplasmic tail of JSRV envelope protein first focused on the VR3 region that was described as the least conserved region between exogenous and endogenous forms. The VR3 region includes the putative membrane-spanning domain as well as the cytoplasmic tail, and series of envelope chimeras revealed that mutations in a YXXM motif of the cytoplasmic tail of JSRV env were sufficient to inhibit its transforming abilities (Palmarini et al. 2001). Further mutational amino acid substitutions have proven the tyrosine residue to be essential for transformation of exogenous JSRV. It is noteworthy that the VR3 region of all exogenous stains of JSRV sequences exhibit this tyrosine residue whereas all the enJSRVs envelopes described so far lacked this motif critical for JSRV transformation (Fig. 4.5). However, despite differences in terms of motif and sequence, JSRV and enJSRVs envelopes use the same cellular receptor called HYAL2. Further systematic mutagenesis studies of the cytoplasmic tail of JSRV envelope TM protein have established four categories of mutants that allow the TM to be devised into subdomains with regard to the transformation efficiency. Among them, mutations in the YXXM motif have various effects including the generation of “supertransformers” while the last nine amino acids of the cytoplasmic tail appear not essentials for the envelope-induced transformation (Hull and Fan 2006). 4.3.2.2 Receptor Binding Consistent with the virology paradigms, the comprehensive search for endogenous retroviral envelope functions led to the identification of the associated receptor (or co-receptors) that allows fusion events to be complete. In 2000, state of the art was to consider three main virus receptor types, PiT-1 and PiT-2, two independent inorganic-phosphate symporters for GaLV and MLV viruses, respectively, and the RDR/Type D receptor, a neutral-amino acid transporter for the cat endogenous retrovirus RD114 and type D simian retroviruses. When we first attempted to check for the right one that could trigger cell fusion, evidence in favor of the RDR/Type D hASCT2 mammalian receptor involvement was revealed through receptor-blocked experiments in cell lines transfected with Syncytin-1 gene (formerly HERV-W Env in Blond’s report) (Blond et al. 2000). Two years later, cell–cell fusion and pseudotypes virion infection assays demonstrated that Syncytin-1 efficiently uses both hASCT2 and the related hASCT1 transporter as receptors, and could recognize the mouse mASCT2 and mASCT1 transporters lacking their N-glycosylation sites removed by mutagenesis (Lavillette et al. 2002). Very interestingly, Syncytin-Ory1 also uses the hASCT2 transporter as specific receptor (Heidmann et al. 2009). AntiRDR serum used for histochemical and flow cytometric biodistribution analyzes, further unveiled a broad expression pattern of RDR/hASCT2 in many normal tissues

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including colon, testis, ovary, bone morrow and skeletal muscle (Green et al. 2004). To move forward into the comprehensive relationship between Syncytin-1 and its hASCT2 receptor, Cheynet and coworkers designed a truncated series of HERV-W SU subunits and performed cell fusion assays. This led to the identification of a minimal N-terminal 124 amino acids of the mature SU glycoprotein required as a receptor-binding domain (RBD) (Cheynet et al. 2006). Furthermore, within this domain, one especially conserved sub-domain among retroviruses belonging to the interference group, the SDGGGX2 DX2 R motif, was proved to be essential for Syncytin-1-hASCT2 interaction (Cheynet et al. 2006). The human Syncytin-2 receptor has been identified more recently. By using an old powerful approach based on a human/Chinese hamster radiation hybrid panel mapping, one candidate gene was identified to encode a putative transmembrane protein (Esnault et al. 2008). The major facilitator superfamily domain containing 2 (MFSD2) was showed to confer cell–cell fusogenicity in the presence of Syncytin-2 and infectivity to Syncytin-2 pseudotypes. MFSD2 belongs to a large family of presumptive carbohydrate transporters, and is highly conserved among mammalian genomes. Although RDR and MFSD2 are two different receptors, they belong to the ion channels and small molecules transporters category, exhibiting a classical hydrophobic profile composed of transmembrane helices. The situation is outstandingly different for the HYAL2 JSRV receptor. Interestingly, early experiments to localize the JSRV receptor gene region were also based on pseudotyping and radiation panel screening (Rai et al. 2000). After identifying a set of overlapping clones, genetic analyses confirmed that HYAL2 was the only protein that functions as a JSRV env receptor (Rai et al. 2001). If the name HYAL2 first suggests a mainly strong hyaluronidase function, studies indeed showed low but detectable hyaluronidase activity pH-dependant (Lepperdinger et al. 1998) Actually, amino acid sequence analyses established one transmembrane domain as well as an hydrophobic carboxyl terminus and upstream signal, indicating that HYAL2 is likely attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor (Rai et al. 2001). As compared to other GPI-anchored proteins, HYAL2 can potentially be involved in signal transduction and mitogenic responses, strongly suggesting a role of HYAL2 in JSRV Env-mediated oncogenesis (Rai et al. 2001). No receptors for mice Syncytin-A and -B have been identified to date. However, fusogenic experiments showed that each Syncytin-A and Syncytin-B expression vectors triggered fusion in two different cell lines, respectively. This result argues in favor of a divergent receptor usage for these two envelopes proteins (Dupressoir et al. 2005). 4.3.2.3 Incorporation in Particles Although one may speculate that domesticated endogenous envelopes are not supposed to spread using viral particle carrier, in an initial attempt to test the fusion capacity of HERV-W Env/Syncytin-1, we sought to generate retroviral pseudotypes in which MLV core particles were coated with HERV-W Env glycoproteins.

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The absence of infectivity of HERV-W Env pseudotypes was due to the inability of HERV-W Env glycoproteins to be incorporated on MLV particles (Blond et al. 2000). Conversely, pseudotyping of HIV-1 virions with the HERV-W envelope resulted in infectious viruses, although with a poor efficiency (An et al. 2001). Such a difference in incorporation efficiency could be attributed to the lenght of the cytoplasmic tail of Syncytin-1, which is longer than the MLV one, but closer to the HIV-1 one. On line with this, shortening of the Syncytin-1 cytoplasmic tail (Fig. 4.5) significantly enhances pseudotyping of HIV-1 viral cores (Lavillette et al. 2002).

4.3.3 Retroviral Envelopes Are Involved in the Placenta Development Syncytins or envelope glycoproteins, originated from ancient retroviral infections, are now fully integrated in mouse, rabbit, sheep and human genomes. They are naturally expressed in the placenta, following a biological protein maturation process and interacting with specific receptors. Based on these observations, the purpose is now to understand how they contribute to the placenta morphogenesis. As bona fide genes, their transcription is strongly regulated by different cellular mechanisms, they are under local and temporal expression control, and finally they cooperate to an integrative way in a biological system as a whole, involving numerous co actors. The next part aims to develop these points. 4.3.3.1 Envelope and Receptor Localization Throughout Mammalian Gestation In humans, the embryo implantation process and the placental development are driven by fetal cytotrophoblasts stem cells. Placental growth is characterized by proliferation and differentiation of the villous cytotrophoblast pool into a multinucleated syncytiotrophoblast layer upon fusion events. This polarized layer constitutes the main fetomaternal interface in direct contact with the maternal blood (Fig. 4.6a). The general attempt to finely localize Syncytin-1 protein within the

Fig. 4.6 (continued) detected either in CT or ST and MFSD2 receptor is detected in ST. b Immunohistochemical detection of Syncytin-1 protein (SC-Syn1) at the apical syncytiotrophoblast microvillus membrane of a 10 weeks gestation normal placenta (upper). Note that desmoplakin, a protein of the desmosomiale plaque involved in intercellular junctions, is absent from the syncytiotrophoblast fused tissue and lines the plasmatic membranes of the cytotrophoblasts (CT-d). The hASCT2 receptor is observed at the membrane of cytotrophoblastic cells (CT-hASCT2) underlying the syncytiotrophoblast (ST). c Transcriptional and epigenetic control of Syncytins and associate receptors during human gestation. Promoter regions are indicated as boxes and CpG schematized by circles. TSE, trophoblast specific enhancer, U3, LTR promoter, R, transcription initiation site. CpG methylation is determined by bisulfite sequencing PCR in cytotrophoblasts (CT) at different times of gestation. Each line represents an independent molecule. Methylated CpGs are schematized by black circles and unmethylated CpGs by white circles

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Fig. 4.6 Local and temporal expression of Syncytins in human placenta. a Schema of human chorionic villi. Cytotrophoblastic cells (CT, in yellow) differentiate by fusion to generate the syncytiotrophoblast (ST, in green). In the anchoring villi the cytotrophoblast cells proliferate and invade the decidua. The extravillous cytotrophoblast cells (ECT) invade the uterin stroma and differentiate into multinucleated giant cells and invade also the lumen of uterine arteries (UA). M: mesenchyme. Sites of expression of Syncytins and receptors are symbolized in the lower box: Syncytin-1 protein is detected in ST and ECT, hASCT2 receptor is detected in CT and ECT, Syncytin-2 protein is

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placenta led the authors to use homemade antibodies that resulted in variable conclusions. With full knowledge of that facts, we decided to focus on the hypothesis that Syncytin-1 is preferentially detected at the apical membrane of the syncytiotrophoblast (Frendo et al. 2003b; Muir et al. 2006) (Fig. 4.6b). Indeed, two convergent studies mentioned that Syncytin-1 is located on specific membrane areas, enriched in cholesterol and called detergent-resistant membrane (DRMs) or rafts (Cheynet et al. 2005; Strick et al. 2007). Moreover, in primary trophoblast cells, Syncytin-1 detection was likely to be associated with cell-to-cell contact zones (Vargas et al. 2009). According to several authors, the level of Syncytin-1 protein increases during early pregnancy but remarkably decreases in very late pregnancy (Smallwood et al. 2003; Muir et al. 2006). The hASCT2 receptor expression is restricted to the cytotrophoblast compartment (Hayward et al. 2007) (Frappart, Cheynet, Mallet, unpublished data), being largely absent in the syncytiotrophoblast (Fig. 4.6b) and no spacial or temporal changes in the hASCT2 expression has been associated with the proliferative status of cytotrophoblast cells (Hayward et al. 2007). On another hand, divergent observations mentioned Syncytin-2 expression to be either restricted to villous cytotrophoblast cells (Malassine et al. 2007) or in the syncytiotrophoblast (Chen et al. 2008). Compromising positions accordingly associated Syncytin-2 with cell-to-cell contact regions, likely at the interface between the cytotrophoblast and the syncytiotrophoblast (Malassine et al. 2007; Vargas et al. 2009). Remarkably, the level expression pattern of Syncytin-2 follows an inverse correlation compared to Syncytin-1, since a significant increase in Syncytin-2 mRNA and protein is monitoring through pregnancy time and primary trophoblast culture evolution (Chen et al. 2008; Vargas et al. 2009). Finally, the MFSD2 receptor expression is unambiguously reported at the level of the syncytiotrophoblast (Esnault et al. 2008). In addition to the villous phenotype, the cytotrophoblastic cells of the anchoring villi can proliferate and invade the endometrium to be in contact with the spiral arterioles of the mother uterus. In these cells that do not fuse, both Syncytin-1 and his receptor hASCT2 have been detected (Malassine et al. 2005; Muir et al. 2006), suggesting that the trophoblastic cell fusion is indeed a complex multifactorial process (Malassine et al. 2005). The mouse placenta is composed of spongiotrophoblasts, giant cells and a socalled labyrinth zone. In this placenta labyrinth, two layers of multinucleated syncytiotrophoblast cells, resulting from cell–cell fusion, function as the major transport surface for nutrient and gas exchange between the maternal and fetal circulation. Early in situ hybridizations showed that Syncytin-A and Syncytin-B are expressed at the level of syncytiotrophoblats, all over the labyrinth zone (Dupressoir et al. 2005). In coherence with that, latter studies more precisely indicated a clear expression of Syncytin-A in the syncytiotrophoblast while expression in trophoblast stem cells and in trophoblast giant cells could hardly be detected (Gong et al. 2007). The rabbit placenta can be divided into the maternal decidua (the uterus modifications after implantation) and the placental lobe, in which a labyrinthine structure results from the fetal invading process. At this interface, a junctional zone presumably formed by cellular cytotrophoblasts takes place and defines a broad syncytial front. In situ hybridizations on paraffin sections of rabbit placenta have shown Syncytin-Ory1 expression to be restricted at the junctional zone and limited

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to the trophoblast cells surrounding the invading fetal vessels (Heidmann et al. 2009). The authors also suggest that the labeling is compatible with an expression of Syncytin-Ory1 in the cytotrophoblast just before fusion takes place and so is consistent with a role for Syncytin-Ory1 in the formation of the syncytiotrophoblast (Heidmann et al. 2009). In the sheep conceptus, trophoblast binucleated cells are in many respects analogous to the trophoblast giant cells of the human syncytiotrophoblast (Hoffman and Wooding 1993). They first appear at day 14 post-coitum and progressively form the outer layer of the fetal placental cotyledon giving rise to the syncytial plaques (Wooding 1984; Palmarini et al. 2001). The plaques then may cover the surface of the endometrial carunucles and aid in development of placentomes that are required for hematrophic nutrition exchanges from the maternal uterus. RTPCR analyses have showed that endogenous JSRV envelopes and HYLA2 were expressed in the trophoblast giant binucleated cells and in the multinucleated syncytial plaques (Dunlap et al. 2006). If both endogenous JSRV envelopes and HYAL2 are detected in placentome throughout gestation, HYAL2 expression was not detected in endometrium (Dunlap et al. 2006). Endogenous JSRV envelopes were first detected in the day 12 conceptus, whereas HYAL2 first detection appeared at day 16, in a coherent way with the initial differentiation start of the binucleated cells at day 14. 4.3.3.2 Splicing Strategy, Transcription Factors and Epigenetic Control For a brief reminder, Syncytins’s chromosomal localizations are 7q21.2 (ERVWE1/Syncytin-1) (Blond et al. 1999) and 6p24.1 (ERVFRDE1/Syncytin-2) (Blaise et al. 2003) in human, and 5qG2 (Syncytin-A) (Dupressoir et al. 2005) and 14qD1 (Syncytin-B) in mouse (Dupressoir et al. 2005). To date the rabbit genome is not available in a definitive assembly to check the Syncytin-Ory1 localization and several integrations sites for enJSRV exist in the sheep genome. Putative splice donor and acceptor sites have been identified for all of them (Blond et al. 1999), although only specific transcripts have been detected depending on the biological context. ERVWE1 produces three major singly-spliced transcripts in placenta (Blond et al. 1999). The first one, 7.4-kb long, containing the gag and pro/pol pseudogenes and env gene, is found both in testis and placenta (Mi et al. 2000), while the 3.1 kb long, strictly including the open reading frame for the envelope protein Syncytin-1, is exclusively detected in the placenta. Early northern blot experiments also detected a 1.3-kb largely-spliced transcript in placenta (Blond et al. 1999), indeed containing the cytoplasmic tail of Syncytin-1. These observations are similar to lentivirus or oncovirus transcription patterns such as human immunodeficiency virus (HIV), the mouse mammary tumor virus (MMTV) or the human T-cell leukemia virus (HTLV), in which several genomic and subgenomic transcripts derive from a single locus by alternative splice variations. Like any other classical retrovirus, endogenous retroviruses may display all the signals required for the transcription initiation and regulation within their long terminal repeat sequences (LTRs) (subdivided in three regions named U3, R and U5). Typically, the U3 region of the 5 LTR possesses a promoter activity. Fine studies

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have well described how Syncytin-1 is under upstream regions control. The core promoter domain within the U3 region contains CAAT box and TATA box located upstream of the CAP site marking the beginning of the R region (Prudhomme et al. 2004). Mutant analyses have confirmed the functional role of these boxes. Moreover, the 5 end of the U3 region harbors multiple binding sites contributing to overall promoter efficiency including GATA, Sp-1, AP-2, Oct-1, and PPAR-γ/RXR. Although Sp-1 and Ap-2 binding sites remain putative, they have been found to be essential for LTR activity (Prudhomme et al. 2004). It is noteworthy that Syncytin-1 regulation elements not only include the 5 LTR but also a so-called upstream regulatory element (URE), a cellular 436 bp sequence immediately upstream the Syncytin1 proviral integration site, that define together with the 5 LTR a bipartite control element (Prudhomme et al. 2004). This URE is composed of two main domains: (i) a distal regulatory region, including the previously putative binding sites found in the promoter core as well as binding sites for the NF-κB and AP-1 important for the stimulation by TNFα, IFNγ, IL-1β, IL-6, and the inhibition by IFNβ (Mameli et al. 2007) (ii) a MaLR retrotransposon with binding sites for glucocorticoid and progesterone receptors (Bonnaud et al. 2005), and including a trophoblast specific enhancer (TSE) with putative sequences for ubiquitous Ap-2, Sp-1 and placenta-specific GCMa binding sites (Prudhomme et al. 2004). Glial cell missing (GCM) is a transcription factor family that has gradually attracted the attention of placenta researches. Originally isolated from a Drosophila melanogaster mutant line, two GCM homologues (GCMa and GCMb) have then been reported in mice, rats and humans (Keryer et al. 1998). GCMa is characterized by a zinc-coordinating DNA binding domain of β-sheets that recognizes an octomeric GCM binding motif 5 -ATGCGGGT-3 (Cohen et al. 2003). GCMa is primarily expressed in placenta in humans and highly expressed in the labyrinthine trophoblast cells in mice (Basyuk et al. 1999). Two binding sites by which GCMa can specifically transactivate Syncytin-1 have been described (Yu et al. 2002). Moreover, GCMa regulation has been linked to the cyclic AMP (cAMP) and protein kinase A signaling pathways (Chang et al. 2005; Knerr et al. 2005). In agreement with these observations, the Syncytin-1 5 LTR core promoter is cAMP-inducible (Prudhomme et al. 2004). Interestingly, a recent microarray approach that aimed to identify GCMa target genes reported Syncytin-A to be downregulated in murine GCMa-deficient placenta (Schubert et al. 2008), and siRNA GCMa inhibition in BeWo cells led to a decrease in syncytialization upon fusion events (Baczyk et al. 2009). Altogether, these data argue that GCMa acts as a major regulator in the humans and mice Syncytins expression as well as in placenta maintenance and development. To conclude with this regulation mechanisms overview, the imprinting hypothesis and the influence of the methylation level is briefly discussed. Genomic imprinting in mammals is though to be a rescue mechanism that maintains balanced growth and development through monoallelic expression of genes in placenta and embryo. Although very little is known about the regulation of most imprinted genes, in 2003 the observations that Syncytin-1 maps very closely to two neighboring maternally imprinted retroelements, SGCE and PEG10, and according to

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their temporally coordinated regulation, the hypothesis that Syncytin-1 could be paternally expressed emerged (Smallwood et al. 2003). On the other hand, methylation pattern studies of the Syncytin-1 5 LTR revealed an inverse correlation between CpG methylation and locus expression indicating that demethylation of the promoter is a prerequisite for the Syncytin-1 expression in trophoblasts cells (Matouskova et al. 2006). In an attempt to generalize this epigenetic characterization, Gimenez and colleagues recently compared different HERV promoters methylation profiles and showed that Syncytin-1 and Syncytin-2 5 LTR are widely hypomethylated in cytotrophoblasts during pregnancy, although in a distinct and pregnancy-stage-dependant manner (Gimenez et al. 2009) (Fig. 4.6c). For instance, the Syncytin-2 locus remains unmethylated throughout pregnancy, whereas methylation of the Syncytin-1 locus appears increased in the last trimester. Thus, the selective and temporal unmethylation of the Syncytin-1 locus in placenta during the first trimester may allow Syncytin-1-mediated cell differentiation and fusion, while, in contrast, increased methylation at term may limit Syncytin-1 production and consequent cell fusion or putative anti-apoptotic protection (Knerr et al. 2007) in accordance with cytotrophoblast limited fusion and higher apoptosis rate. 4.3.3.3 Additional Factors The complete inhibition of cytotrophoblast fusion can’t be reached by blocking the Syncytin-1 protein (Mi et al. 2000; Frendo et al. 2003b), and as we mentioned above, extravillous cells that express Syncytin-1 and hASCT2 do not necessarily fuse. This indicates so far that Syncytin-1 plays a major role in fusion, but also strongly suggests that other elements may contribute to this event. We proposed here to review some of them, with special interest focuses on plasma membrane dynamics, cell– cell communication and immunity as a link between the various events leading to a coordinated tissue function. Human placenta only will be discussed. Before many retroviral receptors were known, observations emerged that an infected cell could not be superinfected by the same retrovirus, and sometimes even not being superinfected by a different one (Kim et al. 2004). Thus, interference groups were defined as set of retroviruses that cannot infect a cell at the same time. Indeed, all the members of an interference group utilize the same receptor for cell entry, and when a cell is infected once, cellular receptors are blocked by the envelope proteins of the first retrovirus and no longer available for infection of the second one. It is obvious that the balance between the envelope and its local receptor availabilities needs to be taken under consideration before predicting anything, as illustrated by FLV (Sommerfelt and Weiss 1990). Yet, given that Syncytin-1 is an endogenous envelope protein, we can expect the same rules to be valid. Indeed, together with spleen necrosis virus (Ponferrada et al. 2003), Syncytin-1 belongs to the RDR/Type D mammalian receptors interference group (Blond et al. 2000). Consequently, the hASCT2 receptors would no longer be available for type D retroviruses infection as soon as Syncytin-1 is co-locally expressed in massive doses. This is the core hypothesis to discuss different models that integrate local and temporal expression data (for a complete review, see (Potgens et al. 2004)). Overall it is a clear indication that the

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cytotrophoblast fusion is guided through local availabilities of both envelopes and their corresponding receptors. We previously mentioned how precisely Syncytin-1 seems to be associated with a restricted part of the polarized cell membrane, i.e. mainly detected at the apical part of the syncytiotrophoblast with strong suspicions that turn around a membrane subdomain enriched in cholesterol and defined as DRMs/rafts. Plasma membranes are lipid bilayers with an asymmetrical distribution of phospholipids between the inner and the outer leaflet. They are fluidic structures that permanently reorganized themselves, through dynamic lateral diffusivity, rotations, and flippase-mediated flip-flop switches. Thus, membrane-anchored proteins are part of this flow. This would allow them to reach and stay at a plasma membrane sub-localization, as well as to contribute to their functional transmembrane stable insertion. Flippases are ATP-dependant translocase enzymes that assure the asymmetrical distribution of phospholipids between the inner and the outer leaflet of the membrane. Remarkably, the loss of asymmetry triggered by a redistribution of phosphatidylserine from the inner to the outer leaflet has been described to be a prerequisite for fusion in skeletal muscle (van den Eijnde et al. 2001) and in placenta derived BeWo cells (Lyden et al. 1993). In agreement, the loss of membrane asymmetry in a cell has been associated with early stages of the apoptosis cascade. Caspase 8 is a caspase initiator involved in early apoptosis that inactivates the flippases, resulting into the asymmetry loss. When antisense and peptide inhibition strategies against caspase 8 are used, fusion of trophoblast cells is inhibited (Black et al. 2004). In addition, cholesterol-enriched domains are associated with weak fluidic properties. Although the direct relation between membrane dynamics and fusogenic proteins still remains to be elucidated, we can speculate that the positioning of Syncytin-1 that leads to the fusion event, may occurs within a well-controlled membrane subdomain with physical properties that form a stringent envelope environment compatible with receptor binding and subsequent events. The different points we just mentioned mainly focused on the syncytiotrophoblast. Syncytin-1 membrane localization, phospholipids dynamics and early stages of apoptosis were presented as part of the multinucleated cell life. The problem here is that the syncytiotrophoblast is presumed to have a very low transcriptional activity and likely depends on the input of RNA to avoid necrosis, as indicated by different in vitro experiments (Bernirschke and Kaufmann 2000; Huppertz et al. 1999). This suggests the importance of an effective cell– cell communication and material supply systems. Gap junctions are transmembrane channels composed of connexin that provide a diffusion system for small proteins such as cAMP, IP3 or Ca2+ . In primary trophoblast culture, the inhibition of connexin 43 (Cx43) resulted in a fusion inhibition (Frendo et al. 2003b) and a decrease of Syncytin-1 mRNA expression (Frendo et al. 2003a). Moreover, the hASCT2 receptor is a Na+ -dependent amino acid transporter that can carry amino acids such as L-glutamine, L-alanine, L-leucine and L glycine, through the membrane. The clear localization of the receptor within the membrane of cytotrophoblastic cells underlying the syncytiotrophoblast (Hayward et al. 2007) (Frappart, Cheynet, Mallet, unpublished data), suggests that hASCT2 could efficiently change

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the amino acid balance between the cytotrophoblast and the syncytiotrophoblast. Altogether, material flow that involves small amino acids and molecules, along with numerous electric charges balance changes (Ca2+ through gap junctions, anionic phosphatidylserine via flip flop events) appear to play a direct or indirect role in the fusion regulation. In such a model, the syncytiotrophoblast is indeed described as one element, part of a global turnover system which includes a generative pool of cytotrophoblast cells that can feed the multinuclear layer upon fusion events and thus maintain placental growth. Athough Syncytin-1 and Syncytin-2 and their receptors play major roles, they are probably not the only proteins involved in this cooperative mechanism. Given that the placenta is an extra-embryonic tissue, half paternal and half maternal genetically inherited, the past decades have gathered reproductive immunologists researches to solve the fetal allograft problem. The contact zone between mother uterus and fetus extravillous cells of spiral arterioles appears to be one of these predictive immunological conflict zones. In direct connection with our topic on retroviral fusogens, note that the simian retrovirus (SRV), that induces immunodeficiency, belongs to the same interference group that Syncytin-1, i.e. it binds to the ASCT2 amino acid receptor. The link between immune response and amino acid balance has been seriously explored, and interestingly the involvement of HERV in immune response has already been suggested (Espinosa and Villarreal 2000). We present here some points of discussion in such a way. During pregnancy, maternal tryptophan is required for the T lymphocytes activation and “immunosuppression by starvation” is the consequence of tryptophan depletion experiments (Mellor et al. 1999). Besides, a tryptophan-catabolizing enzyme, the indoleamine 2,3-dioxygenase (IDO), is particularly expressed in the syncytiotrophoblast. Thus, the lymphocyte regulation appears to be strongly mediated by the ability of the apical membrane to incorporate the tryptophan into the syncytiotrophoblast (Kudo and Boyd 2001). In other words, the tolerance towards the allograft is conditioned by the CD98/LAT1 tryptophan transporter and the resulting amino acid balance changes. Even if the Syncytin-1 and Ory-1 hASCT2 receptor only mediates the transport of small amino acids (and consequently probably not tryptophan), considerations about balance changes that could impact the immune system response are maybe not so far. Indeed, glutamine is a necessary substrate for the nucleotide synthesis of lymphocyte cells. In peripheral blood an optimal glutamine level is required to influence the switch within the sub-populations of T lymphocytes, Th1 and Th2, through a predominantly Th1 host response (Chang et al. 1999). In sepsis mice, glutamine supplementation changes the production of IL-6, IL-4 and IFN-γ, and thus may reverse or re-equilibrate the Th1/Th2 balance response during sepsis (Yeh et al. 2005). Although it is still in debate, the Th1/Th2 switch appears to play a critical role during pregnancy, especially through a Th1 bias in recurrent pregnancy loss (Chaouat 2007). This allows us to speculate a direct or indirect involvement of amino acids balance changes as a consequence of the envelope protein fixation on its receptor. Yet, another answer to the allograft tolerance during pregnancy emerged after it was reported that MSRV particles (related to the HERV-W family) induce T lymphocyte response (Perron et al. 2001), the analysis of the putative

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immunosuppressive domain in the TM subunit of different Syncytins has revealed a immunosuppressive activity for Syncytins-2 and -B but surprisingly not for the Syncytins-1 and -A (Mangeney et al. 2007) We can conclude this discussion by briefly reporting different abnormalities that occur in pathological contexts in relation with some elements just mentioned above. Pre-eclampsia (PE) and HELLP syndrome (hemolysis, elevated liver enzymes and low platelets) are disorders associated with abnormal placentation, including defects in syncytiotrophoblast formation. Numerous studies have associated PE and HELLP with Syncytin-1 and Syncytin-2 significant reduction (Lee et al. 2001; Knerr et al. 2002; Chen and Olson 2005; Strick et al. 2007; Chen et al. 2008). Interestingly, a redistribution of the Syncytin-1 within the syncytiotrophoblast polarized cell layer was observed for patients with PE (Lee et al. 2001). Moreover, PE is associated with a predominant Th1 immunity type (Jianjun et al. 2010), that could hypothetically make the bridge with Syncytins defects, unbalanced amino acids flux and immunity. Hypoxia is overall important in the differentiation and fusion steps, since these conditions reduce the Syncytin-1 transcriptional level and inhibit cytotrophoblast fusion, whereas hASCT2 mRNA level remains unchanged (Kudo et al. 2003; Knerr et al. 2003; Chen and Olson 2005). Finally, higher apoptotic rates are observed in cultured cytotrophoblast cells from PE and HELLP (Strick et al. 2007).

4.3.4 Syncytin-1 Expression Outside of Its Privileged Tissue As illustrated above, the multi levels-control of expression of Syncytin-1 suggests that for all Syncytins, expression is tightly regulated to be constrained to placenta. Among Syncytins, only the expression of Syncytin-1 has been so far described outside from its privileged tissue. Syncytin-1 is expressed in astrocytes, glial cells and activated macrophages in brain regions affected by multiple sclerosis (MS). Syncytin-1 expression in astrocytes mediates neuroimmune activation and death of oligodendrocytes by inducing the release of cytotoxic redox reactants (Antony et al. 2004). In astrocytes, Syncytin1 induces the expression of OASIS (old astrocytes specifically induced substance), an endoplasmic reticulum stress sensor, which in turn increases the expression of inducible NO synthetase and concurrent suppression of cognate hASCT1 receptor, resulting in diminished myelin protein production (Antony et al. 2007). What mechanisms reactivate Syncytin-1 in the brain in MS is still not clear. It could be the result of viral infection of the brain, such as herpes simplex virus, which has previously been shown to transactivate Syncytin-1 expression, or cytokine deregulation (Perron et al. 1993). Indeed it has been shown in astrocyte cultures that MS detrimental cytokines, IFN-γ and TNF-α are able to induce Syncytin-1 expression through NF-κB activation, while MS protective IFN-β inhibits its expression (Mameli et al. 2007). In addition Syncytin-1 induction by exogenous TNF-α into the corpus callosum, a region of the brain frequently exhibiting demyelination in MS, leads to neuroinflammation, reduction of myelin proteins level and neurobehavioural deficits in Syncytin-1-transgenic mice, as observed in MS (Antony et al. 2007).

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Interestingly as a parallel between MS and cancers, NO production in tumor vessels correlates with an increase of the over-all survival as well as the decrease of metastatic potency in experimental systems (Mortensen et al. 2004). On line with this, the level of Syncytin-1 expression represented a positive prognostic indicator for recurrence-free survival of breast cancer patients (Larsson et al. 2007). Conversely, increased Syncytin-1 expression was associated with decreased overall survival in rectal but not in colonic cancer patients (Larsen et al. 2009). The situation appears unclear in endometrial carcinoma (EnCa) where Syncytin-1 expression increase in normal endometrium of patients may possibly influence the development of endometriosis (Oppelt et al. 2009). Thus, the prognostic impact of Syncytin-1 expression appears to vary with the tumor type potentially, due to different functions associated with different pathways of reactivation. In breast cancers, Syncytin-1 expression was observed for about one-third of patients, and additionally, neighbouring endothelial cells were shown to express hASCT2 receptor (Bjerregaard et al. 2006). In vitro studies confirmed the involvement of Syncytin-1 in the fusion process between breast cancer cell lines and endothelial cells (Bjerregaard et al. 2006). Syncytin-1 associated cell–cell fusion was also identified in EnCa tumors in vivo, but interestingly, in vitro studies showed the implication of Syncytin-1 in both the fusion and the proliferation of EnCa cells (Strick et al. 2007). Syncytin-1 up regulation via the cAMP pathway leads to cell–cell fusion while induction by steroid hormones (estradiol) leads to proliferation. This molecular switch is apparently controlled by TGF-β1 and TGF-β3 which are induced by steroid hormones and may override Syncytin-1 mediated cell–cell fusions (Strick et al. 2007).

4.4 Conclusion Our life begins with fusion as a successful pregnancy in mammals appears to depend on Syncytin(s), retroviral members of a family of single-pass transmembrane proteins which contribute at least to cell–cell fusion necessary for placental syncytiotrophoblast morphogenesis. As we have seen, works of the last 10 years showed that Syncytins may represent extreme examples of foreign genes domestication as different elements were preserved during (parallel) evolution to assume (partly) similar roles in various species such as rodents, lagomorphs, sheep, and primates including human. These domesticated elements represent apparently a tremendous but very minor part of endogenous retroviruses (ERV) which colonized mammalian genomes. Can we consider that gift as a pay-back of retroviruses, as they emerged from our ancestors genome by transcomplementation of retrotransposons with viral envelopes (Xiong and Eickbush 1990; Malik et al. 2000), or is there a “price to pay”? From studies in animal and human cancers, there is little doubt that tumor hybrids/fused cells are generated in vivo and that at least in animals they can be a source of metastasis (Pawelek and Chakraborty 2008). Interestingly, fusion between cancer and normal cells can lead to restoration of the apoptosis cascade or to cell differentiation, inducing a reduced tumorigenicity. However cancerous cells fusion may also lead on the contrary to a more aggressive phenotype, and, if fusion occurs

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with vascular endothelial cells, to metastasis. Consequences of Syncytin-1 expression in various cancers may reflect such a diversity. Although retrovirology is a 100 years old discipline, it is less than 5 years ago that a role for viruses in cell fusion and its importance in the overall evolution of cancer was proposed (Duelli and Lazebnik 2007). Altogether, these findings strongly support a comparative analysis of the modalities and consequences of infectious retroviruses and endogenous retroviral Syncytins expression on the cell–cell fusion processes. HERV expression/activation seems to be a common feature in cancers, a phenomenon that has been linked to deregulation of methylation (Schulz et al. 2006). Global hypomethylation of transposable elements may be a prerequisite of chromosomal instability. Similarly, viral induced fusion might result in the chromosomal instability observed in cancer cells (Duelli and Lazebnik 2007). Hypomethylation of the ERVWE1/Syncytin-1 in placenta and in seminoma (Gimenez et al. 2010) as compared to HERV-W family hypermethylation in placenta and hypomethylation in seminoma may reflect both situations (Gimenez et al. 2009). At the protein level, clarifying the interactions between Syncytin-1 and TGF-β may contribute to elucidate the regulation of cell– cell fusions occurring in development and in other syncytial cell tumors. Thus, the increase of cholesterol efflux from cellular membrane by TGF-β could modify membrane location and function(s) of Syncytin-1. Overall, fusion/differentiation, proliferation and suggested anti-apoptotic capacities of Syncytin-1 delineate the portrait of an oncogene. Acknowledgments We are grateful to Sarah Prudhomme, Frederick Arnaud and Juliette Gimenez for their critical comments which contributed significantly to the improvement of this chapter. We thank Danièle Evain-Brion, Thierry Heidmann, Thomas E. Spencer, and François-Loïc Cosset for providing pictures and photographs.

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Chapter 5

Syncytins: Molecular Aspects Hungwen Chen and Mei-Leng Cheong

Abstract Cell–cell fusion is essential for many physiological events such as egg-sperm fertilization, muscle differentiation, viral entry, and placental development. The envelope genes of human endogenous retrovirus (HERV)-W and -FRD, named syncytin-1 and -2, are primarily expressed in placenta and mediate trophoblastic fusion for formation of the multinucleated syncytiotrophoblast layer, which is essential for fetal growth. Recently, envelope genes of retroviral origin encoding fusogenic proteins have also been identified from the genomes of other mammals. These novel envelope genes, named mouse syncytin-A and -B, rabbit syncytin-Ory1, and sheep enJSRV envelope, are required for placental development and trophoblastic fusion. Although syncytins are critical for placental development, abnormal expression of syncytins is associated with placental disorders, cancers, and neurological diseases. Here we provide a general description of the biological functions of syncytins and the regulation of syncytin gene expression at the molecular level. In addition, the pathological functions of syncytins are also discussed. Keywords cAMP · cancer · cytotrophoblast · envelope proteins · GCM1 · HERV-FRD · HERV-W · membrane fusion · multiple sclerosis · preeclampsia · receptors · syncytin · syncytiotrophoblast Abbreviations AKT ASCT CBP CREB CTM en Env

Protein kinase B Alanine, serine and cysteine selective transporters CREB binding protein cAMP response element binding protein C-terminal part of TM Endogenous Envelope

H. Chen (B) Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan e-mail: [email protected] L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_5, C Springer Science+Business Media B.V. 2011

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FBW GCM GSK HA HDAV HELLP HERV HIV HR IL JSRV LTR MFSD2 MLV MS OASIS PC PI-3 K PKA RDR SCF SKP SU SUMO SynT TM

H. Chen and M.-L. Cheong

F-box/WD repeat-containing protein Glial cell missing Glycogen synthase kinase Hemagglutinin Histone deacetylase Hemolysis, elevated liver enzymes and low platelets Human endogenous retrovirus Human immunodeficiency virus Heptad repeat Interleukin Jaagsiekte sheep retrovirus Long terminal repeat Major facilitator superfamily domain containing 2 Murine leukemia virus Multiple sclerosis Old astrocytes specifically induced substance Proprotein convertase Phosphatidyl inositol-3 kinase Protein kinase A Retrovirus D-type receptor Skp, Cullin, F-box S-phase kinase-associated protein Surface unit Small Ubiquitin-like Modifier Syncytiotrophoblast Transmembrane unit

Contents 5.1 Cell Fusion in the Placenta . . . . . . . . . . . . . . . . . . 5.1.1 Development of the Placenta . . . . . . . . . . . . . . 5.1.2 Human Syncytin-1 . . . . . . . . . . . . . . . . . . . 5.1.3 Human Syncytin-2 . . . . . . . . . . . . . . . . . . . 5.1.4 Mouse Syncytin-A and -B . . . . . . . . . . . . . . . 5.2 Structure and Functional Studies of Syncytins . . . . . . . . . 5.2.1 Biosynthesis of Syncytins . . . . . . . . . . . . . . . . 5.2.2 Functional Domains and Motifs in Syncytins . . . . . . . 5.2.3 Syncytin Receptors . . . . . . . . . . . . . . . . . . 5.2.4 Mechanism of Membrane Fusion . . . . . . . . . . . . 5.3 Regulation of Syncytin Expression . . . . . . . . . . . . . . 5.3.1 GCM1 Regulation of Syncytin-1 and -2 Gene Expression . 5.3.2 Regulation of GCM1 Activity . . . . . . . . . . . . . . 5.3.3 Epigenetic Regulation of Syncytin-1 and -2 Gene Expression 5.4 Syncytins and Disease . . . . . . . . . . . . . . . . . . . . 5.4.1 Syncytins in Placental Disorders . . . . . . . . . . . . .

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Syncytins: Molecular Aspects

5.4.2 Syncytin-1 in Malignancies . . . . 5.4.3 Syncytin-1 in Neurological Diseases 5.5 Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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5.1 Cell Fusion in the Placenta The first differentiation event in the early stage of embryonic development is the formation of the trophectoderm, which later develops into specialized placental trophoblasts essential for implantation and placentation. The primary physiological functions of placenta include mediation of fetal-maternal nutrient and gas exchange, synthesis of hormones and growth factors, and protection of the fetus from maternal immune surveillance. Recent epidemiological studies have revealed a highly significant association between undernourished fetuses and a greater risk of developing stroke, heart disease, and type II diabetes in the adult (Barker 2004). Therefore, a functional placenta is essential for both a successful pregnancy outcome and a healthy adult life.

5.1.1 Development of the Placenta The definitive structure of the human placenta becomes apparent as early as day 21 of pregnancy (Benirschke and Kaufmann 2001b). In the early gestation stage, cytotrophoblast stem cells facing the maternal decidua proliferate and fuse to form a syncytial mass. Later, vascular spaces called trophoblastic lacunae appear in the syncytium around day 8–9. The cytotrophoblast cell layer under the syncytium rapidly proliferates into lacunae and forms the primary chorionic villi. Subsequently, proliferation of the cytotrophoblasts, growth of mesenchymal cells under the cytotrophoblast cell layer, and development of blood vessels transform the primary villi into secondary and tertiary villi. The fetal placental arteries and veins develop within the chorionic villi and are surrounded by an inner layer of cytotrophoblasts and an outer layer, the multinucleated syncytiotrophoblast, formed by cell–cell fusion of underlying mononucleated cytotrophoblasts (Fig. 5.1a) (Benirschke and Kaufmann 2001a). The physiological functions of syncytiotrophoblast include gas and nutrient exchange between fetus and mother, hormone production, and immunomodulation. For mouse placental development, the trophectoderm cells overlying the inner cell mass proliferate both inwards to form the extraembryonic ectoderm and outwards to form the ectoplacental cone. As the development of the mouse placenta proceeds, the extraembryonic ectoderm expands to form the chorionic epithelium. The allantois arises from the mesoderm at the posterior end of the embryo and makes contact with the chorion later at embryonic day 8.5 (E8.5), an event termed chorioallantoic attachment (Cross et al. 2006, Hemberger and Cross 2001). The interaction of the chorion with the allantois folds into a villous tree-like structure called the labyrinth, which is analogous to the chorionic villi in human placenta. In the labyrinth, fetal blood vessels are separated from the maternal circulation by three

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Fig. 5.1 Placental anatomy in human and mouse. (a) Structure of the human placental villus. The human placental villus is composed of mesenchymal cells, fetal blood vessels, a mononuclear cytotrophoblast layer, and a multinuclear syncytiotrophoblast layer. The latter is in contact with the maternal blood and is essential for gas and nutrient exchange between mother and fetus. Note the migration and invasion of cytotrophoblasts into the maternal endometrium, which remodels the endometrial arteries to increase maternal blood flow to the placenta. (b) Structure of murine placental labyrinth. The murine placental labyrinth contains juxtaposed maternal blood sinuses and fetal blood vessels separated by a trilaminar trophoblast cell structure, which is composed of a single layer of mononuclear trophoblast cells and two layers of multinuclear syncytiotrophoblasts (I and II)

layers of trophoblast cells, including a layer of mononuclear trophoblast cells that is in contact with the maternal blood sinusoids, and two syncytiotrophoblast layers, SynT-I and SynT-II. SynT-I also faces the maternal blood space, whereas SynT-II is in contact with the fetal blood vessel (Fig. 5.1b) (Rossant and Cross 2001, Simmons et al. 2008).

5.1.2 Human Syncytin-1 Approximately 8% of the human genome contains sequences of retroviral origin, including gag (structural proteins), pol (viral enzymes), env (envelope proteins), and long terminal repeats (LTRs). Collectively, these sequence elements are named human endogenous retroviruses (HERVs). The gag gene encodes structural proteins for viral genome encapsidation and particle formation. The pol gene encodes viral enzymes for reverse transcription of the viral genome into a double-stranded DNA and subsequent integration of this DNA into the host genome. The env gene encodes a viral envelope protein that recognizes the host cell surface receptor and mediates viral-host membrane fusion. The LTRs contain regulatory elements for

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transcription initiation and termination. Most HERVs are defective in replication due to accumulated mutations in their genomes. Cell–cell fusion of cytotrophoblasts is essential for formation of thesyncytiotrophoblast. Syncytin is the first identified fusogenic protein mediating trophoblastic fusion in human placenta. As a functional envelope protein of the HERV-W family, syncytin was independently identified and characterized by two groups. Blond and coworkers (Blond et al. 1999) investigated the expression of HERV sequences related to the multiple sclerosis-associated retrovirus in healthy tissues and identified a novel envelope gene of HERV-W that is highly expressed in placenta. At about the same time, using a yeast signal sequence trap for identification of novel secreted proteins, Mi and colleagues (Mi et al. 2000) identified a cDNA fragment that is also primarily expressed in human placenta and named syncytin. Syncytin turns out to be identical to the HERV-W envelope gene reported by Blond et al. Further studies demonstrated that the syncytin gene is specifically expressed in the placental syncytiotrophoblast layer and that ectopic expression of syncytin protein in non-placental cells such as COS cells promotes cell–cell fusion (Mi et al. 2000). It has long been known that elevation of cAMP level or activation of PKA can stimulate cell–cell fusion of human placental cells (Keryer et al. 1998). Correspondingly, the transcript level of syncytin gene is positively stimulated by the cAMP stimulant, forskolin, in the human BeWo placental cell line. In addition, cell–cell fusion of BeWo cells is blocked in the presence of antiserum against syncytin, suggesting that syncytin is involved in mediation of trophoblastic fusion (Mi et al. 2000). For nomenclature purpose, syncytin has been renamed syncytin-1 to distinguish it from a second HERV envelope gene named syncytin-2, described in the next subsection.

5.1.3 Human Syncytin-2 In silico analysis of human genome databases has revealed an additional 15 envelope genes predicted to encode candidate fusogenic proteins. Functional screening of these envelope genes by cell fusion assays is performed by transient expression experiments in a panel of mammalian cell lines. Of these candidate genes, one HERV-FRD envelope gene demonstrated to encode a fusogenic protein is also highly expressed in placenta. This HERV-FRD envelope gene is named syncytin-2, for encoding the second placental fusogenic protein (Blaise et al. 2003). Interestingly, similarly to syncytin-1, syncytin-2 gene expression is also upregulated in placental cells treated with forskolin. Therefore, both syncytin-1 and -2 are likely the downstream effectors in the cAMP/PKA signaling pathway that stimulates placental cell fusion. Sequence comparison indicated that the syncytin-2 sequence is highly conserved in primates from humans to New World monkeys. Phylogenetic analysis suggests that syncytin-1 and -2 genes entered the primate genome about 25 and 40 million years ago, respectively (Blaise et al. 2003). Interestingly, unlike syncytin-1, syncytin-2 possesses potential immunosuppressive activity. This is evidenced by the fact that overexpression of syncytin-2 in tumor

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cells prevents their rejection in allogenic mouse hosts (Mangeney et al. 2007). Therefore, the immunosuppressive activity of syncytin-2 may protect the fetus against the maternal immune system. Expression of syncytin-2 transcript has been detected in placental cytotrophoblasts by in situ hybridization (Esnault et al. 2008, Kudaka et al. 2008).

5.1.4 Mouse Syncytin-A and -B About 10% of the sequences in the murine genome are of retroviral origin. A systematic in silico search of mouse genome databases has identified two murine endogenous retroviral envelope genes named syncytin-A and -B that encode placental fusogenic proteins. Evolutionarily, syncytin-A and -B entered the rodent/murine genome about 20 million years ago. Expression of syncytin-A and -B transcripts can be detected from 9.5 days postcoitum, increasing till 14.5 days postcoitum (Dupressoir et al. 2005). In situ hybridization further localized the syncytin-A and -B transcripts to the SynT-I and SynT-II layers of the labyrinth, respectively (Simmons et al. 2008). A recent gene targeting study of syncytinA demonstrates that syncytin-A is critical for formation of the SynT-I layer and for proper placental development. Poor placental vascularization was found in the syncytin-A knockout mice due to overexpansion of unfused trophoblast cells, which reduced fetal blood vessel spaces (Dupressoir et al. 2009). This study also suggests that syncytin-A and -B are not functionally redundant and syncytin-B may be indispensable for the development of the SynT-II layer. In addition, as with human syncytin-2, the ectodomain of the syncytin-B transmembrane subunit is also immunosuppressive (Mangeney et al. 2007). Further investigation is warranted to elucidate the physiological functions of syncytin-B in the mouse placenta. Endogenous retroviral envelope genes encoding placental fusogenic proteins have also been identified from the genomes of other mammals. For example, a rabbit syncytin gene named syncytin-Ory1 has recently been reported after mining the rabbit genome (Heidmann et al. 2009). In addition, an envelope gene of endogenous Jaggsiekte sheep retroviruses (enJSRVs) is crucial for ovine placental development because antisense oligonucleotides targeting the enJSRV envelope gene blocked trophoectoderm outgrowth and trophoblast giant binucleate cell differentiation (Dunlap et al. 2006).

5.2 Structure and Functional Studies of Syncytins Both syncytin-1 and -2 genes encode a polypeptide of 538 amino acids, whereas syncytin-A and -B genes encode a polypeptide of 617 and 618 amino acids, respectively (for sequence alignment, see Fig. 5.2a). Like the conventional retroviral envelope proteins, the syncytin polypeptide is also composed of functional domains and motifs that are required for conformational integrity, interaction with receptors on target cell surface, and mediation of membrane fusion.

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Fig. 5.2 Human and murine syncytins. (a) Protein sequence alignment of syncytin-1, -2, -A, and -B. Sequence alignment is based on MULTALIN (http://npsa-pbil.ibcp.fr/cgibin/align_multalin.pl). The CX2 C and CX6 CC motifs for disulfide bond formation are underlined. Of note, the position of CX2 C motif in syncytin-1 is away from the N-terminus of syncytin-1 polypeptide. The furin cleavage site is boxed. Conserved residues in all of four syncytins are shown in bold. Numbers on the right indicate the numbering of the last residue in each line of the indicated syncytin protein sequence. (b) Schematic representation of the domain structures of syncytins. SP, signal peptide; FP, fusion peptide; TMD, transmembrane domain. (c) Model of cell–cell fusion mediated by syncytin-1 and -2. The SU subunits of the matured syncytin-1 and -2 proteins interact with their cognate receptors, ASCT2 and MFSD2, respectively, on the target membrane. It is generally believed that the interaction induces a conformational change to expose the fusion peptide in the TM subunit and facilitate its insertion into the target membrane. An intramolecular interaction between HRA (A) and HRB (B) in the TM subunit brings the two membranes closer together to fuse with each other. Note that only one mature protein of the syncytin trimer is depicted. Topology analysis of ASCT2 and MFSD2 is based on TOPCONS (http://topcons.cbr.su.se)

5.2.1 Biosynthesis of Syncytins Syncytin polypeptides are post-translationally cleaved into two subunits, surface (SU) and transmembrane (TM) subunits during biosynthesis (Chang et al. 2004, Chen et al. 2008, Cheynet et al. 2005). Furin is a member of the proprotein convertase family of serine proteases, including PC1/3, PC2, and PC4, to name a few. It is generally believed that furin is involved in the cleavage reaction because treatment of furin inhibitor I blocks cleavage of both syncytin-1 and -2, and consequently cell–cell fusion mediated by both proteins (Chen et al. 2008). The consensus furin cleavage site is positioned after the C-terminal R residue in the sequence R/K-R or R-X2-R. Accordingly, the R-N-K-R and R-V-R-R sequences in syncytin1 and -2, respectively, and the R-R-K-P and R-P-K-R sequences in syncytin-A

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and -B, respectively, are the predicted furin cleavage sites critical for generation of functional proteins (Fig. 5.2a, b). Indeed, mutation of the R-N-K-R motif in syncytin-1 into A-A-A-R generates mutant syncytin-1 proteins on the cell surface with no fusion activity, supporting the idea that proteolytic cleavage is essential for biosynthesis of functional syncytin-1 protein (Cheynet et al. 2005). When transiently expressed in the furin-defective LoVo cells, the processing of syncytin-1 and -2 polypeptides was not efficient enough to confer cell–cell fusion. However, enhanced processing of both syncytin precursor proteins and significant cell–cell fusion were detected when functional furin was coexpressed in LoVo cells (Chen et al. 2008). Therefore, this complementation study also demonstrates that furin is a major protease in the conversion of syncytin proproteins into mature fusogenic proteins. Moreover, cell–cell fusion events may take place only when a sufficient level of mature fusogenic protein is present on the cell surface. Transient expression experiments have been performed to study the biosynthesis of syncytin-1 in BeWo cells. This study demonstrates that syncytin-1 is subjected to N-glycosylation, which is critical for generation of functional syncytin-1 protein. This notion is also supported by the finding that syncytin-1 proteins were absent on the cell surface of transfected BeWo cells after treatment with tunicamycin, an inhibitor of N-glycosylation (Cheynet et al. 2005). Furthermore, syncytin-1 proteins undergo trimerization during biosynthesis, which may be facilitated by an N-terminal heptad repeat region in the ectodomain of the TM subunit (see Section 5.2.4).

5.2.2 Functional Domains and Motifs in Syncytins The functional domains and motifs and structure-functional relationships in syncytins have been identified and characterized by linker-scanning, site-directed, and deletion mutagenesis of syncytin-1 and -2 proteins. Random in-frame insertions of a five-amino acid linker into the syncytin-1 backbone indicates that the SU subunit and the ectodomain of TM subunit maintain a rigid conformation, which does not allow linker insertion in almost all sites tested. Exceptionally, Ser51, Val139, and Glu156 in the SU subunit and the CTM domain have a flexible enough conformation to allow linker insertion that does not abolish the fusogenicity of mutants. Deletion of the CTM domain of syncytin-1, but not syncytin-2, further enhanced its fusogenicity (Chang et al. 2004, Chen et al. 2008). A peptide region of 16 amino acids in the CTM of murine leukemia virus (MLV) envelope protein, termed R-peptide, has been demonstrated to inhibit its fusogenicity. Deletion of this R-peptide in the MLV envelope protein caused extensive fusion by formation of large syncytia. In fact, the MLV R-peptide is cleaved by a viral protease during virion maturation (Yang and Compans 1997). Apart from MLV, R-peptide cleavage has been demonstrated in other viruses such as Mason-Pfizer monkey virus and spleen necrosis virus, which are type D and C retroviruses, respectively (Bobkova et al. 2002). Since syncytin-1 has a longer CTM domain than syncytin-2, the possibility of an R-peptide-like region in the CTM domain of syncytin-1 cannot be ruled out.

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Critical domains for regulation of cell–cell fusion in the ectodomain of syncytin TM subunit include a fusion peptide at the N-terminus of the ectodomain and two heptad repeat regions (HRA and HRB) downstream of the fusion peptide. The fusion peptide is believed to insert into the target membrane, followed by intramolecular interaction between the two heptad repeats, to facilitate membrane fusion (see Section 5.2.4). This notion is supported by the fact that peptides derived from the heptad repeat region of an envelope protein are able to block its fusogenic activity. For example, synthetic peptides derived from the HRB of syncytin-1 and the HRA of syncytin-A have been shown to specifically inhibit cell–cell fusion mediated by syncytin-1 and -A, respectively (Chang et al. 2004, Peng et al. 2007). It is generally believed that the processed SU and TM subunits of an envelope protein initially form an unactivated complex on the cell surface. When the SU subunit binds its cognate receptor, a conformational change may occur to allow the TM subunit to resume its fusion activity. In this regard, formation and rearrangement of disulfide bond between a disulfide isomerase motif, CX2 C, in the SU subunit and a CX6 CC motif in the TM subunit are required for activation of TM after the interaction between SU and its receptor. For the unactivated MLV envelope protein, a disulfide bond is formed between the CX2 C motif and the last cysteine residue in the CX6 CC motif. Interestingly, receptor binding induces isomerization of the disulfide bond between the SU and TM subunits to form a disulfide bond in the CX2 C motif of SU (Wallin et al. 2004). As a result, the fusogenicity of TM is activated to promote membrane fusion via the fusion peptide and the heptad repeat regions (see Section 5.2.4). Because syncytin proteins contain the CX2 C and CX6 CC motifs (Fig. 5.2a, b), it is highly possible that formation and isomerization of a disulfide bond between CX2 C and CX6 CC is essential for the biosynthesis and fusion function of syncytin proteins. Indeed, mutagenesis of the last cysteine of the CX6 CC motif in syncytin-1 into alanine abolished its fusogenic activity (Cheynet et al. 2005). Furthermore, mutagenesis of the cysteine residues in the CX2 C motifs of syncytin-1 and -2 also abolished their fusogenic activities (Chen et al. 2008). Importantly, the syncytin-1 and -2 CX2 C mutants imposed a dominant negative effect on their wild-type counterparts. Moreover, mutation of the first cysteine in the CX2 C motif (C186 in syncytin-1 and C43 in syncytin-2) had a stronger dominant negative effect than mutation of the second cysteine (C189 in syncytin-1 and C46 in syncytin-2) (Chen et al. 2008). These observations suggest that the C186 and C43SU mutants may compete effectively with the wild-type SU for association with TM during biosynthesis. It is also possible that the C186SU-TM and C43SU-TM complexes fail to interact with the cognate receptors for syncytin-1 and -2 or undergo proper isomerization of disulfide bond in order to activate the fusion activity of TM. On the other hand, domain swapping experiments indicate that the SU and TM subunits of syncytin-1 and -2 are not interchangeable in terms of fusion activity. The positions of CX2 C motifs in the SU subunits of syncytin-1 and -2 are relatively different, i.e. in the middle of the syncytin-1 SU subunit and close to the N-terminus of the syncytin-2 SU subunit. It is very likely that swapping the SU and TM subunits between syncytin-1 and -2 affects the formation and isomerization of disulfide

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bonds that are essential for biosynthesis and fusion function of syncytin-1 and -2. This speculation is further substantiated by the observation that the syncytin-1 C186 mutant had no effect on inhibition of biosynthesis and fusogenicity of syncytin-2, and neither did the syncytin-2 C43 mutant on syncytin-1 (Chen et al. 2008).

5.2.3 Syncytin Receptors Envelope-mediated membrane fusion first requires an interaction between envelope and its cognate receptor on the target membrane. Receptors for syncytin-1 and -2 have been identified. Because envelope proteins sharing sequence homology may use the same cellular receptor, it is reasonable to speculate that syncytin-1 may interact with the same receptor for those retroviral envelope proteins with sequence similarity to syncytin-1. Syncytin-1 shares sequence homology with a large interference group of retroviruses that use a type D mammalian retrovirus receptor (RDR) as their common receptor. RDR is a synonym for human sodium-dependent neutral amino acid transporter type 2 (ASCT2). Therefore, ASCT2 is a candidate receptor for syncytin-1. This speculation can be tested by receptor interference assays for the fusogenicity of syncytin-1 in an array of TE671 human cells expressing a panel of retroviral envelope proteins. If syncytin-1 binds ASCT2, cell–cell fusion mediated by syncytin-1 will be blocked in TE671 cells expressing an envelope protein that also uses ASCT2 as its receptor. This is the case and supports ASCT2 as the syncytin-1 receptor (Blond et al. 2000). Interestingly, functional studies further indicate that syncytin-1 is able to induce cell–cell fusion of CHO cells expressing ASCT2 or a related transporter ASCT1 (Lavillette et al. 2002). These observations indicate that both ASCT2 and ASCT1 are the functional receptors for syncytin-1. Structure-function studies further identified the ASCT2-binding domain in syncytin-1. Soluble full-length SU proteins were compared with the N- and C-terminal truncation mutants for binding to cells expressing ASCT2. Accordingly, a region consisting of the N-terminal 124 amino acids of the mature SU subunit was found to be the minimal receptor-binding domain of syncytin-1 (Cheynet et al. 2006). Identification of syncytin-2 receptor was much more labor-intensive. Complementation experiments were conducted to identify the cellular receptor for syncytin-2, based on the principle that cells originally refractory to syncytin-2 pseudotypes will become susceptible when a piece of human chromosome carrying the syncytin-2 receptor gene in introduced. The Chinese hamster fibroblast A23 cell line is not susceptible to syncytin-2 pseudotypes. Accordingly, a panel of human/hamster hybrid cell lines harboring different human chromosomal fragments was screened for their susceptibility to syncytin-2 pseudotypes. This screen mapped the candidate syncytin-2 receptor to chromosome 1p34.2. Functional characterization of candidate genes in this chromosome region identified the major facilitator superfamily domain containing 2 (MFSD2) as the receptor for syncytin-2 (Esnault et al. 2008). In terms of sequence and structural similarities, MFSD2, like the bacterial permeases and symporter proteins, belongs to the 10–12

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transmembrane domains protein superfamily. Of note, two alternatively spliced variants of human MFSD2 have been identified, with the variant 1 containing extra 13 amino acids in the first extracellular loop. Nevertheless, cell fusion assays for both MFSD2 variants do not reveal a significant difference in cell–cell fusion mediated by syncytin-2 (H.C. unpublished results). Human MFSD2 gene is highly expressed in placenta and its transcript level is upregulated in BeWo cells in response to forskolin. In situ hybridization indicates that MFSD2 gene is primarily expressed in the placental syncytiotrophoblast layer (Esnault et al. 2008). Because syncytin-2 transcripts are expressed in cytotrophoblasts, this suggests that syncytin2-expressing cytotrophoblasts are restricted to fuse into the syncytiotrophoblast layer.

5.2.4 Mechanism of Membrane Fusion The molecular mechanisms underlying the cell–cell fusion mediated by syncytin-1 and -2 are currently unknown. Nevertheless, structural studies of influenza hemagglutinin (HA) and human immunodeficiency virus (HIV) envelope protein (gp160) have provided important insights into the fusion processes mediated by retroviral envelopes. HA and gp160 proteins are arranged into a homotrimer conformation after synthesis. The HA polypeptide is cleaved into HA1 and HA2 subunits, whereas the gp160 polypeptide is cleaved into gp120 and gp41 subunits. After cleavage, the fusion peptide located at the N-terminus of HA2 and gp41 is shielded from exposure in a pocket. The fusion peptide springs out towards the target membrane when HA2 undergoes a conformational change at acidic pH in endosomes or when gp120 interacts with its cognate receptor to induce a conformational change in gp41. Subsequently, two heptad repeat regions (HRA and HRB) in HA2 and gp41, whose primary sequences are predicted to be extended amphipathic α-helices, form coiledcoils and bring the host and target membranes closer together to facilitate membrane fusion (Colman and Lawrence 2003, Frey et al. 2006). By analogy with the working model for HA and gp160, interaction between the SU subunit of syncytin and the syncytin receptor may facilitate isomerization of disulfide bonds in SU and TM subunits and cause a conformational change such that SU is released and the fusion peptide in the ectodomain of the TM subunit is exposed to insert into the target membrane. Moreover, HRA and HRB in the ectodomain of TM subunits interact with each other and bring together the apposed membranes to facilitate membrane fusion (Fig. 5.2c). As mentioned above, integration of syncytin-2-containing HERV-FRD into the primate genome occured at an early stage of primate evolution. Moreover, sequence analysis of the syncytin-2 gene demonstrates limited polymorphism in humans and high sequence identity among primate species. Theses observations suggest that the sequence of syncytin-2 gene is well conserved and may be very close to the ancestral gene. Therefore, structural analysis of syncytin-2 may help to reveal the structure of the oldest mammalian retroviral envelope protein. A central domain of 54 amino acids in the ectodomain of the syncytin-2 TM subunit, which primarily contains the

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HRA, has been subjected to crystallographic analysis. The solution of the structure of this domain discloses a trimeric conformation with three α-helices associated with each other, which may be involved in oligomerization of syncytin-2 during biosynthesis. In addition, the backbone structure of this central domain is very similar to that of present-day retroviruses such as MLV and the human T-cell lymphotropic virus, given that the distribution of surface charges and peptide sequence are different (Renard et al. 2005). Therefore, structural similarity is well preserved in retroviral envelope proteins, and they may all undergo a similar fusion process.

5.3 Regulation of Syncytin Expression The human syncytiotrophoblast layer undergoes apoptosis and sheds into the maternal circulation. Therefore, replenishment of new syncytiotrophoblast layer is required to maintain the functional integrity of placental villi. As syncytin-1 and -2 are crucial for formation of syncytiotrophoblast layer, tight regulation of syncytin gene expression is imperative to maintain proper placental development and function.

5.3.1 GCM1 Regulation of Syncytin-1 and -2 Gene Expression The underlying mechanism for placenta-specific expression of syncytin-1 and -2 has been investigated at transcriptional and epigenetic levels. GCM (glial cell missing) was originally isolated from a fly mutant line that produces additional neurons at the expense of glial cells (Hosoya et al. 1995, Jones et al. 1995). Two GCM-like genes (GCM1 and GCM2) have been reported in mouse, rat, and human. GCM proteins share sequence homology in the N-terminal region that constitutes a DNAbinding domain called the GCM motif, which has a preferred binding sequence of 5 -(A/G)CCC(T/G)CAT-3 or its complement (Akiyama et al. 1996, Schreiber et al. 1998). Moreover, two zinc ions tightly coordinated by cysteine and histidine residues in the DNA-binding domain of GCM1 are essential for its DNA-binding activity (Cohen et al. 2003). Sequence homology is less preserved outside the GCM motif; a transactivation domain has been identified in the C terminus of GCM proteins. Ontogeny of murine GCM1 demonstrates that GCM1 gene is expressed in a subset of trophoblast cells at the chorionic plate at E7.5-8. When chorioallantoic attachment takes place at E8.5, expression of GCM1 is detected in clusters of trophoblast cells of the chorioallantoic surface, where invaginations begin, and this is accompanied by development of fetal blood vessels to form the labyrinth (Basyuk et al. 1999, Stecca et al. 2002). It is evident that GCM1 is required for this branching morphogenesis during labyrinthine development as these events are impaired in GCM1-knockout mice. Moreover, GCM1-knockout trophoblasts fail to undergo cell–cell fusion to form syncytiotrophoblasts (Anson-Cartwright et al. 2000, Schreiber et al. 2000). Expression of human GCM1 transcripts can be detected

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in the cytotrophoblasts and syncytiotrophoblasts of term placenta as well as in villous sprouts and cytotrophoblast cell columns of first trimester placenta, further supporting an important role for GCM1 in human placental development (Baczyk et al. 2004, Nait-Oumesmar et al. 2000). The studies of GCM1-knockout mice suggest that GCM1 may control trophoblastic fusion by regulation of syncytin-1 gene expression at the transcriptional level. Indeed, GCM1-binding sites (GBSs) have been identified upstream of the 5 -LTR of syncytin-1 promoter and are required for transactivation of syncytin-1 promoter activity by GCM1. Moreover, overexpression of GCM1 stimulates cell– cell fusion in BeWo and JEG-3 cells (Yu et al. 2002). Functional GBSs have been identified in the 5 -LTR of syncytin-2 promoter and the MFSD2 promoter, and these are also responsive to GCM1. In addition, ectopic expression of GCM1 stimulates expression of syncytin-2 and MFSD2 transcripts in BeWo cells and MCF-7 breast cancer cells (H.C. unpublished results). Correspondingly, functional assays further demonstrate that silencing GCM1 expression by RNA interference or antisense oligonucleotides suppresses BeWo cell fusion and inhibits syncytiotrophoblast formation, but promotes proliferation of cytotrophoblasts in villous explants (Baczyk et al. 2009). On the other hand, studies in GCM1-knockout mice and GCM1knockout trophoblast stem cells indicate that GCM1 is involved in regulation of syncytin-B, but not syncytin-A, gene expression (Simmons et al. 2008). Overall, these observations clearly show that GCM1 is a critical for syncytin gene expression and is required for the differentiation of human trophoblast cells.

5.3.2 Regulation of GCM1 Activity Because the cAMP/PKA signaling pathway regulates syncytin-1 and -2 expression and human placental cell fusion, it is reasonable to speculate that GCM1 is a downstream effector of the cAMP/PKA signaling pathway in stimulation of placental cell fusion. Indeed, GCM1 activity is regulated by cAMP/PKA signaling pathway at both transcriptional and post-translational levels. Elevation of cAMP level and activation of PKA increase the GCM1 transcript level in BeWo cells (Knerr et al. 2005). In addition, activation of cAMP/PKA signaling enhances the association between GCM1 and CBP, a transcriptional coactivator with histone acetyltransferase activity. As a result, CBP acetylates GCM1 at Lys367, Lys406, and Lys409 to prevent GCM1 from ubiquitination and thereby increase GCM1 stability. CBP also enhances the transcriptional activity of GCM1 and colocalizes with GCM1 at the syncytin-1 promoter (Chang et al. 2005). As the syncytiotrophoblast layer is a highly dynamic structure, its maintenance as a steady-state structure during pregnancy is crucial. Accordingly, GCM1 activity is subjected to tight regulation in order to maintain proper trophoblastic fusion in the placenta. In this regard, GCM1 is a labile protein with a half-life of about 90–120min and is subject to degradation by the ubiquitin-proteasome system (Yang et al. 2005). Ubiquitination of target proteins involves a cascade of reactions, i.e. (1) activation of ubiquitin by an E1 ubiquitin-activating enzyme in an ATP-dependent

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process, (2) transfer of activated ubiquitin to an E2 ubiquitin-conjugating enzyme, and (3) transfer of ubiquitin from E2 to a substrate protein with or without the aid of an E3 ubiquitin-protein isopeptide ligase. The SCF E3 ligase is a complex composed of Skp1, Cullin1, Rbx1, and a substrate-recognition F-box protein. A screening of F-box proteins expressed in placenta has identified FBW2 as the functional F-box protein for mediation of GCM1 ubiquitination and degradation (Yang et al. 2005). Recognition of GCM1 by FBW2 primarily depends on phosphorylation of Ser322 in GCM1, which is mediated by GSK-3β (Chiang et al. 2009). Of interest, acetylation of GCM1 at Lys367, Lys406, and Lys409 by CBP protects GCM1 from ubiquitination. Given that there are 28 Lys residues in GCM1, just how acetylation of the three Lys residues prevents the remaining Lys residues in GCM1 from ubiquitination remains an interesting question. An additional mode of regulation of GCM1 activity is sumoylation of GCM1 Lys156, an activity mediated by Ubc9, the E2 component of the sumoylation machinery. Conjugation of SUMO-1 to Lys156 impedes the DNA-binding activity of GCM1, and therefore downregulates GCM1 activity in placenta (Chou et al. 2007). Sumoylation of endogenous GCM1 can be detected in JAR placental cells. At present, it is not clear whether sumoylation of GCM1 is a constitutive modification or is regulated by upstream signaling pathways. Overall, regulation of GCM1 activity is subjected to fine-tuning by different types of post-translational modification, which may be crucial for the functional and structural integrity of the syncytiotrophoblast layer.

5.3.3 Epigenetic Regulation of Syncytin-1 and -2 Gene Expression Epigenetic regulation of gene expression involves modification of cytosines in the context of CpG dinucleotides, i.e. methylation at the C5 of the cytosine pyrimidine ring. Methylated cytosines recruit methyl-cytosine-binding proteins, HDACs, and transcriptional corepressors to silence gene expression. Epigenetic regulation of syncytin-1 and syncytin-2 plays an important role in maintaining the placentaspecific expression pattern of both genes. Analysis of the CpG methylation pattern in the U3 region of the 5 -LTR of syncytin-1 promoter reveals hypermethylated CpGs in non-placental cells such as skin fibroblasts, HeLa cells, and breast carcinoma cells (Matouskova et al. 2006). Instead, hypomethylation of these GpGs is detected in placental tissues and BeWo cells. Of interest, treatment of HeLa cells with 5 -azacytidine and trichostatin A, which are inhibitors of DNA methylation and histone acetylation, respectively, does not significantly affect the methylation pattern of these CpGs, not to mention stimulation of syncytin-1 expression (Matouskova et al. 2006). Likewise, CpGs in the 5 -LTR of syncytin-2 promoter are hypermethylated in non-placental cells, but hypomethylated in placental tissues and BeWo cells (Gimenez et al. 2009). However, these methylated CpGs are less resistant to treatment of 5 -azacytidine and trichostatin A in non-placental cells (H.C. unpublished results). It is very likely that CpG hypomethylation in the 5 -LTRs of syncytin-1 and -2 promoters may facilitate GCM1-mediated transcriptional activation of syncytin-1

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and -2 in placenta. Interestingly, ectopic expression of GCM1 in MCF7 breast cancer cells promotes CpG demethylation in the 5 -LTR of syncytin-2 promoter, and therefore stimulates syncytin-2 expression and cell fusion (H.C. unpublished results). A possible mechanism underlying CpG hypomethylation in syncytin-2 promoter by GCM1 may involve active DNA demethylation. Because a GBS is within the 5 -LTR of syncytin-2 promoter, it is possible that GCM1 may recruit proteins with demethylation activity to promote CpG demethylation and then transactivate syncytin-2 gene expression.

5.4 Syncytins and Disease Expression of endogenous retroviral sequences in host species can be advantageous by induction of resistance to exogenous retrovirus infection or detrimental by causing cancer or contribution to disease development, as in multiple sclerosis and schizophrenia (Griffiths 2001). As syncnytin-1 and -2 are specifically expressed in placenta and are essential for placental function, decreased expression of syncytin-1 and -2 is found in placental disorders. Unexpectedly, aberrant expression of syncytin-1 is also found in multiple sclerosis and breast and colorectal cancers (Antony et al. 2004, Bjerregaard et al. 2006, Larsen et al. 2009).

5.4.1 Syncytins in Placental Disorders The proper interaction and transformation process between maternal decidua, myometrium, and fetal trophoblasts is essential for a successful pregnancy outcome. Abnormal placentation incurring insufficient oxygen and nutrient exchange has been reported in pregnancy disorders including preeclampsia, HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and fetal growth restriction. Preeclampsia is a major pregnancy-specific disorder affecting around 5% of pregnancies and is one of the major causes of maternal deaths and stillbirths worldwide. The diagnostic criteria of preeclampsia are new-onset or aggravated hypertension and proteinuria in the latter half of a pregnancy. Besides the high mortality, clinical impacts include cerebral or visual disturbances, oliguria, pulmonary edema, HELLP syndrome, fetal growth restriction, and grand mal seizures. Although the etiologic factors of preeclampsia are currently unknown, shallow trophoblast invasion and insufficient maternal vascular remodeling are observed in preeclamptic placentae (Maynard et al. 2008). It is thought that these defects impair the development of the fetal-maternal vasculature and result in placental ischemia and hypoxia, which then contributes to the pathogenesis of preeclampsia in the late second or third trimester of pregnancy. Recently, decreased expression of syncytin-1 and -2 has been reported in preeclampsia. A possible mechanism for this decreased expression in preeclampsia is disruption of the GCM1 transcription network following placental hypoxia. The activity of GSK-3β is elevated in preeclamptic placentae and hypoxic placental cells due to inhibition of the upstream

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Fig. 5.3 Regulation of gene expression of syncytin-1 and -2 by GCM1. GCM1-responsive elements have been identified upstream of the 5 -LTR of syncytin-1 promoter and within the 5 -LTR of syncytin-2 promoter. GCM1 is a labile protein subject to ubiquitination (Ub) via FBW2, which recognizes Ser322-phosphorylated GCM1. Phosphorylation of Ser322 is mediated by GSK-3β of which activity can be suppressed by the PI-3K/Akt signaling pathway. In preeclampsia and in hypoxic placental cells, the PI-3K/Akt signaling pathway is inhibited, leading to elevation of GSK3β activity, and therefore a promotion of GCM1 degradation. GCM1 protein is stabilized in the cAMP/PKA signaling pathway by enhancement of GCM1 acetylation (Ac) by CBP. Deacetylation of GCM1 can be mediated by HDAC3. Furthermore, GCM1 is sumoylated in placental cells and conjugation of SUMO-1 (SU-1) at Lys156 in the GCM motif impedes the DNA-binding activity of GCM1. Note that GCM1 phosphorylation by GSK-3β and PKA may take place in the cytosol

PI-3K/Akt signaling pathway. As a result, more GCM1 proteins are subjected to Ser322 phosphorylation by GSK-3β, leading to GCM1 ubiquitination and degradation (Fig. 5.3). Concomitantly, syncytin-1 and -2 expression is downregulated and this may destabilize the structure of the syncytiotrophoblast layer in preeclampsia.

5.4.2 Syncytin-1 in Malignancies The most common form of breast cancer is ductal carcinoma originating from the inner lining of mammary ducts or the lobules that supply the ducts. Although the real mechanism is not clear, breast cancer cells can fuse with normal host cells in vitro and in vivo. When cancer cells migrate, they invade adjacent tissues and are prone to approach and fuse with the endothelium. Human breast cancer cell lines derived from ductal adenocarcinoma and part of ductal adenocarcinoma tissues have recently been discovered to express syncytin-1, which may function

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as a mediator to regulate cancer-endothelial fusion (Bjerregaard et al. 2006). The staining ratio of syncytin-1 in breast cancer tissues is positively correlated with the recurrence-free survival. Here syncytin-1 acts as a good prognostic factor. On the other hand, the expression ASCT2 in endothelial cells also increases in breast cancer tissues, but does not have a significant impact on overall or recurrence-free survival (Larsson et al. 2007). Fusions may occur when syncytin-1-expressing cancer cells make contact with ASCT2-expressing host endothelial cells. Under these circumstances, the host immune system and tumor suppressor genes may be triggered in the endothelium to eliminate the cancer cells. Colorectal cancer is the fourth most common form of cancer and the third leading cause of cancer-related death worldwide. Using a monoclonal antibody against syncytin-1 and -2, overexpression of syncytins was found to be associated with decreased survival for rectal, but not colon cancer (Larsen et al. 2009). These data suggest that syncytin expression might show different prognostic impacts in different types of tumor. Endometrial carcinoma is another major cancer in women, mostly affected at perimenopausal and postmenopausal period. Histologically, endometrioid carcinoma is the most common subtype of endometrial carcinoma, a cancer which often expresses both estrogen and progesterone receptors and is hormone-dependent. Cell–cell fusions can be detected in endometrioid carcinoma tissues by histological examination. One study (Strick et al. 2007), showed upregulated syncytin-1 expression in all benign (polyp, hyperproliferative stage, and hyperplasia) and malignant endometrial tissues, with the most significant change in endometrial carcinoma. Furthermore, syncytin-1 expression was stimulated by estrogen, progesterone, or cAMP in primary cells and cell lines from endometrial carcinoma. Such an increase in syncytin-1 gene expression may be associated with the transformation phenotype of anchorage independence, because knocking down the expression of syncytin 1 by RNA interference reduced the colony number and size in soft agar assay (Strick et al. 2007). In this study, however, statistical analysis of the prognostic impact of syncytin-1 on survival in endometrial carcinoma was not presented.

5.4.3 Syncytin-1 in Neurological Diseases Multiple sclerosis (MS) is primarily an inflammatory disorder of the brain and spinal cord in which focal lymphocytic infiltration leads to damage of myelin and axons resulting in demyelination. In most patients, clinical manifestations indicate the involvement of motor, sensory, visual, and autonomic systems (Compston and Coles 2008). A recent study demonstrated that approximately 3 times as much syncytin-1 is present in brain tissues of MS patients than the controls (Antony et al. 2004). In addition, expression of syncytin-1 in human fetal astrocytes resulted in a significant increase of proinflammatory cytokine IL-1β. The culture medium from the above condition contained redox reactants that were highly cytotoxic to human oligodendrocytes. In a mouse model of MS, syncytin-1-mediated neuroinflammation and death of oligodendrocytes were prevented by the antioxidant ferulic acid, indicating

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syncytin-1 may be involved in the pathogenesis of active demyelination through redox reactant-mediated cellular damage in the brain. A follow-up study further demonstrated that syncytin-1 induces ER stress and the expression of old astrocyte specifically induced substance (OASIS), an ER stress sensor, in cultured astrocytes. Expression of OASIS leads to downregulation of ASCT1 expression, which may cause oligodendrocyte injury and death through dysregulated transport of trophic and toxic amino acids (Antony et al. 2007). The pathologic role of syncytin-1 has also been studied in the patients with motor neuron diseases, neurological disorders that progressively destroy cells controlling essential muscle activity such as speaking, swallowing, breathing, walking, and general movement. The levels of syncytin-1 transcript were significantly elevated in biopsies from the most affected muscles compared to the control tissues (Oluwole et al. 2007). Nevertheless, whether the role of syncytin-1 in the pathophysiology of motor neuron diseases is similar to that in MS needs further investigation.

5.5 Conclusion The multinucleated syncytiotrophoblast layer is a specialized epithelium lining the outer surface of the placental villus and is responsible for placental transport and hormone production during human pregnancy. Trophoblastic fusion is essential for differentiation of syncytiotrophoblast, which is mediated by syncytin-1 and -2 derived from HERV envelope genes and is regulated by GCM1 transcription factor and its upstream kinases such as PKA and GSK-3β. Recent studies have identified syncytin-like proteins in mouse and rabbit. Importantly, a transgenic study demonstrated that mouse syncytin-A is essential for trophoblastic fusion and syncytiotrophoblast differentiation, confirming that syncytin-like proteins are also required for placental development in other mammals. Being fusogenic proteins for trophoblastic fusion, human syncytin-2 and mouse syncytin-B possess some immunosuppressive activity via the ectodomains in their TM subunits. This additional function suggests that syncytin-2 and -B may contribute to immune tolerance of the developing embryo, though further studies are needed. Sequence comparison and phylogenetic analysis suggest that human and mouse syncytin genes are not related and were independently adopted by these species during evolution. Nevertheless, functional domains and motifs characteristic of a retroviral envelope protein have been identified in both human and mouse syncytins and these are essential for their fusogenic activities. Therefore, the fusion process mediated by syncytins may be similar to the known processes for influenza HA and HIV gp160. As the integrity of the syncytiotrophoblast layer is maintained by a dynamic balance between ongoing apoptosis and syncytial fusion, tight regulation of syncytin gene expression is imperative. Interestingly, expression of both syncytin-2 and its receptor MFSD2 genes are regulated by GCM1, and this may be a positive evolutionary selection in coordination of syncytin-2 functions in placenta. Clinically, decreased expression of syncytin-1 and -2 is detected in preeclampsia, whereas syncytin-1 overexpression is found in many other diseases. Defective

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placentation may impair GCM1 activity such that syncytin gene expression is downregulated contributing to the development of preeclampsia. In multiple sclerosis, overexpression of syncytin-1 in neurons and glial cells incurs an inflammatory response, ER stress, and reduced transporter activity that damage cells in the central nervous system. As the study of syncytin continues, new findings about the pathological roles of syncytins in human diseases and cancers are anticipated, which may provide new therapeutic avenues against the diseases. In addition, understanding the immunosuppressive roles of syncytin-2 and -B during pregnancy may significantly improve the success rate of pregnancy. Acknowledgments We thank Dr. Tso-Pang Yao for critical reading of this manuscript. Work in Hungwen Chen’s laboratory is supported by grants from National Science Council of Taiwan (grant 96-2311-B-001-034) and Academia Sinica of Taiwan.

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Chapter 6

Role of the Actin Cytoskeleton Within FuRMAS During Drosophila Myoblast Fusion and First Functionally Conserved Factors in Vertebrates Susanne-Filiz Önel, Christine Dottermusch, Angela Sickmann, Detlev Buttgereit, and Renate Renkawitz-Pohl

Abstract The larval musculature of Drosophila arises by fusion of two types of myoblasts: the founder cells (FCs), which determine the identity of every individual muscle, and fusion competent myoblasts (FCMs). Cell–cell recognition and adhesion is mediated by the Ig class of transmembrane proteins. They form an adhesion ring/belt at the contact sites of FCMs and FCs/growing myotubes to establish a Fusion Restricted Myogenic Adhesive Structure (FuRMAS). FuRMAS are postulated to trigger myoblast fusion, with the formation, and dissolution of F-actin foci/plugs at the sites of cell–cell contact. Electron-dense vesicles accumulate at opposing membranes of FCMs and FCs/growing muscles, and form a pre-fusion complex (1 μm2 ). This is hypothesised to take place in the centre of the FuRMAS. The vesicles are thought to be exocytosed, followed by membrane vesiculation and removal of membrane remnants to achieve cytoplasmic continuity over an area of 12 μm2 . The FCM can then be integrated into the growing myotube. This last step depends on Arp2/3 mediated F-actin reorganisation. The data on cell adhesion, signalling and actin regulation in zebrafish, C2C12 cells and mice strongly indicate conserved factors and principles between Drosophila and vertebrate myoblast fusion. Keywords FuRMAS · myoblast fusion · actin cytoskeleton · signalling cascades Abbreviations A Ants Arf Blow CA Cdc42

Acidic domain Antisocial ADP ribosylation factor Blown fuse Cofilin-homologous, and acidic domain Cell division control protein 42 homolog

R. Renkawitz-Pohl (B) Fachbereich Biologie, Entwicklungsbiologie, Philipps-Universität Marburg, 35043 Marburg, Germany e-mail: [email protected]

L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_6, C Springer Science+Business Media B.V. 2011

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CED DOCK Duf EM F-actin FC FCM FuRMAS GEF Gfl Hbs IgSF IrreC Kirre Lmd Mbc Minc Mib2 PIP2 Rac Rols Rst S2 cells Sing Sltr Sns TEM VCA vrp1 WASP Wip

S.-F. Önel et al.

Caenorhabditis elegans death gene Dedicator of cytokinesis Dumbfounded Electron microscopy Filamentous actin Founder cell Fusion competent myoblast Fusion restricted myogenic adhesive structure Guanine nucleotide exchange factor Gleefull Hibris Immunoglobulin superfamily Irregular optic Chiasma Kin of irre Lameduck Myoblast city Myoblast incompetent Mind bomb 2 Phosphatidylinositol 4,5-bisphosphat Ras-related C3 botulinum toxin substrate Rolling pebbles Roughest Schneider cells Singles bar Solitary Sticks and stones Transmission electron microscopy Verprolin-homologous, cofilin-homologous, and acidic domain Verprolin 1 Wiskott-Aldrich syndrome family protein WASP-interacting partner

Contents 6.1 Introduction to the Cell Biology and Topology of Myoblast Fusion in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1 Founder Cells, Fusion-Competent Myoblasts, Progenitors and Myofibres . . . . . . . . . . . . . . . . . . . . . . . 6.1.2 Two Phases of Myoblast Fusion . . . . . . . . . . . . . . . . . . . . . 6.2 Pre-fusion Complexes Form at Opposing Membranes, the Membranes Vesiculate, and FCMs Are Integrated into the Growing Myotube . . . . . . . . . 6.2.1 Electron-Dense Vesicles and the Pre-fusion Complex . . . . . . . . . . . 6.2.2 Electron-Dense Plaques and Vesiculating Membranes . . . . . . . . . . . 6.3 Cell Adhesion and Signalling Cascades . . . . . . . . . . . . . . . . . . . .

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Role of the Actin Cytoskeleton Within FuRMAS

6.3.1 Cell Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 Duf/Kirre Very Likely Acts via Rolling Pebbles in FCs and Growing Myoblasts . . . . . . . . . . . . . . . . . . . . . 6.3.3 Signalling on the FCM Side . . . . . . . . . . . . . . . . . . . . 6.4 Actin Regulation at the Site of Adhesion During Drosophila Myoblast Fusion 6.4.1 Molecular Mechanisms of F-Actin Regulation at the Site of Drosophila Myoblast Fusion . . . . . . . . . . . . . . . . . . 6.4.2 Possible Roles for Arp2/3-Based F-Actin Formation at the Site of Drosophila Myoblast Fusion . . . . . . . . . . . . . 6.4.3 Actin Regulation During Vertebrate Myoblast Fusion . . . . . . . . 6.5 The FuRMAS Model and the Topology of Myoblast Fusion . . . . . . . . 6.5.1 Fusion Pores, Membrane Vesiculation and the Size of Cytoplasmic Continuities . . . . . . . . . . . . . . . . . . . . 6.5.2 FuRMAS as Signalling Centres . . . . . . . . . . . . . . . . . . 6.6 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141

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150

. . . . . . . . .

151 152 153

. . .

154

. . . . . . . . .

156 159 161

. . . .

162 163 165 165

. . . .

. . . .

6.1 Introduction to the Cell Biology and Topology of Myoblast Fusion in Drosophila As in vertebrates, muscles of Drosophila are multinucleated and arise from fusion of myoblasts. In Drosophila, genetic screens identified genes, which when mutated, lead to failure of myoblast fusion. Recently, it turned out that a number of the essential factors identified in Drosophila are functionally conserved in vertebrates. Here, we briefly introduce the larval musculature in Drosophila before concentrating on myoblast fusion. The complete Drosophila musculature is established within a few hours, and following successful cell adhesion, a multiprotein complex, called FuRMAS (Fusion Restricted Myogenic Adhesive Structure) assembles at the site of fusion (Kesper et al. 2007, Önel and Renkawitz-Pohl 2009). F-actin plugs/foci accumulate at the fusion site (Kesper et al. 2007, Richardson et al. 2007), and alterations in the actin cytoskeleton are thought to be required for myoblast fusion (reviewed by Chen and Olson 2004, Kim et al. 2007, Önel and Renkawitz-Pohl 2009, Önel et al. 2004, Rochlin et al. 2010). Since individual fusion events take place within a matter of minutes (Beckett and Baylies 2007), a FuRMAS is a very transient structure (Kesper et al. 2007). Here, we describe the ultrastructural features of myoblast fusion, F-actin branching at the site of fusion, the essential molecules and their role during fusion. We discuss the topology at the individual fusion site and suggest that FuRMAS not only act as a platform to recruit fusion-relevant molecules, but also limit the area of membrane breakdown. We provide a comprehensive review of the key molecular players in Drosophila and functionally conserved proteins in zebrafish, C2C12 cells and mice.

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6.1.1 Founder Cells, Fusion-Competent Myoblasts, Progenitors and Myofibres During the Drosophila life cycle, the musculature develops twice: once during embryogenesis leading to the musculature of the larvae, and a second time during metamorphosis to form the adult musculature of the holometabolic insect (reviewed by Maqbool and Jagla 2007). In this review, we focus on the well-studied formation of larval muscles during embryogenesis, since this has provided the greatest insights into myoblast fusion so far. Larval muscles comprise individual myotubes with 4–24 nuclei (Baylies et al. 1998), forming a stereotypic pattern per segment (Fig. 6.1a) that is determined by early processes within the mesoderm. A combination of ectodermal and mesodermal regulators specifies the somatic mesoderm shortly after mesoderm formation (Riechmann et al. 1997). Subsequently, NotchDelta-mediated lateral inhibition determines fusion-competent myoblasts (FCMs) and progenitor cells, the latter defined by the interplay of intrinsic and extrinsic regulators (Maqbool and Jagla 2007). The progenitor divides once more to yield two FCs, or alternatively one FC plus one adult muscle progenitor. The FCs, determining the identity of individual muscles, are localised at the external layer of the somatic mesoderm. They seed the characteristic pattern of the

Fig. 6.1 Myoblast fusion mutant Drosophila embryos lack the stereotypic muscle pattern. (a) The stereotypic pattern of larval musculature is visualized by β3 Tubulin (green) (Buttgereit et al. 1996, Leiss et al. 1988) in wild-type embryos. (b) Embryo of a fusion mutant (homozygous for blow2 ) is characterized by many unfused FCMs (green, β3 Tubulin) and FCs (red nuclei, rP298 enhancer trap in the duf/kirre gene (Nose et al. 1998), green cytoplasm β3 Tubulin)

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larval musculature directed by combined sets of transcriptional regulators (reviewed by Baylies et al. 1998, Frasch 1999, Maqbool and Jagla 2007, Paululat et al. 1999, Taylor 1998). All FCMs express the Gli-related Zn-finger protein Lameduck (Lmd)/Myoblast incompetent (minc)/Gleefull (Gfl) (Duan et al. 2001, Furlong et al. 2001, Ruiz-Gomez et al. 2002). However, recent evidence suggests that FCMs are not a unique population but contain molecular diversity (discussed in Rochlin et al. 2010). After fusion Lmd/Minc/Gfl is degraded and the nuclei of the integrated FCMs become reprogrammed to the fate of the individual FC. A likely candidate for degrading Lmd/Minc/Gfl is Mind bomb 2 (Mib2, E3 ubiquitin ligase) (CarrascoRando and Ruiz-Gomez 2008) which is also required for muscle integrity and stability (Nguyen et al. 2007).

6.1.2 Two Phases of Myoblast Fusion Myoblast fusion progresses in two temporal phases (Bate 1990, Bate and Rushton 1993, Beckett and Baylies 2007). First, one FC fuses with one or two FCMs to form a muscle precursor (Fig. 6.2a). During the second phase of fusion, the precursor fuses with further FCMs until reaching the number of nuclei characteristic for an individual myofibre (for nomenclature and histology see review by Bate 1993). In addition to temporal differences, one model is that genetic differences also exist between the two phases of fusion, meaning some genes play an important role in progression from the precursor stage to the mature muscle (Berger et al.

Fig. 6.2 FCs and FCMs express Ig-class of cell adhesion molecules. (a) In the first phase of fusion a single FC (blue nucleus) fuses with FCMs (yellow nuclei) to form a precursor. After fusion the nuclei adopt the fate of the FC. Further fusion events lead to the final size of a mature muscle fibre. (b) The FC/growing myotube expresses Duf/Kirre (blue) and Rst/IrreC (green) as membrane spanning molecules. Both contain five Ig loops in the extracellular domain. (c) FCMs express Rst/IrreC (green), Sns (yellow) and Hbs (orange). Sns and Hbs are characterised by eight extracellular Ig–domains. Plasma membrane (PM); extracellular (EC), transmembrane (TM) and intracellular (IC) domains of the proteins are indicated

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2008, Massarwa et al. 2007, Rau et al. 2001, see Önel and Renkawitz-Pohl 2009 for discussion). In the larval musculature of Drosophila, a single myotube/myofibre corresponds to an individual muscle specifically attached to their individual epidermal attachment sites yielding the stereotypic muscle pattern (see Fig. 6.1a). This pattern is severely disrupted in fusion mutants, where one can distinguish FCs, precursor cells and FCMs (Fig. 6.1b). To date, screening and analysing fusiondefective mutants have identified a number of fusion-relevant genes. Their gene products include transmembrane proteins, signalling molecules and actin regulators (summarised in Table 6.1). Ultrastructural analyses of wild-type embryos and fusion-defective mutants have provided mechanistic insights into myoblast fusion and helped create a topological model.

Table 6.1 Myoblast fusion relevant proteins of Drosophila and their functional conservation in vertebrate myoblast fusion Mice

Mouse C2C12 cells

Transmembrane proteins Duf/Kirre Kirrel

Neph1

–

Hbs

–

–

–

Rst/Irre C

–

–

–

Singles bar Sns

– Nephrin

– Nephrin

– –

Signalling molecules Arf6a –

–

Arf6

Blow

–

–

–

Cdc42b

–

Cdc42

–

Crk

Crk, Crkl

–

–

Mbc

Dock1, Dock5

Dock1, Dock5

Dock180

Mib2

–

–

–

Drosophila

Zebrafish

References Ruiz-Gomez et al. (2000), Srinivas et al. (2007), Sohn et al. (2009) Artero et al. (2001), Dowark et al. (2001) Strünkelnberg et al. (2001) Estrada et al. (2007) Bour et al. (2000), Sohn et al. (2009) Chen et al. (2003), Pajcini et al. (2008) Doberstein et al. (1997), Artero et al. (2003), Schröter et al. (2003) Schäfer et al. (2007), Vasyutina et al. (2009) Erickson et al. (1997), Balagopalan et al. (2006), Moore et al. (2007) Rushton et al. (1995), Erickson et al. (1997), Moore et al. (2007), Laurin et al. (2008), Pajcini et al. (2008) Nugyen et al. (2007), Carrasco-Rando and Ruiz-Gomez (2008)

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Table 6.1 (continued) Mouse C2C12 cells

Drosophila

Zebrafish

Mice

Rac1, Rac2

Rac1

Rac1

Rols

–

–

–

Schizo/Loner

–

–

Brag-2

Actin regulators ArpC1 – Arp3 –

– –

– –

Kette/Nap-1

–

–

Nap-1

Scar

–

–

WAVE2

Vrp1/Sltr

WASP

Wip

–

–

WASP

References Hadeka-Suzuki et al. (2002), Srinivas et al. (2007), Vasyutina et al. (2009) Chen et al. (2001), Menon et al. (2001), Rau et al. (2001) Chen et al. (2003), Pajcini et al. (2008) Massarwa et al. (2007) Richardson et al. (2007), Berger et al. (2008) Schröter et al. (2004), Nowak et al. (2009) Richardson et al. (2007), Berger et al. (2008), Nowak et al. (2009) Kim et al. (2007), Massarwa et al. (2007), Berger et al. (2008) Kim et al. (2007), Massarwa et al. (2007), Schäfer et al (2007)

a Arf6 probably shares functional redundancy with another Arf-GTPase, since maternal and zygotic

loss of function mutants show wild-type muscle development (Dworak et al. 2001). seems not to be relevant for myoblast fusion in the Drosophila embryo (Schäfer et al. 2007). b Cdc42

6.2 Pre-fusion Complexes Form at Opposing Membranes, the Membranes Vesiculate, and FCMs Are Integrated into the Growing Myotube In the first phase of fusion (during stage 12), when one mononucleated FC fuses with one or two FCMs to form a precursor, myoblasts differ only slightly in size. Each myoblast is approximately 4 μm wide (light microscopy: Bate 1990 TEM: Schröter et al. 2006). The contact site of two fusing myoblasts and the area of membrane vesiculation is about 1.7–1.9 μm, with initial studies revealing no further particular features (Schröter et al. 2006). Thus, the area of membrane breakdown and cytoplasmic continuity roughly correspond to the diameter of the cells (for an example see Fig. 6.3, first phase of fusion). Thereafter, several FCMs often attach laterally to growing myotubes, with their ends extending filopodia towards

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first phase of fusion

a N

FCMs

FC

N

second phase of fusion

b N

pre-fusion complex

N

c

electron-dense vesicle

N

electron-dense plaque

N

d

N

electron-dense plaque

membrane vesiculation

mature muscle N

Founder Cell (FC)/Fusion-Competent Myoblast (FCM)

Fig. 6.3 Characteristic features of myoblast fusion at the ultrastructural level in the first and second phase of fusion. Myoblast fusion is characterised by opposing membranes, a pre-fusion complex consisting of paired electron-dense vesicles, electron-dense plaques and vesiculating membranes. The left column illustrates the fusion events schematically at the ultrastructural level (drawings of representative photographs from transmission electron microscopy). The right column visualises the position of ultrastructural features relative to FCs (growing myotubes) and FCMs. The opposing membranes are drawn as lines, electron-dense vesicles (red in b), electron-dense plaques (green in c) and vesiculation of the membrane (a, d) with large cytoplasmatic continuities marked by arrows in left column a, d. M labels the mitochondria; N labels the nuclei (blue in FCs, yellow in FCMs)

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individual attachment sites. At this point, the FCM is small in comparison to the growing myotube (Fig. 6.3, second phase of fusion). Therefore, a mechanism to restrict the area of fusion might be more important in the later rather than the initial phase of fusion. For the second phase of fusion, Doberstein et al. (1997) pioneered ultrastructural analyses of embryos at stage 13/14. The majority of fusion events occurs at this stage, progressing from precursor to multinucleated myotubes. The serial sections of Doberstein et al. (1997) revealed that several pre-fusion complexes can form simultaneously between one cell (presumably a precursor cell) and several contacting myoblasts (presumably FCMs). Myoblasts are closely aligned with their membranes directly opposite each other. Interestingly, ultrastructural analyses revealed distinct membrane-associated structures such as paired vesicles, electrondense plaques and vesiculating membranes (for a summary scheme see Fig. 6.3, second phase of fusion).

6.2.1 Electron-Dense Vesicles and the Pre-fusion Complex Electron-dense paired vesicles (40 nm in diameter) accumulate at opposing membranes. All the paired vesicles at an individual contact site comprise a pre-fusion complex. Up to 15 such paired vesicles per contact site have been detected (Doberstein et al. 1997). The number of paired vesicles varies from section to section (Doberstein et al. 1997, Kim et al. 2007); on average, sections contain 1.4 vesicles at opposing membranes (Estrada et al. 2007), which we think depends on the level of the section. Understanding the spatial distribution of vesicles of the pre-fusion complex is highly informative in terms of the topology of fusion sites. The serial sections of Doberstein et al. (1997) show that an individual pre-fusion complex spreads over an area of 1 μm2 at both sides of the opposing membranes (schematically depicted in Fig. 6.4).

6.2.2 Electron-Dense Plaques and Vesiculating Membranes Although rare, perhaps implicating a transient nature, electron-dense plaques (Fig. 6.3) with a length of 500 nm and width of 10 nm were observed at areas of intact membranes, and sometimes membrane vesiculation was seen nearby (Doberstein et al. 1997). However, neither the molecular components and function of these electron-dense plaques, nor the spaciotemporal relation between pre-fusion complexes and the electron-dense plugs are as yet clear. A frequently observed feature of fusion is that opposing membranes vesiculate, producing numerous membrane remnants. This area of membrane vesiculation often spans 3–4 μm in the second phase of fusion (for an example of an EM section see Fig. 6.5a). It is generally thought that membranes fuse by undergoing hemifusion between the outer leaflets of opposing membranes (reviewed by Martens

148 Fig. 6.4 Serial sections reveal the distribution of the pre-fusion complex over 1 μm2 . (a) Schematic drawings of an individual pre-fusion complex according to serial sections of 100 μm each (Doberstein et al. 1997, Fig. 6.3). Electron-dense vesicles accumulate at the FCM side (fusion competent myoblasts, yellow vesicles) and at the FC side (founder cell, blue vesicles)

S.-F. Önel et al.

FCM

N

FC N

N

N

M

M

Founder Cell (FC)/Fusion-Competent Cell (FCM)

and McMahon 2008). During myoblast fusion, the opposing membranes are first brought into close apposition (Fig. 6.5b). Then, we propose that multiple hemifusions occur between the outer leaflets of the FC/growing muscle and the FCM (Fig. 6.5c). Subsequent fusion of the inner leaflets leads to membrane vesiculation and thus to multiple areas of cytoplasmic continuity (Fig. 6.5d). Removal of membrane remnants is required to achieve full cytoplasmic continuity and integration of the FCM into the growing myotube. However, again the mechanism behind this is unclear. In summary, at the ultrastructural level myoblast fusion can be divided into distinct steps: e.g. cell–cell recognition and adhesion, alignment of opposing membranes, formation of pre-fusion complexes over an area of 1 μm2 and transient electron-dense plaques, vesiculation of opposing membranes, removal of membrane remnants to achieve cytoplasmic continuity, and finally integration of the FCM into the growing myotube (summarised in Fig. 6.3).

6.3 Cell Adhesion and Signalling Cascades Founder cells and fusion-competent myoblasts express a dedicated gene, and it is the dialogue between these cells that ultimately leads to myoblast fusion. This dialogue includes chemo-attraction, cell migration, recognition, adhesion and signalling. FCs/growing muscles express specific chemo-attractive signals that are proposed

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Role of the Actin Cytoskeleton Within FuRMAS a

149 b

N

3.6 μm N

767 nm growing myotubes

FMC

c

close apposition

d

hemifusion (stalk formation)

content mixing

Fig. 6.5 Multiple hemifusions lead to vesiculation of the FC and FCM membranes. The model visualizes how hemifusion of the outer lipid-bilayers and subsequent fusion of the inner lipidbilayers of an FCM and FC/growing myotube might lead to vesiculation of the cell membranes. (a) The vesiculating membrane is visualized by transmission electron microscopy over 3.6 μm (ends marked by blue arrows), areas of cytoplasmic continuity are marked by red arrowheads. Adjacent, the individual membranes of the myoblasts are clearly distinguishable. The blue arrows also mark the position of the horizontal bars in b, c and d. The scale bar corresponds to 767 nm. In b, c and d, the processes at the membrane are shown schematically, the membranes of FCM is drawn in yellow, the membrane of the FC/growing myotube in blue. The vertical bars (blue) correspond to the positions indicated by blue arrows in (a). This scheme visualizes the phosphobilayers closely opposed. (b) Hemifusion of the outer leaflets of the membranes is proposed to lead to stalk structures. (c) Subsequent fusion of the inner leaflet of the plasma membrane of FCM and FC leads to membrane vesiculation and multiple areas of cytoplasmic continuity, which allows content mixing

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to direct the FCMs towards the FCs. In both cell types, the relevant transmembrane molecules have multiple roles since they also mediate cell recognition, transient adhesion and signalling.

6.3.1 Cell Adhesion The contact sites between FCMs and FCs/growing muscles are characterised by the expression of four transmembrane molecules of the immunoglobulin superfamily (IgSF), Dumbfounded/Kin of irre (Duf/Kirre), Roughest/Irregular optic Chiasma (Rst/IrreC), Sticks and stones (Sns) and its paralogue Hibris (Hbs). These are involved in establishing and maintaining specific membrane contacts in various cell interactions (reviewed by Fischbach et al. 2009). During myoblast fusion, Duf/Kirre is expressed uniquely on the FC/growing muscle side (Ruiz-Gomez et al. 2000), Sns (Bour et al. 2000) and Hbs (Dworak et al. 2001) on the FCM side, while Rst/IrreC (Strünkelnberg et al. 2001) is expressed in both cell types (summarised in Fig. 6.2b, c, and Table 6.1). Duf/Kirre and Rst/IrreC share functional redundancy in FCs (Strünkelnberg et al. 2001). Expression of Duf/Kirre and Rst/IrreC at ectopic sites attract FCMs to these sites – at least in the absence of competing Duf/Kirre and Rst/IrreC in the FCs – thus it was hypothesized that Duf/Kirre and Rst/IrreC act as chemo-attractants (Ruiz-Gomez et al. 2000, Strünkelnberg et al. 2001). This idea was supported by time-lapse microscopy showing that FCMs direct their filopodia towards FCs and migrate towards them (Beckett and Baylies 2007), although the mechanism is unknown. Hbs acts in partial redundancy to Sns but less efficient (Shelton et al. 2009). Each individual muscle has its characteristic number of nuclei, resulting from a different number of fusion events. Several distinct enhancer modules are postulated to regulate duf/kirre expression in specific FCs. These modules might be a way to regulate the number of fusion events of an individual muscle at a molecular level (Guruharsha et al. 2009). The Ig repeats in the extracellular domains of all these transmembrane molecules suggest that they mediate heterologous cell adhesion between FCs/growing myotubes and FCMs. Indeed, individual transfections of Drosophila Schneider cells (S2 cells) with Sns and Duf/Kirre followed by mixing these cells leads to Sns and Duf/Kirre localising in opposing cells, and presumably as a consequence to cell adhesion, suggesting that Sns and Duf/Kirre indeed physically interact (reviewed by Abmayr et al. 2008). In the embryo, Duf/Kirre, Rst/IrreC and Sns expression is very dynamic (Kesper et al. 2007). Duf is limited to FCs and has functional redundancy to Rst/IrreC, which is reflected by enrichment of Rst/IrreC in FCs/growing myotubes (Fig. 6.6). However, Rst/IrreC is only expressed in a few FCMs at a given time point (Buttgereit and Renkawitz-Pohl, unpublished) while Sns is limited to FCMs. Duf/Kirre, Rst/IrreC and Sns appear in dots at the contact sites of FCs/growing myotubes and FCMs (shown for Duf in Fig. 6.6a). Higher magnifications reveal that Duf/Kirre, Rst/IrreC and Sns localise in a ring-shaped pattern at the contact sites of FCMs and growing myotubes (Fig. 6.6b–d). Their cytodomains are implicated in

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Fig. 6.6 At their contact sites FC and FCM express Ig-class of cell adhesion molecules as a belt/ring. (a) Expression of Duf/Kirre at stage 13 of embryogenesis, a time with numerous fusion events. Anterior part of the embryo is left, dorsal site to the top. In all hemi-segments Duf/Kirre is expressed in many FCs/growing muscles in a dot like manner. In b, c and d higher magnifications of individual FCMs (circumferences are marked with dotted lines) are shown contacting growing muscles. Duf/Kirre, Rst/IrreC and Sns show a ring shaped distribution at the sites of cell contact

monitoring successful cell adhesion and inducing signalling cascades into both cell types. The cytodomains are further proposed to trigger the formation of transient F-actin plugs/foci at the contact sites (see below for details), subsequently leading to membrane vesiculation and cytoplasmic continuity (for a summary of known fusion-relevant components, see Table 6.1).

6.3.2 Duf/Kirre Very Likely Acts via Rolling Pebbles in FCs and Growing Myoblasts The intracellular domains of Duf/Kirre and Rst/IrreC are 15% homologous and contain two highly conserved motifs (Strünkelnberg et al. 2001). A cellular adaptor protein, Rolling pebbles (Rols)/Antisocial (Ants) is expressed in FCs, precursors and growing myotubes, similar to Duf/Kirre and Rst/IrreC (Chen and Olson 2001, Menon and Chia 2001, Rau et al. 2001). Myoblasts in rols/ants mutant embryos often form small syncytia, implicating that Rols is not absolutely necessary for the first fusion events (Beckett and Baylies 2007, Rau et al. 2001). Rols colocalises with Duf/Kirre in the FuRMAS (Kesper et al. 2007) and is required for the enlargement of myoblasts (Menon et al. 2005). This membrane-associated localisation of Rols is lost in the mutant Df(1)w67k30, in which both duf/kirre and rst/irreC genes are deleted (Chen and Olson 2001, Menon and Chia 2001). The myoblast fusionrelevant isoform (Pütz et al. 2005) of Rols7/Ants is a 1900 amino acid protein with multiple putative protein–protein interaction domains, such as a ring finger

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in the N-terminal region, eight ankyrin repeats, and three tetratricopeptide repeats in the C-terminal region. Co-immunoprecipitation experiments with protein lysates from transfected cells, or yeast-two hybrid assays indicate that Duf/Kirre and Rols interact physically (Chen and Olson 2001, Kreisköther et al. 2006). Besides its role in myoblast fusion, Rols7 is a component of the Z-discs and terminal Z-discs (Kreisköther et al. 2006). Because of multiple functions interactions of Rols shown by in vitro methods need to be functionally correlated to the specific process. Also the Arf-GEF (Guanine nucleotide exchange factor) Schizo/Loner (Chen et al. 2003, Önel et al. 2004) expressed in FCs is recruited to the membrane depending on Duf/Kirre or Rst/IrreC, at least in cell culture assays (Chen et al. 2003). The GTPase Arf6 is thought to be the target for the GEF Schizo/Loner (Chen et al. 2003). Arf 6 presumably acts in redundancy to another Arf-GTPase, since lossof-function Arf6 mutants only show a cytokinesis defect in male meiosis (Dyer et al. 2007). In contrast, Richardson et al. (2007) detected Schizo/Loner also in FCMs, where it is localised outside of FuRMAS. Thus Schizo/Loner’s role in myoblast fusion requires further clarification (see Rochlin et al. 2010 for detailed discussion).

6.3.3 Signalling on the FCM Side Sns is the major cell adhesion molecule of FCMs and has been studied extensively. In S2 cells, the extracellular domain of Sns mediates heterotypic cell adhesion with Duf/Kirre or Rst/IrreC expressing cells (Galletta et al. 2004). The sns20–5 mutant isoform (formerly rost5 : Paululat et al. 1995) lacking the cytodomain of Sns allows cell adhesion t o take place, but fusion does not progress (Bour et al. 2000). This proves that the cytodomain of Sns – functionally partially redundant to Hbs (Shelton et al. 2009) – is essential for fusion to proceed. It further indicates that the cytodomain transfers the signal into the FCM after the extracellular domains of Sns and Duf/Kirre interact. The cytodomain of Sns is particularly well characterised and contains 14 putative sites for tyrosine phosphorylation. Mutagenesis of these sites and testing the rescue capabilities of such modified Sns molecules in a sns mutant background revealed considerable functional redundancies between these putative phosphorylation sites (Kocherlakota et al. 2008). There are several candidates for relaying the signal into the cell. rac1, rac2 double mutants are severely defective in myoblast fusion (Hakeda-Suzuki et al. 2002, Luo et al. 1994). An unusual GEF for Rac GTPases, Myoblast City (Mbc, also known as Dock180 in vertebrates), is required in FCMs; its function depends on its SH3 and Docker domains but is independent of its Crk-binding site. Therefore, it has been suggested that PXXP sites in the cytoplasmic domain of Sns interact with Mbc (Balagopalan et al. 2006). Rac activation is often indicative of actin polymerisation (see Section 6.4). Blown fuse (Blow) is a PH domain protein and essential for myoblast fusion (Doberstein et al. 1997). Blow is solely expressed in FCMs (Schröter et al. 2006) and is recruited to the area of F-actin foci/plugs within the

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FuRMAS (Kesper et al. 2007). It also interacts genetically with Kette/Nap1/Hem (Schröter et al. 2004), implicating that Blow plays a role in F-actin regulation (see Section 6.4 and Fig. 6.9).

6.4 Actin Regulation at the Site of Adhesion During Drosophila Myoblast Fusion At the light microscopic level, fusion is accompanied by the accumulation of transient F-actin plugs/foci that appearing at the contact sites of myoblast fusion within the adhesion belt/ring formed by adhesion molecules (Fig. 6.7) (Kesper et al. 2007, Richardson et al. 2007). Recent functional data have demonstrated that dynamic regulation of filamentous actin (F-actin) is critical for myoblast fusion (reviewed by Önel 2009, Önel and Renkawitz-Pohl 2009, Richardson et al. 2008b, Rochlin et al. 2010). In duf/kirre, rst/irreC double and sns single mutants, these actin foci/plugs are significantly reduced in size (Richardson et al. 2007), indicating that the adhesion receptors may cooperate with the cytoskeleton during membrane remodelling. Furthermore, the actin foci/plugs are highly dynamic. Live-imaging data revealed that they reached a maximum size of 4.5 μm2 in approximately 2 min (Richardson et al. 2008a), subsequently dissolving rapidly. In this section, we discuss recent molecular understanding of F-actin formation during myoblast fusion and provide possible insights into the role of F-actin forming at the site of fusion. We then compare F-actin formation in Drosophila and vertebrate myoblasts to explore possible conserved mechanisms.

Fig. 6.7 An actin focus forms in an adhering FCM attached to a growing myotube. (a) Drosophila stage 15 embryo expressing GFP-actin (green) under the control of the mesoderm-specific twist promoter. Double immunostaining with the anti-β3-Tubulin antibody (red) which marks all muscles and anti-GFP to visualize the actin foci (scale bar 10 μm). An actin focus (green) is visible at the contact site between an FCM and growing myotube (yellow arrow). (b) Schematic drawing and enlargement of the actin (green) plug/focus

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6.4.1 Molecular Mechanisms of F-Actin Regulation at the Site of Drosophila Myoblast Fusion The principles of F-actin polymerisation are well known from cell culture systems. However, until recently only little was known about the mechanism of F-actin regulation and its role at the site of fusion. In general, cells require actin nucleators to catalyse de-novo assembly of filaments. To date, three classes of nucleation factors have been identified, including the Arp2/3 complex, formins and the spire family. The evolutionary conserved Arp2/3 complex was one of the first nucleators identified and promotes formation of a new, branched actin filament on a mother filament at an angle of 70◦ (Pollard 2007, Welch and Mullins 2002). In order to perform its diverse cellular roles, the complex must first be activated by nucleation-promoting factors (NPFs), such as members of the WASP and Scar/Wave family (Millard et al. 2004). These proteins link signal transduction pathways and Arp2/3-based actin polymerisation (Stradal and Scita 2006). Recent genetic studies in Drosophila revealed that myoblast fusion depends on both Arp2/3 regulators WASP and Scar/Wave (Berger et al. 2008, Gildor et al. 2009, Massarwa et al. 2007, Richardson et al. 2007, Schäfer et al. 2007). In Drosophila, the mother supplies the egg with sufficient scar/wave and wasp mRNA to complete embryogenesis. For this reason, scar/wave and wasp zygotic mutants do not display a severe myoblast fusion defect. Defects are only apparent when both maternal and zygotic expression of scar/wave or wasp is removed (Massarwa et al. 2007, Richardson et al. 2007). Figure 6.8 illustrates the evolutionary conserved domain structure of WASP and Scar/Wave. Unlike mammals that contain two WASP and three Scar/Wave proteins, Drosophila possesses only one WASP and one Scar/Wave protein. Both proteins bind via their functional VCA domain to the Arp2/3 complex to trigger

Fig. 6.8 Schematic representation of Drosophila WASP and Scar proteins. WASP and Scar/Wave share similar regulatory and functional domains. Drosophila WASP possesses an N-terminal WASP homology domain (WH1) that can bind the WASP-interacting partner Wip (known as Vrp1 or Sltr in Drosophila); a regulatory region that contains binding sites for PIP2 (B-domain) and a GTPase-binding domain (GBD) as well as a proline-rich region (PPP) that can bind SH2-SH3 adaptor proteins. Drosophila Scar also has a B- and PPP-region. Both proteins further possess a common functional VCA domain consisting of one to two Verprolin (V) and a Cofilin (C) homologous domain as well as an acidic tail (A)

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a conformational change leading to Arp2/3 complex activation. Two different mechanisms are known to regulate the activity of NPFs themselves. WASP proteins are normally folded in an auto-inhibited conformation, which masks the VCA domain. Proteins that bind to the regulatory region of the WASP protein are able to activate WASP (Fig. 6.8). Although unmasking mammalian WASP depends on the synergistic binding of phosphatidylinositol 4,5-bisphosphat (PIP2 ) and GTP-bound Cdc42, neither the PIP2 and Cdc42 binding domains (B and GDB) of WASP, nor Cdc42 itself seem to be important during Drosophila myoblast fusion (Massarwa et al. 2007, Schäfer et al. 2007). However, removing conserved A or CA domains in WASP cause severe defects in myoblast fusion (Schäfer et al. 2007). Another protein binding to the N-terminal WH1 domain of WASP (Fig. 6.8) is the WASP-interacting partner Wip, which is also known as verprolin 1 or solitary (vrp1/sltr) in Drosophila. The loss of Vrp1/Sltr/Wip function in Drosophila disrupts myoblast fusion (Berger et al. 2008, Kim et al. 2007, Massarwa et al. 2007). Furthermore, Vrp1/Sltr/Wip is exclusively expressed in FCMs at the beginning of myoblast fusion, localising at the tip of the FCM attached to the FCs/growing myotubes (Berger et al. 2008). Upon fusion Vrp1/Sltr/Wip has also been observed in growing myotubes (Massarwa et al. 2007). Unlike WASP, a protein complex keeps mammalian Scar/Wave proteins in an inactivated state. Members of this complex are Sra-1/PIR121, Nap1, Abi and HSPC300 (reviewed by Takenawa and Suetsugu 2007). The first clue that members of the Scar/Wave complex are crucial for the fusion process in Drosophila came from mutant analyses of the Drosophila Nap1 homologue Kette. kette mutant embryos show clear defects in myoblast fusion and interact genetically with the FCM-specifically expressed gene blow (Schröter et al. 2006). How the Scar/Wave complex becomes activated is not yet clear and may differ between mammalian Wave and Wave2 [reviewed by Pollard 2007). One study found that GTP-bound Rac leads to dissociation of the Wave complex and thus activation of Wave (Eden et al. 2002). For Wave2, however, it was reported that Rac-GTP binds to the Wave complex without leading to dissociation of the complex (Innocenti et al. 2004). A third group reported that Rac-GTP stimulation must be enhanced by another protein (Suetsugu et al. 2006). In Drosophila two rac (rac1 and rac2) and a rac-related (mtl) genes are known. The rac1 and rac2 double mutants as well as rac1, rac2 and mtl triple mutants show severe defects in myoblast fusion, indicating that rac1 and rac2 share functional redundancy during fusion (Hakeda-Suzuki et al. 2002). Recent data from Gildor et al. (2009) suggest that Rac and Kette, and thus the Scar/Wave signalling pathway, not only play a role during myoblast fusion, but are also important for myoblast migration occurring prior to fusion. Localisation of Rac to the plasma membrane seems to depend on Drosophila Schizo/Loner and the Arf6-GTPase (Chen et al. 2003). Additionally, activation of Rac relies on Mbc (Côté and Vuori 2006). The loss of mbc in Drosophila causes a block in myoblast fusion (Erickson et al. 1997, Rushton et al. 1995). Mbc is a member of the CDM superfamily of GEFs, other members being the vertebrate protein Dock180 and the C. elegans protein CED-5 (Wu and Horvitz 1998). Dock180 is known to form a signalling complex with

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CrkII/CED-2 and ELMO/CED-12 (reviewed by Meller et al. 2005). Interestingly, Drosophila Elmo was identified as an interaction partner for Mbc in myoblasts (Geisbrecht et al. 2008). Moreover, the Drosophila SH2-SH3 adaptor protein Crk was shown to interact with the cell adhesion molecule Sns to activate the Wip-WASP complex (Kim et al. 2007). Some of the actin components described above apparently overlap with the F-actin plugs/foci observed at the site of fusion, e.g. Drosophila Titin, Blow, Mbc and Scar (Kesper et al. 2007, Richardson et al. 2007). Scar/Wave is virtually undetectable in kette mutant embryos, suggesting that Kette might be involved in the localisation and stabilisation of the Scar/Wave protein (Richardson et al. 2007). The same study reported that F-actin foci/plugs between adhering myoblasts in kette, scar/wavematernal/zygotic , blow, and mbc single mutants and rac1, rac2, mtl triple mutants are enlarged. This implies that the membrane breakdown of adhering myoblasts signals dissolution of the F-actin foci. In addition, experiments with double mutants shed light on the interplay between WASP and Scar/Wave in Arp2/3 activation during the fusion process (Berger et al. 2008, Schäfer et al. 2007). These experiments indicate that WASP and Scar play distinct roles in activating the Arp2/3 complex (Fig. 6.9). Phenotypic analyses of double mutants suggest that scar wip mutants stop fusing during the first phase, whereas scar wasp mutants stop fusing during the second phase of fusion. Thus, it is possible that Vrp1/Sltr/Wip may act independently of WASP together with Scar/Wave during the first fusion phase, while Scar/Wave, WASP and Vrp1/Sltr/Wip control the second fusion phase (Berger et al. 2008). In support of this notion, waspmaternal/zygotic mutant embryos were still able to form three-nucleated myotubes (Massarwa et al. 2007). Genetic epistasis experiments with kette and wasp further imply crosstalk between Scar-dependent and WASP-dependent Arp2/3 activation. Intriguingly, reducing the gene dosage of wasp in a kette mutant background suppressed the kette mutant phenotype (Schäfer et al. 2007). All actin components involved in Drosophila myoblast fusion and a model for Scar- and WASP-dependent regulation during the first and second phase of fusion are summarised in Fig. 6.9 and Table 6.1.

6.4.2 Possible Roles for Arp2/3-Based F-Actin Formation at the Site of Drosophila Myoblast Fusion Although many molecular components leading to Arp2/3-based actin polymerisation are apparently crucial for myoblast fusion, the intriguing question still remains as to the actual role of actin polymerisation in myoblast fusion. This has been investigated by transmission electron microscopy (TEM) studies on mutant embryos. Ultrastructural analyses of diverse fusion-defective mutants have shown that the characteristic ultrastructural features (Section 6.2; Fig. 6.3) are crucial for Drosophila myoblast fusion (examples in Fig. 6.10). Doberstein et al. (1997) observed that the genetic interaction partner of kette (Section 6.3.3), blow, ceases fusion after the formation of electron-dense vesicles (Fig. 6.11). In contrast,

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Fig. 6.9 The Arp2/3 complex is regulated differently during the first and second phase of fusion. Single and double mutant analyses suggest that the Arp2/3 complex is regulated differently during the two fusion phases. The cell adhesion molecules Duf/Kirre and Sns are required for myoblast recognition and adhesion during both fusion phases and seem to trigger a signalling cascade leading to the formation of actin foci at the site of fusion. (a) In Berger et al. (2008) we proposed that during the first fusion phase the Scar/Wave pathway induces formation of F-actin at the site of fusion. Components of this pathway known to be essential for the fusion process are the small GTPase Rac and its activator Mbc as well as the Nap-1 homologue Kette. kette is further known to interact genetically with the FCM-specifically expressed gene blow. Localisation of Rac seems to depend on Sec7-GEF Schizo/Loner by activating Arf6-GTPase. (b) During the second phase the WASP signalling pathway is also important for fusion. In the growing myotube the multidomain protein Rols/Ants interacts directly with the intracellular domain of the cell adhesion molecule Duf. Biochemical assays suggest that Sns transfers the fusion signal via the SH2-SH3 adaptor protein Crk. In turn, Crk activates the Vrp1/WIP-WASP complex. Prior to fusion Vrp1 is only present in FCMs. Whether it also plays a role in the growing myotube after fusion is not yet clear. Dosage experiments further indicate that Kette and WASP act antagonistically during fusion as indicated by black line. Thus, there might be crosstalk between the Scar and WASP signalling pathways

kette mutant embryos arrest fusion after the formation of electron-dense plaques (Fig. 6.11) (Gildor et al. 2009, Schröter et al. 2004). Doberstein et al. (1997) further observed that embryos expressing activated Rac1G12V in myoblasts sometimes form small fusion pores, leading to mixing of cytoplasmic content between fusing myoblasts. Taken together, these findings along with new EM data on kette wasp double mutant embryos from Gildor et al. (2009) may indicate that the Scar-dependent signalling pathway is required to form small fusion pores during myoblast fusion. As kette wasp double mutants, kette single mutants show no membrane breakdown (Gildor et al. 2009, Schröter et al. 2004). Phenotypic analyses of wasp and vrp1/sltr/wip mutant embryos have produced two different results. Kim et al. (2007) observed accumulation of electron-dense vesicles in vrp1/sltr/wip mutant embryos using the sltrS1946 allele. This allele, a

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Fig. 6.10 wasp and vrp1 mutant myoblasts stop fusion during membrane breakdown whereas Arp3 mutant myoblasts fail to integrate the FCM into the growing myotube. Conventional electron microscopy of Drosophila stage 14 embryos. (a) Fusing wild-type myoblasts. The membranes of the myoblasts have fused (arrows) and membrane remnants are visible in between, numerous cytoplasmic continuities (arrow heads) have been formed and content mixing of both myoblast types has occurred. (b and c) wasp3D3-035 and wipD30 mutant myoblasts. The membranes of the myoblasts have fused at the edge of the contact sites (arrows) and the membranes in between are in the process of breaking down. (d) Arp3Schwächling mutant myoblasts show a fully open but small cytoplasmic continuity (arrows) with no visible membrane remnants

1.2 kb deletion of genomic DNA spanning three exons, produces a truncated version of the Vrp1/Sltr/Wip protein. In contrast, Massarwa et al. (2007) reported that the vrp1/sltr/wip loss-of-function allele wipD30 arrests fusion after forming multiple small fusion pores (compare Fig. 6.11). A GFP-leakage assay where GFP was expressed specifically in FCs/growing myotubes supports the idea that cytoplasmic mixing occurs in wipD30 mutant myoblasts, since GFP can only leak into attached FCMs. The existence of small fusion pores in wipD30 mutant myoblasts was confirmed by Berger et al. (2008). Similarly to wipD30 , wasp mutant myoblasts also arrest fusion after forming multiple fusion pores (Fig. 6.10b, c) (Berger et al. 2008, Massarwa et al. 2007). Based on these findings it was proposed that WASPdependent actin polymerisation coordinates the clearance of membrane remnants between fusing myoblasts (see Fig. 6.11). Interestingly, no membrane remains are seen between fused myoblasts carrying the mutant allele of the Arp2/3 subunit Arp3

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Fig. 6.11 Myoblast fusion mutants arrest fusion in different ways. (a) In sing23 mutants, prefusion complexes (grey vesicles and red arrow in a) accumulate (Estrada et al. 2007), in blow2 mutants the pre-fusion complex forms and dissolves, then fusion seems to arrest (Doberstein et al. 1997). (b) In kette mutants, the electron dense plaques are observed (c) Mutation of vrp1/wip and Rac1G12V expression lead to arrest of membrane fusion when membranes vesiculate (red arrow). (d) In Arp3schwächling mutants, membranes dissolve (red arrow), but the FCM is not integrated into growing myotubes. Drawings according to representative ultrastructural analysis (see Fig. 6.10)

(Arp3Schwächling ) (Fig. 6.10d) (Berger et al. 2008). Since Arp3schwächling mutants show cytoplasmic continuity (removal of membrane remains) between FCMs and growing myotubes, but fail to incorporate the FCM content into the growing syncytium, this may indicate that Arp2/3-based actin formation is additionally required to integrate the FCM into the growing myotube (Berger et al. 2008).

6.4.3 Actin Regulation During Vertebrate Myoblast Fusion Vertebrate skeletal muscles form like Drosophila muscles via two phases of myoblast fusion (Horsley and Pavlath 2004). In the first phase, de-novo fusion of myoblasts generates nascent myotubes that serve as scaffolds in the second

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phase, when additional mononucleated myoblasts fuse with these myotubes until a syncytial myofibre with several nuclei is formed. Also on the cellular level, Drosophila and vertebrate muscle formation seem to be identical, in that myoblasts must recognise each other and adhere, finally leading to the breakdown of the membrane. Furthermore, the same ultrastructural features that occur during Drosophila myoblast fusion have been identified during skeletal muscle formation, namely electron-dense vesicles and electron-dense plaques (Doberstein et al. 1997, Knudsen and Horwitz 1977, Rash and Fambrough 1973). However, for a long time it seemed that skeletal muscle formation partially differs at a molecular level from Drosophila myoblast fusion. Although the cell adhesion molecules Duf/Kirre and Sns share structural similarities with Neph1 and Nephrin, Nephrin proteins were simply associated with maintaining the kidney filtration barrier (Tryggvason 1999). Only recently, the expression of Nephrin proteins and their function during fusion was also reported in vertebrate skeletal muscles (see Table 6.1) (Sohn et al. 2009, Srinivas et al. 2007). Moreover, mixed cell culture experiments suggest that Nephrins must be present in mononucleated myoblasts, but not in nascent myotubes, for secondary myotube formation (Sohn et al. 2009). This suggests that Nephrin may play a role in vertebrate myoblasts similar to Sns in FCMs. Since Arp2/3-based actin polymerisation appears essential for Drosophila myoblast fusion, this has raised the question as to whether components of the Arp2/3 machinery are also crucial for skeletal muscle formation. The first evidence that some of the molecular components are also conserved in vertebrates came from studies using zebrafish as a model organism (Table 6.1). The Rac-GTPase and its activator Mbc (Dock1 and Dock5 in zebrafish), as well as the SH2-SH3 adaptor proteins Crk and Crk-like (Crkl) play a role in forming multinucleated fasttwitch muscles in zebrafish (Moore et al. 2007, Srinivas et al. 2007). Knockdown of Dock1, Dock5, Crk and Crkl significantly compromises myoblast fusion (Moore et al. 2007). Interestingly, it was further reported that simultaneous knockdown of Crkl and Dock5 blocks fusion almost completely in zebrafish. Both human Crkl and Drosophila Crk interact with the WASP-interacting partner Wip (Kim et al. 2007, Sasahara et al. 2002), which is known to be essential for Drosophila myoblast fusion. Additionally, there is evidence that Drosophila Crk interacts with the cell adhesion molecule Sns (Kim et al. 2007). Whether zebrafish Crkl can also interact with zebrafish Nephrin and Wip still needs to be elucidated. However, there also seem to be functional differences between some of these genes during Drosophila and zebrafish myoblast fusion. For example, expression of activated Rac and overexpression of Crk and Crkl in zebrafish lead to hyperfusion in multinucleated fast-twitch muscles (Moore et al. 2007, Srinivas et al. 2007). In contrast, expression of activated Rac leads to a block in myoblast fusion Drosophila (Fig. 6.11c) (Doberstein et al. 1997, Luo et al. 1994) and no hyperfusion was reported for Crk expression in myoblasts. Up to now the conserved function of most actin components has mainly been tested in mouse C2C12 myoblasts (Table 6.1). RNAi knockdown of N-WASP and Wip reduces myoblast fusion significantly in murine C2C12 cells (Kim et al. 2007).

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Furthermore, it was demonstrated that targeted knockdown of Rac effector proteins such as MBC, Schizo and Arf6 lead to fusion defects in C2C12 cells (Pajcini et al. 2008). A recent study by Nowak et al. (2009) examined the relevance of the Drosophila Kette homologue Nap1 as well as Wave1 and Wave2 for the fusion of C2C12 cells using short hairpin RNA. Large numbers of Nap1- and Wave2knockdown myoblasts remained unfused. In contrast, Wave1 did not seem to be crucial for C2C12 fusion. In the same study, accumulation of F-actin rich aggregates was observed in adhering Nap1-knockdown myoblasts. Moreover, these aggregates also contained the Mbc homologue Dock 180. Whether these aggregates correspond to enlarged F-actin foci/plugs identified in Drosophila actin mutants such as kette mutant embryos needs further investigation. At an ultrastructural level, Duan and Gallagher (2009) reported that F-actin walls form between fusing C2C12 myoblasts in culture, suggesting that this supports membrane apposition, appearance of fusion pores and regulates vesicle trafficking. Functional analysis of actin components in a developing mammalian embryo is difficult to assess since loss of most components leads to early embryonic lethality. Dock1 or Dock5 are the orthologues of Drosophila Rac1-GEF Mbc. Impaired myoblast fusion was observed for Dock1 loss-of-function mice embryos, but not for Dock5-null-mutant embryos (Laurin et al. 2008). Although homozygous Dock5mutant animals were viable and had no obvious morphological abnormalities, genetic analysis revealed functional redundancy between Dock1 and Dock5 in myoblast fusion. Another elegant study used conditional mutagenesis to demonstrate a role for Rac1 and Cdc42 in mice myogenesis (Vasyutina et al. 2009). The authors used Cre-recombinase under the control of Lbx1, which induces the recombination of floxed Rac1 and Cdc42 in myogenic precursor cells that migrate to targets like the limb, diaphragm and tongue. The fusion index in Rac1 and Cdc42 mutant mice was reduced and most myoblasts remained unfused. It is notable that in contrast to Drosophila, mutations of Rac1 in mice are sufficient to interfere with fusion (see Table 6.1). Primary cell culture experiments further revealed that although myoblasts do adhere, F-actin recruitment is reduced in Rac1 or Cdc42 mutant myoblasts. Furthermore, recruitment of Arp2/3 was different in Rac1 and Cdc42 mutant myoblasts. Arp 2/3 recruitment is remarkably reduced in rac1 mutant cells, whereas Arp 2/3 localisation to the contact sites is unaffected in Cdc42 mutant cells, indicating that Rac1 and Cdc42 may function in non-equivalent manners. In Drosophila, however, Cdc42 appears not to be important (Schäfer et al. 2007).

6.5 The FuRMAS Model and the Topology of Myoblast Fusion Live imaging of myoblasts expressing actin-GFP showed that an individual fusion event takes a matter of minutes (Richardson et al. 2008a). This means membraneassociated events such as cell adhesion, signalling, forming and dissolving the actin plugs/foci and generating the pre-fusion complex are highly dynamic. How are membrane dynamics, the ultrastructural features and cytoskeletal dynamics

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integrated? There is evidence that F-actin regulation within the FuRMAS differs between the first and second phase of fusion (see Section 6.4 and Fig. 6.9). From a morphological point of view the first and second phase of myoblast fusion are certainly different (Fig. 6.9). During the first phase, the area of contact between FC and FCM spans nearly the width of the myoblasts (1.7–01.9 μm) (Schröter et al. 2006). Also during the second phase of fusion, the FCM is as small as in the first phase, but the myotube increases in size with every successful fusion event. This likely requires a mechanism to restrict the area of membrane breakdown in the second fusion phase, and besides signalling, might imply an additional role of Rols (Rau et al. 2001). Furthermore, FCMs attach laterally to the growing myotubes, which then migrate towards their epidermal attachment sites by extending filopodia from the distal and proximal end (Bate 1990), a process that also depends on the cytoskeleton (recently reviewed by Guerin and Kramer 2009). During the second phase of fusion, the ultrastructure data show membrane vesiculation over a large area, for example 3.6 μm2 in diameter as shown for an individual fusion site in Fig. 6.5a.

6.5.1 Fusion Pores, Membrane Vesiculation and the Size of Cytoplasmic Continuities Fusion of membranes has been particularly well studied for the entry of viruses into cells and for synaptic vesicles at neuromusculature junctions (reviewed by Chernomordik and Kozlov 2008, Martens and McMahon 2008). In these cases, the initial fusion pore is predicted to have a radius of 3 nm (Jackson 2009). In contrast, the cytoplasmic continuity (diameter of up to 4 μm) during myoblast fusion stretches over a far larger area of 12 μm2 , which is about 1,000-fold larger than in vesicle fusion. How is this cytoplasmic continuity achieved in such a short time, does fusion pore expansion progress like opening a zipper, or do multiple fusion events join to form wider cytoplasmic continuity? Doberstein et al. (1997) proposed that the vesicles of the pre-fusion complex are exocytosed and might carry a fusogen. At the ultrastuctural level membrane vesiculation was observed after this (see Section 6.2.2), leading to several small cytoplasmic bridges and mixing of the cytoplasm as shown by a diffusion assay (Gildor et al. 2009). The molecules that regulate exocytosis or induce membrane vesiculation are so far unknown. However, loss-offunction mutants existing for a number of fusion-relevant genes provided a unique opportunity to correlate the protein of interest with interruption of myoblast fusion at the ultrastrucural level. Two mutants (Fig. 6.10), blow2 and singles bar23 (sing23 ), do not allow fusion to progress towards membrane vesiculation (Doberstein et al. 1997, Estrada et al. 2007). In contrast to FCM-specific expression of blow (Schröter et al. 2004), sing is expressed in FCs/growing myotubes and FCMs (Estrada et al. 2007). blow2 loss-of-function mutants do form, but do not accumulate pre-fusion complexes, while sing23 mutants (stop codon in C-terminal part of the protein) do accumulate pre-fusion complexes. The failure to go beyond pre-fusion complexes, and the fact that wild-type Sing protein contains a MARVEL domain, a conserved

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domain involved in membrane apposition (Sánchez-Pulido et al. 2002), suggest that Sing allows progression past the pre-fusion complex, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane (Estrada et al. 2007).

6.5.2 FuRMAS as Signalling Centres So far we have discussed that myoblast fusion requires highly coordinated mechanisms for cell recognition, adhesion, signalling, local F-actin regulation, exocytosis, membrane merging, removal of membrane remnants and finally integration of the FCM into the growing myotube. But what is the topology of myoblast fusion at the individual site of fusion? We suggested that cell adhesion leads to establishing a FuRMAS as a signalling centre and thereby triggering myoblast fusion (Kesper et al. 2007, Önel and, Renkawitz-Pohl 2009). This structure is transient and hypothetically regulates spaciotemporal communication between FCs/growing myotubes and FCMs during fusion, i.e. events such as formation and dissolution of F-actin plugs/foci, precise apposition of vesicles of the pre-fusion complex, and finally membrane vesiculation. In Drosophila, fusion of electron-dense vesicles to the target membranes is proposed to occur before membrane merging between FCs/growing muscles and FCMs (Doberstein et al. 1997). This might require two types of fusion: first, vesicles fuse with their target membrane and second, membranes merge between opposing myoblasts. This means membranes have to be brought into tight contact. Bilayers can apparently fuse when they are in close proximity of 1 nm (Kozlovsky et al. 2004). At the pre-fusion complex stage of myoblast fusion membranes are in close apposition over a large area, 1 μm2 (see Fig. 6.4). How is this achieved? At the light microscopic level, the cell adhesion molecules form a belt/ring at the site of fusion (Fig. 6.11, Sns = yellow, Duf = blue). In a lateral view, F-actin is at the centre of the FuRMAS surrounded by a ring of Duf and SNS (Fig. 6.9). Figure 6.12 depicts our FuRMAS model for the topology of myoblast fusion with respect to the described ultrastructural features, and the role of F-actin is discussed within this context. Immuno-EM on wild-type myoblasts implicate that the electron-dense vesicles observed at the plasma membrane of adhering myoblasts emanate from the Golgi and are targeted by actin-directed migration to the plasma membrane to form pre-fusion complexes (Kim et al. 2007) (Fig. 6.12a). Based on the serial section (Fig. 6.4) of Doberstein et al. (1997) and the ring-shaped distribution of Sns and Duf/Kirre (Fig. 6.6), Kesper et al. (2007) proposed that the pre-fusion complex with its clouds of electron-dense vesicles accumulates at the opposing membranes between these cell adhesion belts (Fig. 6.12b). As mentioned above, vesicles accumulate over an area of 1 μm2 membrane vesiculation, however, is observed over an area (12 μm2 ), with a diameter of 4 μm (Fig. 6.5). At the light microscopic level, this is reflected by the expansion of the ring of adhesion molecules from 1 to 4–5 μm in diameter (Kesper et al. 2007). This suggests that the area widens to create this size of cytoplasmatic continuity, which allows integration of the FCM into the growing myotube (Fig. 6.12c). This widening and integration of the FCM

164 Fig. 6.12 The FuRMAS model of myoblast fusion. Model diagrams of the topology at the contact site between a growing myotube (blue nuclei) and an FCM (yellow nucleus). Adhesion rings/belts are formed by Duf (blue) on the growing muscle side and Sns (yellow) on the FCM side. (a) Vesicles are transported from the Golgi (G) to the opposing membranes within the FuRMAS. (b) The pre-fusion complex forms over an area of 1 μm2 . (c) Cytoplasmatic continuity is achieved over an area of 12 μm2 . Features are schematic and not drawn to scale. Plaques observed in the EM are not integrated in this scheme (see Section 6.2.2) since the topological arrangement is unclear. F-actin and signalling molecules are discussed in Fig. 6.9.

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depend on the activation of the Arp2/3 complex (Berger et al. 2008). The Arp2/3 complex, however, is neither essential for vesicle exocytosis nor for membrane merging. The putative fusogens which mediate vesicle exocytosis and merging of the plasma membranes from FCs/growing myotubes and FCMs are unknown. The identification of such a fusogen is one of the important questions to address to in the future. It is of high interest that FuRMAS share molecular and anatomical features such as belts/rings of adhesion molecules, local actin polymerisation, and signalling centre properties with other transient cell–cell adhesions such as the immunological synapse. This synapse connects the antigen-presenting cell with a T-cell (Grakoui et al. 1999, Monks et al. 1997, Shaw and Dustin 1997). Furthermore, there are similarities to cell matrix interactions such as podosomes and invadopodia (reviewed by Linder (2007), in particular many local F-actin regulators are in common (for a detailed comparison see Önel and Renkawitz-Pohl 2009).

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6.6 Outlook Myoblast fusion is a fundamental process for myogenesis in higher organisms. Cell–cell fusion, however, also occurs in several other vital processes, such as zygote formation, yeast mating (reviewed by Ydenberg and Rose 2009) hypodermis and ulca (lid of the vulva) formation in nematodes, oesteoclast development in vertebrates and placenta formation in mammals (reviewed by Oren-Suissa and Podbilewicz 2007). Hypodermis and ulca fusion starts within an epithelium and here fusogens EFF-1 and Aff1, which are both necessary and sufficient for cell– cell fusion, have been identified and functionally characterized (reviewed by Sapir et al. 2008) Besides Syncytins (section X and Y and Larsson et al. 2008) in mammals, fusogens for cell–cell fusion do not seem to be conserved in sequence. The action of fusogens is one of the last events in membrane merging after successful cell–cell recognition and adhesion, apparently signalling into the cell that the actin cytoskeleton can be modified and fusion can commence. Since 2007, several conserved molecular components of these fusion-preparing processes have been found to be conserved between Drosophila and mammalian myoblast fusion (summarised in Table 6.1 and discussed in detail in Section 6.4.3). During myoblast fusion in Drosophila, two types of fusion are required: first the electron-dense vesicles must fuse with the opposing membranes, which should be followed by fusion of the opposing membranes of FCs/growing muscles and individual FCMs. We have an advanced understanding of cell recognition, cell adhesion and F-actin regulation during fusion, but the most central and challenging question of how membranes merge and build a syncytial cell is unresolved. After birth in vertebrates, it is essential that muscles increase the number of nuclei during postnatal growth and that muscles can be repaired after injuries by myoblast fusion events. These myoblasts originate from the activation of satellite cells, the stem cells of skeletal muscles (for a recent review see Rudnicki et al. 2009). The emerging common components in Drosophila and mammalian myoblast fusion prove that ground level research in Drosophila advances investigations in mammals, which in turn opens the way to translational health-relevant research. Acknowledgments We are grateful to Katja Gessner for preparing most of the drawings and for excellent secretarial assistance, Christina Hornbruch for critical reading. This work was supported by the Deutsche Forschungsgemeinschaft with grants to S.Ö. (OE 311/4-1) and R. R.-P. (Re628/14-3, R2628/15-2) and GRK1216 as well as the EU network of Excellence MYORES.

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Chapter 7

Role of CD9 in Sperm-Egg Fusion and Its General Role in Fusion Phenomena Natsuko Kawano, Yuichiro Harada, Keiichi Yoshida, Mami Miyado, and Kenji Miyado

Abstract In fertilization, two types of sex cells or gametes – a sperm and an egg – unite in a stepwise manner to form a mother cell, which is capable of developing naturally into a new individual. Notably, the “membrane fusion” that occurs intercellularly between a sperm and an egg is essential for fertilization. A sperm factor that is delivered into the egg cytoplasm through fusion serves to activate a signaling pathway; this leads to the resumption of meiosis in the egg. In mammals, sperm-egg fusion is partly mediated by two integral membrane proteins, sperm Izumo (Inoue et al. 2005) and egg cluster of differentiation 9 (CD9) (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000), and the roles played by both are critical but yet unknown. A recent study (Miyado et al. 2000) showed that CD9containing vesicles are released from wild-type eggs, and that these exosome-like vesicles induce fusion between sperm and CD9-null eggs in vitro, even though CD9null eggs are highly refractory to sperm-egg fusion. This result provides compelling evidence for the crucial involvement of CD9-containing, fusion-facilitating vesicles in sperm-egg fusion and offers new insight into both gamete fusion and other membrane fusion events. Keywords CD9 · CD81 · cell-cell fusion · egg · exosomes · fertilization · Izumo · membrane fusion · sperm · tetraspanins Abbreviations ADAM CD GM3 EGFP HIV HSP

A disintegrin and metalloprotease Cluster of differentiation Monosialo ganglioside 3 Enhanced green fluorescent protein Human immunodeficiency virus Heat shock protein

K. Miyado (B) Department of Reproductive Biology, National Center for Child Health and Development, Tokyo 157-8535, Japan e-mail: [email protected]


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Intracytoplasmic sperm injection Large extracellular loop Motility-related protein 1 Microvilli Perivitelline space Tetraspanin Zona pellucida

Contents 7.1 Introduction . . . . . . . . . . . . . 7.2 Sperm-Egg Fusion in Fertilization . . . 7.3 CD9 and Its Role in Cell Function . . . 7.4 Tetraspanin . . . . . . . . . . . . . 7.5 Tetraspanin as a Component of Exosomes 7.6 Lessons from “Living Eggs” . . . . . . 7.7 Membrane Fusion and Exosomes . . . References . . . . . . . . . . . . . . . .

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7.1 Introduction Membrane fusion is defined as the merger of two neighboring membranes, which occurs intracellularly in the cytoplasm or intercellularly on cell–cell membranes (Jahn et al. 2003). Especially, intercellular membrane fusion (hereafter, simply referred to as “membrane fusion”) is very exact in relation to the formation of certain kinds of cells and tissues associated with dynamic turning events in development, e.g., myoblast fusion in muscle formation and sperm-egg fusion in fertilization. These fused cells play a central role in the formation of tissues and organs in newborns. Hence, the absence of components necessary for fusion events results in the failure of cell formation, which causes severe muscular atrophy or an extremely decreased rate of fertilization. Moreover, membrane fusion occurs on invasion of viruses into cells. For viruses to inject their genetic materials into host cells, their envelopes need to fuse with the outer membranes of the host cells (Hernandez et al. 1996). Further, when recipient cells are infected with certain types of viruses, such as human immunodeficiency virus-1 (HIV-1) and paramyxovirus, expression of the virus fusion protein at the cell–cell contact sites can induce the fusion of neighboring cells, leading to the formation of multi-nucleate immune cells (or syncytia). Such formation of abnormally fused cells severely impairs normal cell function in terms of antibody production and antigen recognition, and finally disrupts the immune system. Therefore, membrane fusion processes are critical for both the occurrence of biologically essential events and pathologically significant diseases. Thus, understanding how outer membranes meet and merge in cell–cell fusion and virus-cell fusion is a major challenge in the creation of medicines used to treat

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muscular dystrophy, infectious incurable diseases, or infertility in humans and certain endangered species, as well as in basic research for cell biology. Here, we focus on cell–cell fusion and discuss its molecular mechanisms in relation to the role of cluster of differentiation 9 (CD9) and other tetraspanins.

7.2 Sperm-Egg Fusion in Fertilization Fertilization is a complicated event that consists of cell–cell adhesion, activation of cell signaling to allow the resumption of the cell cycle, and cell–cell fusion (Fig. 7.1). In organisms, including animals, fungi, and plants, fertilization occurs when two types of sex cells come into contact and naturally form one unified cell, termed a fertilized cell. This fertilized cell subdivides into a number of cells and eventually becomes the origin of all the tissues and organs necessary for the formation of a new individual. In addition, the eggs have a unique ability to reprogramme DNA methylation patterns in both sperm genomes and their own genomes. Fertilization is a key event that is directly linked to the generation of a new individual. In fertilization, two membrane-spanning proteins necessary for fusion are expressed in the egg and sperm – a cell adhesion molecule “integrin” (Almeida et al. 1995, Chen et al. 1999) and a membrane-anchored protease “ADAM (a disintegrin and metalloprotease)” (Evans 2001) – and have been biochemically identified and

Fig. 7.1 Series of steps from sperm–egg interaction to fusion during mammalian fertilization. This is an overview of mammalian fertilization. Fertilization is divided into multiple steps: interaction of sperm-somatic cells (termed cumulus cells), binding of sperm to the extracellular matrix (termed zona pellucida), and penetration of the egg. After the sperm penetrates the zona pellucida, it can bind and fuse to the egg cell membrane. Successful fertilization requires not only that a sperm and egg fuse, but also that no more than one sperm fuses with the egg i.e., a polyspermy block occurs

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immunocytochemically confirmed to localize on the outer cell membranes of each gamete. Further, antibodies against these proteins have been shown to significantly reduce the rate of sperm-egg binding and fusion in mice. Integrins were initially identified as regulators of muscle cell fusion (Rosen et al. 1992) and have also been well-identified as cell adhesion molecules along with other membrane proteins such as cadherins. Integrins, which are expressed in many types of cells and are conserved in all animals examined, mediate cell–cell and cell–matrix interaction and intercellular communication, including cell adhesion and cell–cell fusion. On the other hand, ADAMs have a characteristic domain that is homologous to an extracellular region of integrins beside a protease-like sequence (Blobel et al. 1992). Thus, the presence of domains conserved between integrins and ADAMs indicated that these two protein families play a role in sperm-egg adhesion and/or fusion (Evans 2001). However, when genetically manipulated mice were produced to facilitate the study of the relevant genes of integrins (He et al. 2003, Miller et al. 2000) and ADAMs (Okabe and Cummins 2007), both male and female mice displayed no overt anomalies in both the sperm-egg fusion and adhesion. Thereafter, other factors participating in sperm-egg fusion have emerged, and contrary to expectations, most were found to be unnecessary, like integrins and ADAMs. From these studies, it is thought that in order to ensure the continuous success of the reproduction cycle in organisms, compatible pathways tuned by overlapping functions of multiple proteins should regulate the mechanism of fertilization. There will be more than one way for a sperm and egg to fuse and the network of multiple pathways may minimize the severity of the malfunction that can occur in sperm or eggs lacking a

Fig. 7.2 Players identified in sperm-egg fusion. Izumo has been found on the sperm membrane, and Izumo-null sperm show a defect in fusion with the egg cell membrane (Inoue et al. 2005). CD9 is expressed on the egg cell membrane and functions in fusion with the sperm (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000). Two membrane proteins, Izumo and CD9, are essential for sperm-egg fusion in mammals. Direct interaction between CD9 and Izumo has not been identified, and the other sperm and egg factors involved in sperm-egg fusion remain unclear. After spermegg membrane fusion occurs, a sperm-specific phospholipase C, termed PLCzeta, triggers Ca2+ oscillations and initiates egg activation in mammals (Saunders et al. 2002)

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single gene. Genetic analyses have confirmed that unlike other factors, CD9 (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000) and Izumo (Inoue et al. 2005) are not exchangeable and that they are considered to be hub proteins in sperm-egg fusion (Fig. 7.2).

7.3 CD9 and Its Role in Cell Function CD9 (also known as DRAP27, MRP-1, MIC3, and Tspan-29) was initially identified as a cell surface protein on platelets and immature B cells, and has now been widely identified in immune cells, growing oocytes, and eggs (full-grown, ovulated oocytes) (Hemler 2008). The CD9 gene encoding a 24-kDa protein is transcribed in all types of cells. The CD9 protein is primarily localized on cell membranes and partially on endosomes, and it is expected to be involved in cell–cell interaction because it has also been identified as an integrin-associated protein. CD9 has also been identified as a motility-related protein 1 (MRP-1), which plays a functional role as a tumor metastasis suppressor (Miyake et al. 1991). CD9 has some structural features: two extracellular loops, four transmembrane domains, and two short cytoplasmic domains; its functional domain is expected to be a large extracellular loop (LEL) (Fig. 7.3). CD9 associates with other membrane proteins via LEL in vitro. In humans and mice, commercially available antibodies recognize the structure of the regions linked by cysteine–cysteine interaction within LEL in a species-specific manner. As mentioned above, CD9 transcripts are expressed in all the cells and tissues examined, whereas its protein is specifically translated in some

Fig. 7.3 Structural features of tetraspanin CD9. CD9 is a member of the tetraspan-membrane protein family, termed tetraspanin, and its molecular mass is 24 kDa. The structural features of CD9 include four transmembrane domains, two extracellular loops, short and large extracellular loops (SEL and LEL), and two short cytoplasmic tails. CD9 has cysteine-cysteine-glycine (CCG) residues (amino acids 152–154) as a specific motif and two other cysteines within LEL. All tetraspanins share a CCG motif and several conserved cysteine residues in LEL

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categorized cells. Due to the significantly higher amounts of CD9 in mesenchymal and embryonic stem cells than in fibroblastic cells, CD9 is considered useful as a cell surface marker for the isolation of undifferentiated cells from cell pools containing fibroblastic cells in mice and humans (Akutsu et al. 2009). Despite two decades of effort, the role of CD9 in immune cells in vivo is not very well understood, although three laboratories independently generated CD9-null mice (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2008). Unexpectedly, the CD9-null female mice were nearly infertile. While sperm from the CD9-null males was fertile, the CD9-null eggs were unable to fuse with the sperm. CD9 has been studied as one of the crucial factors in sperm-egg fusion in vivo and in vitro. In sperm-egg fusion, a functionally essential domain of CD9 was thought to be located within the LEL and to mediate the association of CD9 with other tetraspanins and other membrane proteins (Kaji et al. 2002, Zhu et al. 2002). Even though candidate proteins have been identified in non-gamete cells (Andre et al. 2007, Charrin et al. 2003), proteins related to egg-fusing abilities have not yet been found. Thus, the functional activity of CD9 remains elusive in sperm-egg fusion.

7.4 Tetraspanin CD9 belongs to a membrane protein superfamily, collectively termed “tetraspanin,” which encompasses 35 members in mammals (including CD9, CD37, CD53, CD63, CD81, CD82, and CD151) (Hemler 2008, Le Naour et al. 2006), 30 in nematodes (Tsp15) (Moribe et al. 2004), and 30 in flies (latebloomer) (Kopczynski et al. 1996, Todres et al. 2000). CD151 forms a stable complex with integrin alpha3beta1 and regulates integrin-mediated cell-substrate adhesion, neurite growth, and cell morphology (Shigeta et al. 2003, Yamada et al. 2008). In mice, CD81 is linked to infection of hepatocytes with the malaria parasite. Malaria sporozoites, a cell form that infects new hosts, are transmitted into the liver of the mammalian host through bites from infected mosquitoes. Malaria sporozoites failed to infect CD81-null mouse hepatocytes (Silvie et al. 2003) in vivo and in vitro. These results demonstrated that CD81 is linked to sporozoite entry into hepatocytes as a host factor. Deficiency of CD81 genes also leads to a reduction in the fecundity of female mice: CD81-null eggs had impaired sperm fusion ability, while the fertility of CD9-null mice was severely impaired (Rubinstein et al. 2006, Tanigawa et al. 2008). CD81 is expressed on CD9-null eggs, whereas CD9 is expressed on CD81-null eggs at an expression rate comparable with that of wild-type eggs. From these reports, CD9 and CD81 are speculated to independently function in sperm-egg fusion. Plants have more than 60 tetraspanin members, as do animals (Chiu et al. 2007, Huang et al. 2005). Tetraspanins and tetraspanin-like members have also been identified in fungi (Lambou et al. 2008), and the molecular mass of tetraspanin-like proteins (more than 200 kDa) is greater than that of tetraspanin (20–30 kDa). Fungal plant pathogens differentiate into an infection structure that is specialized for host penetration – the appressorium. On the basis of analysis of a non-pathogenic mutant from the rice blast fungus Magnaporthe grisea, tetraspanin PLS1 (MgPLS1) has been shown to control an appressorrial function essential for the penetration of fungus into host

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leaves (Clergeot et al. 2001). Furthermore, Colletotrichum lindemuthianum PLS1 (ClPLS1), a functional homologue of MgPLS1, has been identified, and a ClPLS1null mutant has been shown to be non-pathogenic to bean leaves as a result of a defect in the formation and/or positioning of the penetration pore (Veneault-Fourrey et al. 2005). These studies suggest that fungal tetraspanins control a conserved appressorial function that determines the correct localization of the site where the penetration peg emerges. The invasion of pathogenic fungi into leaves is an event closely related to membrane fusion events. Tetraspanins are closely related to other cell–cell membrane fusion events, such as muscle (Tachibana et al. 1999) and bone formation (Takeda et al. 2003, 2008), and virus infection (Garcia et al. 2005). Especially, tetraspanins have been reported to regulate cell–cell transmission of HIV-1 (Wiley and Gummuluru 2006). The presence of tetraspanins, CD9, CD63, CD81, and CD82 at HIV-1 budding sites and their enrichment in HIV-1 virions have been demonstrated. The tetraspanin-containing exocytosed vesicle-associated HIV-1 particles from dendritic cells were 10-fold more infectious than cell-free virus particles (Fig. 7.4). However, despite two decades of effort, the physiological

Fig. 7.4 Exosome-mediated HIV-1 transinfection. Dendritic cell-mediated HIV-1 transfection can be mediated by exocytosis of the HIV-1 particles captured. After endocytosis, the captured HIV-1 particles are targeted to a multi-vesicular endosomal body (MVB) in dendritic cells. The virus particles acquire exosomal antigens including tetraspanins (black bars) through the MVB. Although some of the MVB-localized virus fraction is targeted to the lysosome and degraded, fusion of MVB with the plasma membrane results in the release of virus particles along with exosomes

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activities of tetraspanin are still elusive, and its fusogenic activity corresponding to fusogenic transmembrane proteins, such as syncytin identified in human placenta (Mi et al. 2000), and virus envelope proteins (Hernandez et al. 1996) remains unidentified.

7.5 Tetraspanin as a Component of Exosomes Tetraspanin has also been identified as a common component of membrane vesicles, termed exosomes (Simons and Raposo 2009), which are released outside immune cells, e.g., reticulocytes, B cells, and dendritic cells. Although cell-cultured media are know to contain nano-sized membrane fragments (Trams et al. 1981), exosomes cannot be structurally distinguished from the debris of dead cells (Couzin 2005). Recent studies of dendritic cells and B cells have shown that the exosomes are derived from living rather than dead cells; exosomes have been proven to play a significant role in the mediation of adaptive immune reactions to pathogens and tumors through the enhancement of antigen-specific T cell responses (Kesimer et al. 2009). Besides immune cells, the exosomes have reported to be released from a wide range of normal and malignant mammalian cell types, and their diameter is estimated to range from 50 to 90 nm. Exosomes can also contain both functional mRNA and microRNA, which can be shuttled from one cell to another, thereby influencing protein synthesis in recipient cells (Valadi et al. 2007). The protein composition of exosomes varies with the origin of cells, yet the exosomes contain a ganglioside GM3, two heat shock proteins (HSP70 and HSP90), and tetraspanins as common components. In eggs, two earlier observations suggest that CD9 plays a role in the organization of the egg cell membrane: (1) CD9 is transferred from the egg to the fertilizing sperm present in the perivitelline space, suggesting the involvement of a process similar to trogocytosis – a mechanism for the cell-to-cell contact-dependent transfer of membrane fragments from antigen-presenting cells to lymphocytes as demonstrated in immune responses for pathogens (Barraud-Lange et al. 2007); (2) CD9 deficiency alters the length and density of microvilli on the egg cell membrane (Runge et al. 2007). As mentioned above, tetraspanin is known to be a common component of exosomes, yet the involvement of exosome release in sperm-egg fusion was unknown until recently.

7.6 Lessons from “Living Eggs” Our recent study demonstrated the potential of enhanced green fluorescent proteintagged CD9 (CD9-EGFP) as a reporter protein for studying sperm-egg fusion in living mouse eggs (Miyado et al. 2008). Notably, in eggs just before fertilization, we showed that CD9-EGFP is significantly accumulated within a small interspace (perivitelline space) that completely surrounds the eggs and lies between the egg cell membrane and the extracellular matrix (zona pellucida). Consistent with the images from CD9-EGFP, immunoelectron microscopic analysis of wild-type eggs revealed

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Fig. 7.5 Electron microscopic images of CD9-containing egg exosomes. After ovulation, the wildtype eggs collected from the oviducts of C57BL/6 mice were treated with an anti-mouse CD9 antibody and 5 nm of gold particles conjugated with a secondary antibody. According to immunoelectron microscopic analysis, CD9-containing vesicular structures (asterisks), lipid bilayers that are formless and distinct from microvilli (M), are estimated to have diameters ranging from 50 to 200 nm, and are accumulated within the perivitelline space (PVS), an interspace between the zona pellucida (Z) and the cell membrane of an egg. (Modified from Miyado et al. 2008)

that CD9 is not only present in the perivitelline space but also incorporated into vesicles of varying size (50–200 nm in diameter) without a sectional profile of a typical lipid bilayer (Fig. 7.5). Furthermore, in opossums (Talbot and DiCarlantonio 1984) and humans (Dandekar et al. 1992), as well as in mice, membrane vesicles have been detected electron microscopically within the perivitelline space of their eggs. We also showed that the vesicles identified in mouse eggs share CD9, GM3, and HSP90 with exosomes; they are absent in eggs lacking CD9 and are reproduced by CD9-EGFP expression restricted to the eggs (Miyado et al. 2008). Taken together, our results suggest that (1) CD9-incroporated exosome-like vesicles are produced in mouse eggs and are released outside the egg cell membrane just before fertilization; (2) CD9 is essential for formation and/or release of the exosome-like vesicles in mouse eggs (Fig. 7.6).

7.7 Membrane Fusion and Exosomes We have also confirmed that CD9-containing exosome-like vesicles (hereafter referred to as egg exosomes) render sperms capable of fusing with CD9-null eggs (Fig. 7.7) (Miyado et al. 2008). This experiment demonstrates that co-incubation with wild-type eggs reverses a defect in the fusion of CD9-null eggs; this was replicated by two independent groups. CD9-null eggs do not generally fuse with eggs, but the co-existence of wild-type eggs results in 60–70% of the CD9-null eggs

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Fig. 7.6 Schematic diagram for the release of CD9-containing egg exosomes from mouse eggs. Previous studies (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000) demonstrated that eggs lacking tetraspanin CD9 display a defect when fusing with sperms. Moreover, a recent study (Miyado et al. 2008) showed that CD9-containing egg exosomes are released from mature oocytes (eggs), but not from immature oocytes, and share common components with canonical exosomes, identified as intercellular transporters of proteins and RNAs. The egg exosome release is correlated with microvilli formation on the egg cell membrane. In CD9-null eggs, both the egg exosome release and microvilli formation are dramatically impaired. Therefore, the egg exosomes are thought to be released from the eggs through the formation of microvilli

fusing with at least one sperm. Considering every possibility, we have no doubt that the above co-incubation experiments can be repeated. This result shows that sperms can fuse with CD9-null eggs with impaired microvilli via the egg exosomes of wild-type eggs: this means that the egg-derived vesicles, and not the microvilli, are essential for sperm-egg fusion. The close relation between egg exosomes and spermegg fusion raises the question of how egg exosomes facilitate fusion. Exosomes (hereafter referred to as canonical exosomes) derived from dendritic cells have been proven to be capable of inducing and enhancing antigen-specific T cell responses in vivo. According to a previous report (Valadi et al. 2007), canonical exosomes can also contain both functional mRNA and microRNA, which can be shuttled from one cell to another, affecting the recipient cell’s ability to produce protein. Moreover, it has been demonstrated that HIV-1 utilizes the canonical exosome biogenesis pathway for the formation of infectious particles, and that in macrophages, HIV1 assembles into an intracellular plasma membrane domain-containing tetraspanin (i.e., CD9, CD81, CD53, or CD63) (Fig. 7.4) (Garcia et al. 2005). Thus, the canonical exosomes may play at least two closely related roles in regulating cell function:

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Fig. 7.7 Co-incubation with wild-type eggs reverses defective fusion with sperm in CD9-null eggs in vitro. CD9-containing egg exosomes are released from wild-type eggs before any interaction with the sperm. In wild-type eggs, shortly after the sperm penetrates the egg’s perivitelline space, the egg exosomes are transferred onto the acrosome-reacted sperm head, and are predicted, given the nature of canonical exosomes previously identified, to fuse into the sperm. Then, a sperm fuses with the egg cell membrane. Interaction between the sperm and the exosomes is an essential step for sperm-fusing ability. In contrast, CD9-null eggs cannot release the egg exosomes, which are correlated with the formation of microvilli on the egg cell membrane. The sperm cannot fuse to the cell membrane of the CD9-null egg. When the zona pellucida is removed from the eggs, the sperm is able to interact with the egg exosomes released from wild-type eggs and can fuse with the CD9null egg. In fact, by co-incubation with wild-type eggs, the sperm can fuse with a similar number of CD9-null and wild-type eggs. Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization procedure in which a single sperm head is injected directly into an egg. This procedure is most commonly used to overcome male infertility and fusion defects in CD9-null eggs. (Modified from Miyado et al. 2008)

shuttling proteins or RNAs from one cell to another and forming infectious particles. In fertilization, these two roles identified in the canonical exosomes may be required to regulate sperm-egg fusion. Evidence obtained recently shows that the egg-derived CD9 is translocated to the head of the fertilizing sperm (Barraud-Lange et al. 2007, Miyado et al. 2008); this translocation appears to be mediated by egg exosomes, similar to that of the canonical exosomes observed in immune cells. In fact, many microvesicles and amorphous substances were observed, using an electron microscope, on the sperm surface that penetrated into the perivitelline space (Toshimori et al. 1998). Nevertheless, the physiology of egg exosomes remains poorly characterized. Our findings underscore the relevance of CD9 for healthy and pathogenic cell–cell fusion processes, and may present useful strategies for influencing the cell-to-cell spread of specific viruses and fertilization ability.

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Acknowledgments We are grateful to Dr. Masaru Okabe, Dr. Kiyotaka Toshimori, Dr. Chizuru Ito, Dr. Naokazu Inoue, and Dr. Eisuke Mekada for their critical discussions.

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Chapter 8

Gamete Binding and Fusion Young-Joo Yi, Shawn W. Zimmerman, and Peter Sutovsky

Abstract Successful mammalian fertilization results in the union of two gametes, a spermatozoon and a mature oocyte. Membrane fusion events are essential for at least two distinct steps of the fertilization process: (i) the vesiculation of the acrosomal surface membranes during sperm acrosomal exocytosis (AE), induced by sperm binding to the egg-coat, and (ii) fusion of the oocyte plasma membrane, the oolemma, with the sperm plasma membrane that occurs after AE and sperm-egg coat penetration. The rearrangement of sperm plasma membrane domains/membrane lipid raft formation during sperm capacitation in the female reproductive tract is a priming step that enables the fusion and vesiculation of outer acrosomal membranes during AE. The membrane fusion/vesiculation events of AE seem to share similarities with synaptic vesicle fusion, assisted by the membrane proteins of the SNARE hypothesis. The AE exposes the transmembrane receptors on the sperm head equatorial segment in preparation for sperm-oolemma adhesion and fusion. Gene ablation studies indicate that the tetraspanin family proteins CD9 and CD81 on the oolemma interact with the superglobulin family protein IZUMO on the sperm plasmalemma to mediate sperm-oolemma adhesion in mammals. The fusogenicity of IZUMO has not been established, so the involvement of this system in the actual membrane fusion part of sperm–oolemma interaction remains open. Interactions of ADAM family proteins on sperm plasma membrane with oolemma integrins appear non-essential during sperm-oolemma fusion, but integrins may play a supporting role via sustenance of the tetraspanin web in the oocyte cortex. Spermoolemma binding may be reinforced by a cast of other receptors found on the surface of the sperm head (e.g. CRISP and MN9). The present chapter reviews recent progress in the study of these fundamental factors of gamete membrane fusion during mammalian fertilization.

P. Sutovsky (B) Division of Animal Science, University of Missouri-Columbia, Columbia, MO 65211, USA; Departments of Obstetrics, Gynecology & Women’s Health, University of Missouri-Columbia, Columbia, MO 65211, USA e-mail: [email protected]


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Keywords Acrosome · CD9 · CD81 · egg · eqatorin MN9 · exocytosis · fertilization · Izumo · membrane fusion · SNAREs · sperm · vesiculation Abbreviations ADAM AE AFAF CD CEA CRISP IAM ICSI IgSF KO LEL NSF OAM PVS PSG PM PTGFRN PTP SEL SNAP SNARE t-SNARE VAMP v-SNARE ZP

A disintegrin and a metalloprotease Acrosomal exocytosis Acrosome formation associated factor Cluster of differentiation Carcinoembryonic antigen Cysteine-rich secretory protein Inner acrosomal membrane Intracytoplasmic sperm injection Immunoglobulin superfamily (protein) Knock-out Large extracellular loop N-ethylmaleimide-sensitive factor Outer acrosomal membrane Perivitelline space Pregnancy-specific glycoprotein Plasma membrane Prostaglandin F2 receptor negative regulator Protein tyrosine phosphatase Small extracellular loop Soluble NSF attachment protein SNAP receptors Target-SNARE Vesicle associated membrane protein Vesicle-SNARE Zona pellucida

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Membrane Fusion Events During Acrosomal Exocytosis . . . . Essential Role of CD9 in Sperm-Egg Binding . . . . . . . . . IZUMO-the Candidate Sperm Partner of Oolemma Tetraspanins Integrin–Disintegrin Interactions in Sperm-Egg Binding . . . . Eqatorin MN9 and other Sperm Surface Ligands Implicated in Sperm-Oolemma Fusion . . . . . . . . . . . . 8.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8.1 Introduction Successful mammalian fertilization requires the union of two gametes, an oocyte and a spermatozoon. Oocytes reach their full fertilizing potential during the gonadotropin-induced meiotic maturation in the female gonad, the ovary. In contrast, spermatozoa that have completed their morphogenesis in the seminiferous epithelium have to leave the male gonad, the testis, to acquire their fertilizing potential. They do so during sperm passage through the epididymis, an accessory gland in which the spermatozoa undergo maturation and storage. After ejaculation, spermatozoa temporarily mix with seminal plasma contributed by secretions of multiple sex accessory glands, and become fully competent for fusion with the oocyte as they move up the female reproductive tract. They reach full fertilizing ability after they transiently bind to and subsequently detach from the epithelium of the oviductal sperm reservoir during a process termed sperm capacitation. During capacitation, sperm plasma membrane undergoes an extensive remodeling thought to increase its fluidity and fuseability [reviewed by (Gadella and Visconti 2006, Suarez 2002)]. There are two important membrane fusion events involved in mammalian fertilization: The acrosomal exocytosis (AE), induced by the sperm acrosomal surface-binding to the glycoprotein-rich egg coat, zona pellucida (ZP), and the process of sperm-oolemma fusion that ensues after AE and sperm ZP-penetration. During AE, the plasma membrane overlying the sperm acrosome fuses with the underlying outer acrosomal membrane to promote the vesiculation of the acrosomal surface membranes and the release/exposure of the acrosomal matrix. The AE requires an extensive membrane domain rearrangement during capacitation, resulting in the formation of membrane lipid rafts. The process of acrosomal membrane vesiculation, giving rise to the acrosomal shroud, requires membrane vesicle fusion that may share some similarities with synaptic vesicle fusion, assisted by membrane proteins of the SNARE hypothesis, such as the synaptobrevin/VAMP and syntaxin. The completion of acrosomal exocytosis enables the spermatozoon to penetrate through ZP. At the same time, AE exposes the sperm surface antigens involved in the second major membrane fusion event of fertilization, the sperm-oolemma fusion, followed by sperm incorporation in the oocyte cytoplasm. Sperm-oolemma fusion relies on the interaction of complementary receptors on the respective sperm and oocyte plasma membranes, the sperm plasmalemma and oolemma, respectively. While oolemma integrin-sperm disintegrin interactions have been studied extensively, more definitive evidence now points to oolemma tetraspanin-sperm superglobulin mediated interactions being essential for spermoolemma adhesion and fusion. These interactions will be discussed in detail in the present chapter.

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8.2 Membrane Fusion Events During Acrosomal Exocytosis Acrosomal exocytosis is an irreversible exocytotic event triggered in the capacitated, hyperactivated spermatozoa by contact of the sperm acrosomal surface with the branched glycans decorating the molecules of sperm receptor protein(s) on the surface of sperm zona pellucida (Sutton-Smith et al. 2007). The sperm acrosome is a specialized organelle assembled from the Golgi complex and perinulear theca-derived components. Topologically, the acrosome is composed of the outer acrosomal membrane (OAM) and inner acrosomal membrane (IAM) surrounding the acrosomal matrix. Acrosomal exocytosis encompasses the fusion of the sperm plasma membrane with the outer acrosomal membrane, which is thought to produce the hybrid membrane vesicles of the acrosomal shroud (Yanagimachi 1994). The acrosomal matrix contains structural proteins as well as multiple proteases, which are vesicle-associated or soluble components thought to be involved in sperm-ZP binding and penetration (Buffone et al. 2008, Foster et al. 1997, Kim et al. 2001a; Olson and Winfrey 1994, Westbrook-Case et al. 1995). Some of the components of the acrosomal matrix may associate with vesicles forming the acrosomal shroud, a transient structure surrounding the ZP-bound sperm head, or with the surface of the IAM, which remains intact after AE. Some of the protein components of the acrosomal matrix that are released or exposed by AE appear to undergo post-translational modifications by proteolytic processing. For instance, proacrosin, a major component of the acrosomal matrix, is cleaved into acrosin during acrosomal exocytosis (Kim et al. 2001b). The AE, once thought to be a rapid, all-or-nothing exocytotis event is now being re-evaluated as a stepwise process, during which the layers or differentially soluble components of the acrosomal matrix are released one at the time. Kim and Gerton (2003) described four distinct stages of acrosomal exocytosis in the mouse. The first stage is characterized by an intact OAM and plasma membrane (PM). During the second stage, the OAM and PM begin vesiculation. The third stage coincides with the progressive vesiculation of OAM/PM and dispersion of soluble acrosomal matrix components from the sperm head. The fourth stage marks the completion of OAM/PM vesiculation and the loss of most of the acrosomal matrix. Once the soluble components dissipate, only the acrosomal proteins associated with the IAM participate in sperm-ZP interactions during and beyond secondary sperm-ZP binding (Buffone et al. 2008). Acrosomal exocytosis is initiated by Ca2+ influx fueling an intra-acrosomal internal calcium increase (Breitbart 2002). This mechanism is similar to the induction of synaptic vesicle-plasma membrane fusion during neurotransmitter secretion in the neurons, which is also calcium-regulated. The GTPases of the Rab family (Novick and Zerial 1997, Zerial and McBride 2001), and SNARE proteins (Chen and Scheller 2001, Gerst 1999) that participate in neurotransmitter secretion, have also been identified in spermatozoa. The SNARE hypothesis of synaptic vesicle fusion predicts that the soluble N-ethylmaleimide-sensitive factor (NSF)attachment proteins (SNAPs), including syntaxin (Bennett et al. 1992) and SNAP-25 (Oyler et al. 1989), regulate membrane vesicle fusion by associating with their specific receptors, the SNARE proteins (Terrian and White 1997, Weimbs et al.

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1997, 1998). There are two types of SNARE receptors, the t-SNAREs, on the target plasma membrane and the v-SNAREs on the secretory vesicles, such as the synaptobrevin/vesicle-associated membrane protein (VAMP) (Schulz et al. 1997). It is believed that the interaction between a t-SNARE and a v-SNARE results in the binding and fusion of vesicle membrane with the plasma membrane (Rothman 1994). Synaptobrevin/VAMP, syntaxin 1, and the SNARE hypothesisassociated calcium sensor synaptotagmin I have been detected in the acrosomes of sea urchin and mammalian spermatozoa and shown to colocalize with the acrosomal membrane vesicles during AE (Ramalho-Santos et al. 2000, Schulz et al. 1997). Both anti-syntaxin and anti-VAMP antibodies inhibited bovine ZP-induced AE and fertilization without affecting sperm-ZP binding (Ramalho-Santos et al. 2000). Multiple SNARE proteins, including VAMP2, SNAP23, SNAP25, and several syntaxins were also detected in the human sperm acrosome, where the blocking of VAMP by botulinum neurotoxin or saturation of spermatozoa with bacterially expressed SNAP proteins inhibited AE (Tomes et al. 2002). The streptolysin O-permeabilized human sperm model has been instrumental in the study of this pathway during acrosomal exocytosis (Yunes et al. 2000). The general model of SNARE involvement in AE can be summarized as follows, based on the description given by De Blas et al. (2005): Prior to AE, the SNARE proteins are locked in heteromeric cis complexes. Upon Ca2+ entry into the acrosome, Rab3 is activated and triggers NSF/SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane (Hohne-Zell and Gratzl 1996) and the OAM can thus reassemble into loose trans complexes that are conducive to membrane fusion. In the final step, Ca2+ is released from the acrosome, triggering the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. This entire process of snare complex disassembly and reassembly may be triggered by dephosphorylation of the NSF by protein tyrosine phosphatase PTP1B at an early stage of AE (Zarelli et al. 2009).

8.3 Essential Role of CD9 in Sperm-Egg Binding The transmembrane 4 superfamily proteins, the tetraspanins, are widely distributed in various tissues and cell types, including the gametes. Tetraspanins contain two extracellular domains (the large and the small extracellular loops, LEL and SEL, respectively) which function as specific receptors, transmembrane anchors, interacting with transmembrane parts of other proteins (Lefevre et al. 2010). In turn, the cytoplasmic N-terminal and C-terminal tails of tetraspanins interact with cytoskeletal elements such as microfilaments and microtubules, and with cytoskeleton-associated molecules involved in signal transduction processes. The tetraspanins take their name from four transmembrane domains including several conserved amino acid residues (Lefevre et al. 2010). Tetraspanins constitute a large transmembrane complex, the tetraspanin web (Boucheix and Rubinstein 2001, Rubinstein et al. 1996), in which they associate with various cytoskeletal,

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transmembrane and intracellular signaling proteins (Boucheix and Rubinstein 2001, Delaguillaumie et al. 2002). Tetraspanins have been implicated in cell proliferation, adhesion, motility and signaling (Charrin et al. 2009, Hemler 2003, Lefevre et al. 2010). The tetraspanin superfamily contains 32 identified members in humans (Boucheix and Rubinstein 2001, Hemler 2003, Levy and Shoham 2005). In particular, three different tetraspanins, CD9, CD81 and CD151, have been detected, among other cell types, in the mammalian oocytes (Andria et al. 1992, Chen et al. 1999b; Neilson et al. 2000). Gene ablation studies in mice unequivocally demonstrated the essential requirement of tetraspanins for the fertilization process. The Cd9–/– female mice showed severely reduced sperm-egg adhesion/fusion (99.4% of recovered eggs failed to fuse with sperm and form pronuclei during IVF) and near complete infertility (Kaji et al. 2000, Le Naour et al. 2000, Miyado et al. 2000). Similarly, the Cd81–/– mice had reproductive impediments; however, their subfertile phenotype was milder than that of Cd9–/– mice (Higginbottom et al. 2003). Anti-CD81 antibodies suppressed sperm-oolemma binding and fusion (Takahashi et al. 2001) as did recombinant CD81 LEL (Higginbottom et al. 2003). The overexpression of Cd81 after microinjection of CD81 mRNA recovered the fusion ability of Cd9–/– eggs. Microinjections of mouse or human CD9 mRNA efficiently restored the fusion ability of Cd9–/– eggs up to 76 and 81% fertilization rate, respectively (Kaji et al. 2002). These results suggest that CD9 and CD81 play complementary, but not necessarily redundant, roles in sperm–egg fusion. Although Cd151 knockout mice did not display a fertility defect, mouse anti-CD151 antibody partially inhibited sperm–egg fusion in humans (Ziyyat et al. 2006). Recently, it has been suggested that CD9 could support plasma membrane functionality through CD9-associated, immunoglobulin superfamily protein IgSF8 (EWI-2) in the mouse oocyte (Glazar and Evans 2009). Accordingly, the expression level of EWI-2 on the Cd9–/– oocyte surface is 40%

5–20%

Syngenic & allogeneic DC

20–30%

35%

26%

20%

25%

Fusion efficiency

Syngenic DC

Syngenic DC

Syngenic DC

Syngenic DC

Syngenic DC

Syngenic DC

DC

[12]

[11]

[10]

[9]

References

Immunization with FC combined with IL-12 injection eradicated [13] 3-day established pulmonary or subcutaneous tumors in 100 and 80% of the mice, respectively A single vaccination with fusion cells plus IL-12 induced a [14] therapeutic immune response against 3-day established pulmonary metastases. The fusion approach is superior to other DC loading methods [15] Vaccination with fusion cell products prevented tumor growth of Renca, M3 and B16 melanoma cells in 50–75, 80–100 and 20–60% of mice, respectively. In addition, immunization with DC-Renca fusion cells eradicated 3-day established subcutaneous tumors

Prophylactic vaccination of mice that develop spontaneous mammary carcinomas with FC/MMT rendered 57–61% mice free of the disease for up to 180 days. In contrast, the control mice developed tumor between the ages of 65–108 days Immunization with FC expressing IL-18 protected 75% of the mice against challenge with tumor cells. By contrast, the protection for mice immunized with fusion cells without IL-18, irradiated tumor cells with or without expression of IL-18 was 37, 25 or 0%, respectively Immunization with FC or DC pulsed with tumor lysates prevented tumor growth in 75 and 30% of mice, respectively Immunization with FC combined with injection of IL-12 rendered 42% of mice bearing 3-day established subcutaneous tumor free of the disease

Comments

Table 15.1 (continued)

334 J. Gong and S. Koido

References

Immunization with FC combined with injection of CpG ODN [19] provides protection for 60% of mice against challenge with B16BL6 cells and 80% of mice against challenge with Renca cells

30%

Cell Fusion and Dendritic Cell-Based Vaccines

References: 1: Gong et al. (1997b); 2: Gong et al. (1998); 3: Lespagnard et al. (1998); 4: Wang et al. (1998); 5: Cao et al. (1999); 6: Homma et al. (2001); 7: Akasaki et al. (2001); 8: Gong et al. (2002); 9: Xia et al. (2003); 10: Xia et al. (2004); 11: Takeda et al. (2003); 12: Homma et al. (2005b); 13: Hayashi et al. (2002); 14: Tanaka et al. (2002); 15: Siders et al. (2003); 16: Kjaergaard et al. (2003); 17: Shimizu et al. (2004); 18: Phan et al. (2003); 19: Hiraoka et al. (2004); 20: Iinuma et al. (2006).

[20]

Treatment with single dose of 6-h hybrids led to long-term survival [18] of 60% of the mice

35–38.6%

Immunization with FC combined with injection of OX-40R mAb [16] rendered 60% of the mice bearing 3-day established subcutaneous tumor and 50% of the mice bearing 3-day intracranial tumor, respectively, free of tumors Immunization with either fusions of mature or immature DC to [17] tumor cells combined with injection of IL-12 significantly reduced the metastatic nodules in mice bearing 3-day established pulmonary tumors

Comments

D. PEG, Electrofusion or combined PEG and electrofusion A/J mice Neuroblastoma Syngenic DC Electrofusion: Immunization of mice with FC/IL-12/IL-18 provided 100% transduced 12.9% protection against challenge with tumor cells and eradicated with IL-12 and PEG: 34.1% 3-day established tumors in 60% of the mice IL-18 genes Combined fusion: 51.6%

C. Virus-mediated fusion C57BL/6 B16 melanoma Syngenic DC mice transfected with viral fusogenic membrane glycoprotein C57BL/6 & B16BL6 melanoma Syngenic DC BALB/c Renca mice

B16 melanoma cells expressing β-gal

Immature DC or 41.7–60% DC isolated from Flt-3L treated mice

Fusion efficiency

C57BL/6 mice

DC

Syngenic & 40–60% allogeneic DC

Tumor

C57BL/6 & MCA205 sarcoma BALB/c mice

Animal

Fusion partners

Table 15.1 (continued)

15 335

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in subsequent studies in tumor models of B16 melanoma (Cao et al. 1999, Hiraoka et al. 2004, Li et al. 2001, Phan et al. 2003, Shimizu et al. 2004, Tanaka et al. 2002, Wang et al. 1998), mastocytoma P815 (Lespagnard et al. 1998), lung carcinoma (Celluzzi and Falo 1998), hepatocellular carcinoma (Homma et al. 2001, 2005b, Zhang et al. 2003), myeloma (Gong et al. 2002, Liu et al. 2002), mammary carcinoma (Lindner and Schirrmacher 2002), glioma (Akasaki et al. 2001, Hayashi et al. 2002), colon carcinoma (Kao et al. 2003, Suzuki et al. 2005, Takeda et al. 2003), renal cell carcinoma (Siders et al. 2003), and fibrosarcoma (Kjaergaard et al. 2003, Ogawa et al. 2004). In all these experiments, partial or complete tumor eradication was achieved, outcome depending on the tumor model, the age of established tumors, the protocol for preparation of the FC and/or the vaccination regimen. The pulmonary metastasis models are most frequently used with favorable results (Gong et al. 1997b, 1998, 2000c, Cao et al. 1999, Li et al. 2001, Phan et al. 2003, Tanaka et al. 2002, Wang et al. 1998). However, studies show that immunization with FC vaccine is also effective in the treatment of established intracranial and skin tumors when combined with an adjuvant (Akasaki et al. 2001, Shimizu et al. 2004). Shimizu et al. (2004) have demonstrated that intrasplenic immunization with FC combined with intraperitoneal injection of OX-40R mAb renders 60% of mice with established skin tumor and 50% of mice with three-day-old intracranial tumor free of disease.

15.7.2 Clinical Trials Based on the results from animal studies, initial phase I clinical trials with FC immunization were conducted (Table 15.2). Krause et al. (2002) fused DC to autologous melanoma cells by electrofusion and assessed the use of such FC as a vaccine in 17 patients with disseminated melanoma refractory to standard therapy. There were no serious side effects associated with the administration of the vaccine. Localized hair depigmentation occurred in one case. In the 13 evaluable patients, one patient each achieved partial response and one had stable disease for 6 months. In another patient, some of the metastases were regressing despite overall progression of the disease. The authors attributed their findings to several factors, including limited tumor-cell viability, limited numbers of DC and tumor cells, immaturity of DC, and the type of vaccination protocol. In an alternative approach, Trefzer et al. (2005) fused malignant melanoma cells with allogeneic DC. In 17 patients, they observed one complete response, one partial response, and six responses of stable disease with long survival times. In addition, 11 of 14 patients, clinical responders and non-responders alike, mounted high-frequency T-cell responses to various tumorassociated antigens. Kikuchi et al. (2001), in a phase I clinical trial in patients with malignant glioma, found that immunization with DC–glioma fusion vaccine produced partial responses in two of six patients. In a similar trial by the same group, FC in combination with IL-12 was administered to patients with malignant brain tumor, breast cancer, gastric cancer, colorectal cancer, ovarian carcinoma, or melanoma.

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Table 15.2 Results of clinical trial in patients with advanced stage of malignant tumors Fusion partners Clinical Patients (n) responsea

References

Autologous

16

[1]

Melanoma

Autologous Autologous

17

Melanoma

Allogeneic

Melanoma


Glioma


Glioma


rh IL-12

12

Gastric/ Colorectal carcinoma Ovarian carcinoma Breast cancer


rh IL-12

3


rh IL-12

3


rh IL-12

2

Breast cancer


10

Renal cell carcinoma Renal cell carcinoma


13

Allogeneic

Autologous

20

Renal cell carcinoma Renal cell carcinoma

Allogeneic

Autologous or Allogeneic Autologous

12

Tumor

DC

Tumor

Melanoma

Allogeneic

Allogeneic

Autologous

Hepatocellular Autologous Autologous carcinoma

Adjuvant

rh IL-2

11

rh IL-12

4 8

10

1

1 (CR), 1 (PR), 5 (SD), 9 (PD) 1 (PR), 1 (SD), 15 (PD) 1 (SD), 10 (PD) 4 (PD) 2 (PR), 1 (SD), 5 (PD) 3 (PR), 2 (MR), 4 (SD), 3 (PD) 1 (SD), 2 (PD) 2 (SD), 1 (PD) 1 (SD), 1 (PD) 2 (PR), 1 (SD), 7 (PD) 5 (SD), 8 (PD) 2 (PR), 8 (SD), 10 (PD) 4 (SD), 8 (PD) 1 (PR), 6 (SD), 3 (PD) 1 (PD)

[2]

[3] [4] [4]

[4, 5]

[4]

[4] [4] [6]

[6] [7]

[8] [9]

[10]

References:1: Trefzer et al. (2005); 2: Krause et al. (2002); 3: Haenssle et al. (2004); 4: Homma et al. (2005a); 5: Kikuchi et al. (2004); 6: Avigan et al. (2004); 7: Avigan et al. (2007); 8: Marten et al. (2003); 9: Zhou et al. (2009); 10: Koido et al. (2008). a CR, complete response; PR, partial response; MR, mixed response; SD, stable disease; PD, progressive disease.

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Three of 12 patients with malignant brain tumor achieved a partial response and one patient a minor response (Homma et al. 2005a, Kikuchi et al. 2004), but the response to other types of malignant tumors was muted. The relatively favorable response to malignant brain tumors is of particular interest since the central nervous system is generally considered an immunologically privileged site as a result of the lack of lymphatic drainage and the nature of the blood–brain barrier (Homma et al. 2005a, Kikuchi et al. 2004). However, these studies demonstrate that appropriately activated T cells can cross the blood–brain barrier to access tumor and initiate tumor regression. Avigan et al. (2004) tested FC vaccine in 23 patients with metastatic breast and renal cancer. Immunologic and clinical responses to the vaccination were observed in a subset of patients. Most notably, two patients with breast cancer exhibited disease regression, including a nearly complete response of a large chest-wall mass. Five patients with renal carcinoma and one patient with breast cancer had disease stabilization. In a subsequent trial, renal cell carcinoma cells were fused to allogeneic DC using electrofusion with fusion efficiency of 20% (Avigan et al. 2007). The vaccine was well-tolerated, and vaccination resulted in antitumor immune response in 10/21 evaluable patients. Partial clinical response was demonstrated in two patients and stabilization of disease in eight patients. Marten et al. (2003) observed immunologic, but not clinical, responses in 12 patients with metastatic renal cell carcinoma (RCC) treated with DC–tumor fusion vaccine. Zhou et al. (2009) reported that DC-tumor fusion cell vaccine was safe and able to elicit immunological responses in a significant portion of patients with RCC. Haenssle et al. (2004) failed to find unequivocal beneficial effects from fusion cell products generated by fusion of HLA class I-mismatched DC from healthy donor to patient-derived melanoma cells. It is apparent that the antitumor response induced by FC vaccines in humans is muted compared with that in the animal studies. Multiple factors may be involved in the differential antitumor reaction between animal models and humans. One obvious difference is that the FC vaccine has been used in patients with a large burden of cancers and/or metastasis. There is a large body of evidence indicating that tumor exerts immunosuppression through alteration of APC, promotion of inhibitory molecules, and expansion of regulatory T cells in general and in the tumor microenvironment particular (Zou 2005). In patients with advanced stages of tumor progression, the tumor microenvironment favors the production of inhibitory molecules including vascular endothelial growth factor (VEGF), IL-6, IL-10 and transforming growth factor-β (TGF-β). These molecules work individually and in concert to promote the recruitment, differentiation, maturation and survival of inhibitory DC subsets or dysfunctional DC. These DC tend to expand the regulatory T cells, which inhibit the expansion and function of tumor-reactive effector T cells. It is likely that the tumor microenvironment plays a role in the muted clinical response to FC vaccination. Indeed, the supernatant from human hepatocellular carcinoma (HCC) cells induced functional impairment of DC as demonstrated by the down-regulation of MHC class I and class II, CD80, CD86, and CD83 molecules (Koido et al. 2008). Moreover, DC exposed to the supernatant from HCC cells secreting TGF-β failed to undergo full maturation upon stimulation with TLR 4 agonist, OK-432. These

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inhibitory molecules by tumor cells may also exert suppressive effect for DCtumor fusion vaccine. For example, tumor-derived TGF-β produced by tumor cells significantly reduced the potency of DC-tumor fusion cell vaccines (Kao et al. 2003) and the blockade of tumor-derived TGF-β reduced the Treg induction by the DC-tumor fusion cell vaccine and enhanced antitumor immunity (Zhang et al. 2008). It is conceivable that a major obstacle in the development of active immunotherapy for cancer is the immunosuppressive environment and inhibitory molecules produced by the growing tumor. In this context, concomitant immunotherapy and cytoreduction therapy, such as chemotherapy or therapy targeting the induction of regulatory T cells, may represent a promising approach. Taken together, DC–tumor fusion vaccine may have certain advantages over other DCbased vaccines. However, a strategy is needed to enhance the therapeutic efficacy of the vaccine and provide clinical benefit to patients.

15.8 Promotion of Antitumor Immunity 15.8.1 Using Adjuvant with Fusion Vaccine The induction of functional CTL requires the interaction of TCR and antigenic peptide–MHC complex presented on the APC in the context of co-stimulatory molecules. In addition, pro-inflammatory cytokines are also needed to overcome the regulation of tolerance. Fusions of DC and tumor cells generally produce proinflammatory cytokines such as IL-12 at a level comparable to that of DC (Gong et al. 2002, Homma et al. 2005a). However, the amounts of the cytokine produced by FC may be insufficient for optimal induction of sustainable CTL. In fact, a mixed T-cell response characterized by the expansion of both activated and regulatory T cells has been induced by DC–breast cancer fusion cells (Koido et al. 2008, Vasir et al. 2008). The latter may exert inhibitory effect on CTL. To address potential problems, cytokines were used as an adjuvant during the priming phase of T cells by FC. Study shows that coculture of T cells with DC–tumor fusion cells in the presence of IL-12, IL-18 or CPG ODN reduces the expansion of regulatory T cells and promotes the expansion of activated effector cells (Vasir et al. 2008). Alternatively, regulatory T cells can be removed using a low dose of chemotherapy or neoadjuvant chemotherapy (Ladoire et al. 2008, Menard et al. 2008, Zou 2006). The most widely used cytokine is IL-12 (Gong et al. 2002, Homma et al. 2005a, Akasaki et al. 2001, Hayashi et al. 2002, Iinuma et al. 2004b), which is involved in the up-regulation of co-stimulatory molecules in DC, promotion of Th1 immunity, and inhibition of induction of anergy (Coughlin et al. 1995, Grohmann et al. 1997, Mountford et al. 1996). Co-administration of IL-12 potentiated the CTL and T-cell proliferative responses to FC vaccination that were translated into better therapeutic efficacy or protection of mice against challenge with tumor cells (Gong et al. 2002, Homma et al. 2005a, Akasaki et al. 2001, Hayashi et al. 2002, Iinuma et al. 2004b). An alternative approach is the use of TLR agonists, which induce DC activation, leading to the production of pro-inflammatory cytokines and

340


the up-regulation of co-stimulatory molecules (Okamoto et al. 2004). For example, the synthetic oligodeoxynucleotides containing specific bacterial unmethylated CpG motif (CpG ODN) induce Th1 immune responses through TLR 9, resulting in the production of pro-inflammatory cytokines IL-12, TNF-α and IFN-γ that promote the development of antitumor immunity (Heckelsmiller et al. 2002). Hiraoka and colleagues (2004) injected FC admixed with CpG ODN into murine tumor models. Co-administration of FC and CpG ODN resulted in increased production of IFN-γ and IL-12, enhanced induction of tumor-specific CTL, and improved protection of mice against lethal subcutaneous tumor challenge and spontaneous lung metastasis. Importantly, long-term antitumor immunity was maintained in mice immunized with FC and CpG ODN, but not FC or CpG ODN alone. Paired, but not solitary combinations of polyinosine:polycytadilic acid (TLR3 agonist) and CpG DNA (TLR9 agonist) stimulated IL-12 secretion from DCs in vitro and synergized with vaccination to achieve potent tumor rejection Zheng et al. 2008). Another example of the use of TLR agonists in this context is OK-432, a penicillin-inactivated and lyophilized preparation of the low-virulence strain of Streptococcus pyogenes (group A). OK-432 is one of the biological response modifiers and a good manufacturing practice grade agent. It has been demonstrated that the compound promotes activation of DC through TLR4 and β2 integrin to enhance antigen-specific CTL responses to a greater extent than does a previously reported mixture consisting of TNF-α, IL-1β, IL-6, and PGE2 (Nakahara et al. 2003, Okamoto et al. 2004). It has been used without apparent side effects as an adjuvant for patients with cancer (Yamanaka et al. 2005), activates macrophages, lymphocytes, and NK cells by inducing multiple cytokines including IL-12 and IFN-γ, and polarizes the T-cell response to a Th1dominant state (Grohmann et al. 1997, Nakahara et al. 2003). These reports suggest that the combination of FC and OK-432 as an adjuvant may provide a more effective and feasible cancer vaccine and indicate the importance of innate immunity in the promotion of adaptive antitumor immunity.

15.8.2 Combined Approaches Dendritic–tumor fusion cells can be used as a vaccine to activate presumably existing, yet limited, tumor-specific CTL precursors in a host. Alternatively, the fusion cells can be used as stimulators to induce and expand ex vivo to therapeutic numbers tumor-specific CTL, which can then be transferred to the tumor-bearing host for adoptive immunotherapy. This approach, though not yet tested in the clinical setting, has potential to enhance the antitumor immunity induced by FC. Recent studies show that DC–tumor fusion cells are potent T-cell stimulators: DC fused to primary tumor cells from patients with ovarian, breast, malignant glioma, leukemia, melanoma, and multiple myeloma stimulate strong CTL activity against autologous tumor cells (Galea-Lauri et al. 2002, Goddard et al. 2003, Gong et al. 2000a, b, 2004, Kikuchi et al. 2001, Raje et al. 2004). In addition to the induction of CTL, DC–tumor fusion cells also possess properties to activate CD4+ T cells, which are required for the maintenance of CTL

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(Dranoff et al. 1993, Gong et al. 1997b, Hung et al. 1998, Marzo et al. 1999, 2000, Ossendorp et al. 1998) and in the effector/memory phase (Chaux et al. 1999, Greenberg 1991, Mumberg et al. 1999, Qin and Blankenstein 2000). These cells are needed to maintain the numbers and cytotoxic capacity of CD8+ T cells and to promote the infiltration of CD8+ T cells in tumor (Marzo et al. 1999, 2000). We observed that fusion cells migrate to draining lymph nodes after subcutaneous injection and are closely associated with CD4+ and CD8+ T cells (Koido et al. 2002). These properties significantly increase the probability of formation of the three-cell cluster composed of fusion cell, CD4+ and precursor CTL. Although this cluster is not required for the induction of CTL (Ridge et al. 1998), simultaneous recognition of cognate peptides presented by MHC class I and class II molecules on APC is more effective in the induction of CTL (Behrens et al. 2004, Bennett et al. 1997, Mitchison 1990). Based on these results, it is conceivable that T cells stimulated by FC can be used for adoptive therapy. Indeed, the efficacy of FC-activated T cells has been demonstrated in animal models (Tanaka et al. 2004, Wang et al. 1998) in which adoptive transfer of T lymph node cells isolated from FC-vaccinated mice has eliminated established pulmonary metastasis (Gong et al. 2000c, Tanaka et al. 2004). Wang and associates (1998) show that adoptive transfer of T-LNC derived from mice immunized with DC–B16 fusion vaccine into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases. Furthermore, adoptive transfer of DC/RMA-S vaccine-primed, cultured T-LNC eradicated disseminated FBL-3 tumor. Collectively, these findings indicate the therapeutic usage of T cells stimulated by FC either in vitro or in vivo and raise the possibility of dual usage of DC–tumor fusion vaccine in antitumor immunity: FC can be used as a vaccine to activate tumor-specific T cells in a host or as T-cell stimulators to generate ex vivo tumor-specific T cells for adoptive use. The dual usage of FC-based active and adoptive immunotherapy was reported by Tamai and associates (2008). Instead of vaccination with FC, T cells were selected from mice inoculated with 4T1 tumor cells and expanded ex vivo. These T cells were adoptively transferred to host bearing metastatic 4T1 tumor in conjunction with DC–tumor vaccination. Immunization with DC–tumor fusion vaccine or adoptive transfer of T-LNC alone was effective in reduction of tumor metastases but insufficient to eradicate tumor. In contrast, combined active immunization with DC–tumor vaccine and adoptive transfer of activated T-LNC rendered 8 of 17 mice free of tumors. Such FC-based active and adoptive immunotherapy may represent a promising strategy in the management of cancer with metastasis.

15.9 Summary Ample evidence indicates that DC–tumor fusion cells constitute a potent tumor vaccine. Yet, we are still searching for optimal ways to use FC-based immunotherapy to benefit patients significantly. Such a gap reflects, at least in part, the variations

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associated with DC–tumor fusion vaccine. Fusion technique is a versatile approach in the design of tumor vaccine and can be applicable to nearly all types of tumor cells using a variety of methods. However, such versatility and wide applicability lead to variations that make it more difficult to standardize the vaccine. Although there is considerable variation in the phenotype of the parent cells and fusogenicity of different type of tumor cells, certain quality control measures are desirable. These quality controls include FC phenotype, cell viability, fusion efficiency, and stability. For example, the range of fusion cells in the fusion-cell product that constitutes a fusion vaccine needs to be defined. Such quality control may be essential in evaluation of DC–tumor fusion vaccines since a vaccine with poor quality can still elicit antitumor immunity, albeit at a lower level. The second challenge facing FC vaccine is how to benefit patients. We have observed significant therapeutic effect with FC vaccination in animal models, whereas in humans, only limited therapeutic results are obtained. A major reason is that tumor vaccines, including FC-based vaccines, are tested as a therapy in patients with advanced stages of disease and large tumor burden refractory to standard therapy. It is conceivable that in this group of patients the tumor vaccines are engaged in an uphill battle against tumor-induced and/or therapy-induced immunosuppression (Finn 2003). This explains, at least in part, why differential therapeutic efficacy of FC vaccination has been achieved in animal compared with human studies. To circumvent this problem, the FC vaccine may be tested in cancer patients in remission to prevent recurrence and metastasis. Alternatively, FC-based immunotherapy may be used in combination with other therapies including surgery and radiotherapy.

References Ahkong QF, Baldwin JM, O’Reilly R et al (1994) Interactions between metal ions and poly(ethylene glycol) in the fusion of human erythrocytes. Mol Membr Biol 11:171–179 Ahkong QF, Lucy JA (1986) Osmotic forces in artificially induced cell fusion. Biochim Biophys Acta 858:206–216 Akasaki Y, Kikuchi T, Homma S et al (2001) Antitumor effect of immunizations with fusions of Dendritic and Glioma cells in a mouse brain tumor model. J Immunother 24:106–113 Avigan D, Vasir B, Gong J et al (2004) Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. Clin Cancer Res 10:4699–4708 Avigan DE, Vasir B, George DJ et al (2007) Phase I/II study of vaccination with electrofused allogeneic dendritic cells/autologous tumor-derived cells in patients with stage IV renal cell carcinoma. J Immunother 30:749–761 Bateman A, Bullough F, Murphy S et al (2000) Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth. Cancer Res 60:1492–1497 Bateman AR, Harrington KJ, Kottke T et al (2002) Viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells. Cancer Res 62:6566–6578 Behrens G, Li M, Smith CM et al (2004) Helper T cells, dendritic cells and CTL Immunity. Immunol Cell Biol 82:84–90 Bennett SR, Carbone FR, Karamalis F et al (1997) Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help. J Exp Med 186:65–70

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Chapter 16

Cancer Cell Fusion with Myeloid Cells: Implications for Energy Metabolism in Malignant Hybrids Rossitza Lazova, Ashok K. Chakraborty, and John M. Pawelek

Abstract It is abundantly clear that metastasis – the migration of cancer cells from their site of origin to distant organs and tissues – is what makes cancer so deadly. It is therefore surprising that so little is known about its onset. We advocate that the century-old theory of cancer cell fusion with tumor-associated leucocytes such as macrophages is the only complete theory we have – potentially explaining most if not all aspects of metastasis, most notably its initiation. The fusion theory states that acquisition of a metastatic phenotype occurs when a healthy migratory leucocyte fuses with a primary tumor cell. The resultant hybrid adapts the white blood cell natural ability to migrate around the body, all the while continuing to go through the uncontrolled cell division of the original cancer cell. Here we review the evidence supporting these concepts. We further focus on autophagy, a common state of macrophages that is also a signature trait of experimental macrophage-melanoma hybrids in culture. We found autophagy to be widespread in pathology specimens of human malignant melanomas, suggesting that autophagy provides an alternate energy source to these tumors. It is proposed that autophagy in melanoma and other malignancies might be a reflection of fusion with myeloid cells. Thus pathways regulating autophagy as well as the fusion events themselves provide potential new targets for cancer therapy. Keywords Cancer cell fusion · metastasis · aneuploidy · autophagy · phagocytosis · cancer epigenome · macrophage · bone marrow-derived cell Abbreviations ABC ALM BMDC CCL2

ATP-binding cassette Acral lentigenous melanoma Bone marrow-derived cell Chemokine (C-C motif) ligand 2

J.M. Pawelek (B) Department of Dermatology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06520-8059, USA e-mail: [email protected] L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9_16, C Springer Science+Business Media B.V. 2011

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c-Met CSF CXCR4 DEJ EM EMT FISH GnT-V H&E HGF HIF-1 IL LAMP LC3B LPHA MC1 MC1R M-CSF MDR MIS MITF PCC PEG prcc Rcc sialyl lex SPARC SSM TAM TGF-β1 TR uPA uPAR VEGF

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The cognate receptor for HGF Colony stimulating factor Cysteine-X-cysteine chemokine receptor 4 Dermal epidermal junction Electron microscopy Epithelial-mesenchymal transition Fluorescent in situ hybridization N-acetylglucosaminyltransferase V Hematoxylin and eosin Hepatocyte growth factor Hypoxia-inducible factor 1 Interleukin Lysosome associated membrane protein Light chain 3 isoform B (autophagy marker) Leucocytic phytohemagglutinin, Melanocortin-1 Melanocortin-1 receptor Macrophage-colony stimulating factor Multi-drug resistance Melanoma in situ Microphthalmia-associated transcription factor Premature chromosome condensation Polyethylene glycol Primary papillary rcc Renal cell carcinoma Sialyl lewisx antigen Secreted protein acidic and rich in cysteine; osteonectin; BM40 Superficial spreading melanoma Tumor associated macrophages Transforming growth factor-β1 Toll receptor Urokinase-type plasminogen activator uPA receptor Vascular endothelial growth factor

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cancer Cell Fusion In Vivo . . . . . . . . . . . . . . . . . . . . . . . Tumor Associated Macrophages as Candidates for Cancer Cell Fusion Partners BMDCs in Human Cancer and Stem Cell-Like Distribution Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5 Cancer Cell Fusion and the Hybrid Phenotype . . . . . . . . . . . . . . .

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16.6 Macrophage-Melanoma Fusion In Vitro Generates Altered Gene Expression and a Metastatic Phenotype In Vivo . . . . . . . . 16.6.1 SPARC . . . . . . . . . . . . . . . . . . . . . . 16.6.2 MCR1 and c-Met . . . . . . . . . . . . . . . . . . 16.6.3 GnT-V and β1,6-Branched Oligosaccharides . . . . . . 16.6.4 Motility-Associated Integrins . . . . . . . . . . . . . 16.6.5 Cell Surface Expression of Lysosome Associated Protein-1 (LAMP-1) . . . . . . . . . . . . . . . . 16.6.6 Autophagy and Coarse Melanin . . . . . . . . . . . . 16.6.7 Autophagy in Cutaneous Malignant Melanoma . . . . . 16.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . 16.8 Considerations for Studying Fusion In Vivo . . . . . . . . . . 16.9 Implications . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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16.1 Introduction The fusion theory was first proposed in the early 1900s and has attracted a lot of scientific interest over the years (Lu and Kang 2009, Pawelek 2000, 2005, Pawelek and Chakraborty 2008a, b, Pawelek et al. 2006). Its roots lie in the pioneering work of Theodore and Marcella Boveri on aberrant chromosome numbers and abnormal mitoses in sea urchin eggs and the remarkable insights of German pathologist Otto Aichel as to how this might relate to tumor progression (Aichel 1911, Boveri 2008). The Boveri’s observed that sea urchin eggs experimentally fertilized with two sets of spermatozoa underwent abnormal mitosis. They later proposed that deregulated growth of cancer cells might also be a result of chromosome imbalance (Boveri 2008). This work motivated Aichel to first propose fusion and hybridization as a mechanism for the imbalance of chromosomes in human cancer, suggesting that a combination of extra chromosomes and the “qualitative differences” in chromosomes from the two cell types could lead to the metastatic phenotype (Aichel 1911). In his 1911 article ‘About cell fusion with qualitatively abnormal chromosome distribution as cause for tumor formation’ Aichel exhorted future scientists to ‘study chromosomes from all angles’. Decades later, the same hypothesis – that metastasis is caused by leucocyte-tumor cell fusion–was proposed independently by Mekler (1968, 1971) and by Goldenberg (1968) and, Goldenberg and Gotz (1968). Several laboratories have now reported that hybrids produced by fusion in vitro or in vivo were aneuploid and of higher metastatic potential (1–5). In 1984, LaGarde and Kerbel summarized the emerging concepts: “[Tumor cell hybridization] can lead to major changes in gene expression. These processes can lead to the evolution of subpopulations of tumor cells having major losses or gains in their malignant aggressiveness and therefore represents a large-scale genetic mechanism capable of generating genotypic and phenotypic diversification . . . If the normal host cell happens to be a lymphoreticular-hematopoietic cell, it could donate this phenotype to cell types which otherwise do not normally express metastatic traits.” There is now considerable evidence to support these concepts.

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The pathways of invasion and metastasis have been under intense scientific scrutiny and much is now known about the steps involved (Gupta et al. 2005, Chambers et al. 2002). However, the actual genesis of metastatic cells from within populations of non-metastatic cells of the primary tumor is not understood. What are the initiating mechanisms that cause a carcinoma or melanoma cell in the epithelium to free its adhesions to neighboring cells, adapt a migratory phenotype, cross the basal lamina into the dermis, intravasate into the blood circulatory system or lymphatics, extravasate, and form new tumors in lymph nodes and distant tissues or organs? The long-standing view is essentially Darwinian: the unstable cancer genome combined with host selective pressures generates metastatic cells in the otherwise non-metastatic primary tumor (Fidler and Kripke 1977, Nowell 1976). This view continues to provide the best framework for envisioning tumor progression. Yet it is difficult to imagine how this might occur through successive, stepwise mutations since generation of a metastatic phenotype would require activation and silencing of very large numbers of genes in the primary tumor cell (Gupta et al. 2005). One solution to this problem lies in the activation of master regulatory genes that control multiple pathways and initiate pro-metastatic cascades (Ma et al. 2007). This has been highlighted in reports that master regulators of epithelial-mesenchymal transition (EMT) in development, such as Snail, Slug, SPARC (secreted protein acidic and rich in cysteine; osteonectin; BM40), Twist, and others play analogous roles in invasion and metastasis where they activate mesoderm-associated pathways of cellular adhesion and migration (Gupta et al. 2005, Ma et al. 2007. However, the mechanisms through which master regulators such as Twist are themselves upregulated in cancer are not understood. We propose that at least in some cases this could be initiated by fusion of cancer cells with bone marrow-derived cells (BMDCs). While a transition from epithelial to mesodermal gene expression is indeed a characteristic of invasion and metastasis, the expressed genes are often remarkably similar to those associated with migratory BMDCs such as macrophages and other myeloid-lineage cells (Pawelek 2005, Pawelek et al. 2006, Chakraborty and Pawelek 2003). Fusion of migratory BMDCs and cancer cells with co-expression of both fusion partner genomes provides a potential explanation for this phenomenon as first proposed by Munzarova et al. (1992). In our opinion the fusion theory comes closer to a unifying explanation of tumor progression than any yet proposed. Fusion represents a non-mutational mechanism that could explain the aberrant gene expression patterns associated with malignant cells. Studies of macrophage-tumor cell fusions have demonstrated that genes from both parental partners are expressed in hybrid cells (Chakraborty et al. 2001a). Gene expression in such cells reflects combinations of myeloid lineage genes along with those of the cancer cell lineage, all in a background of de-regulated cell division. In fact, many molecules and traits associated with tumor progression are expressed by healthy myeloid lineage cells, for example, angiogenesis, motility, chemotaxis and tropism, immune signaling, matrix degradation and remodeling, responses to hypoxia, and multidrug resistance to chemotherapy (Pawelek 2005, Pawelek et al. 2006). Tumor fusion could also account for aneuploidy and genetic rearrangements in metastatic cells (Duelli and Lazebnik 2007, Lu and Kang 2009, Pawelek 2000, 2005, Pawelek and Chakraborty 2008a, b, Pawelek et al. 2006). It is further possible

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that tumor-BMDC fusions are a source of cancer stem cells (Bjerkvig et al. 2005, Dittmar et al. 2009). This chapter reviews the molecular and cellular pathways activated following fusion of tumor cells with BMDCs, their expression in macrophages and other BMDCs, and their similarities to those governing tumor progression in animal and human cancer.

16.2 Cancer Cell Fusion In Vivo From studies in animal and human cancers there is little doubt that tumor hybrids are generated in vivo and that at least in animals they can be a source of metastases (Pawelek 2000, 2005, Pawelek and Chakraborty 2008a, b, Pawelek et al. 2006). Cancer cells fuse with many cell types in vivo including stromal cells (Jacobsen et al. 2006), epithelial cells (Rizvi et al. 2006) and endothelial cells (Bjerregaard et al. 2006, Mortensen et al. 2004, Streubel et al. 2004). There are more than 30 reports of tumor cell fusion with host cells and many of these implicate macrophages or other BMDCs as host fusion partners (Lu and Kang 2009, Pawelek 2000, 2005, Pawelek and Chakraborty 2008a, b, Pawelek et al. 2006). Though these earlier proposals were not widely cited, by the early 1980s experimental evidence for hybridization in cancer had been reported by several laboratories (Rachkovsky et al. 1998). The results fell largely into three categories: (a) tumor-host hybrids were observed naturally within tumors; (b) some naturally-occurring hybrids found within tumors showed enhanced metastatic potential; and (c) experimental hybrids produced in the laboratory between healthy white blood cells and weakly metastatic tumor cells also showed enhanced metastatic potential. The first report of spontaneous hybrid formation came from Barski and Cornefert (1962) who mixed together two separate lines of tumor cells and co-injected them into host C3H mice. Hybrid clones of cells were isolated from the resultant tumors and were found to be tumorigenic when injected back into C3H mice. Miller et al. (1988, 1989) also observed tumor x tumor hybrids after injecting mixed populations of tumor cells into host mice. Janzen et al. (1971) co-injected a mixture of two tumorigenic cell lines, a sarcoma and a lymphoma, into host mice and found by chromosome analyses that 3% of the cells within the developing tumor were hybrids. Similarly, Hart (1984) co-injected two separate sub-lines of B16 melanoma cells into host C57/B6 mice and isolated melanoma-melanoma hybrids from within the developing tumors. Hart was unable to detect fusion hybrids between host cells and the injected B16 melanoma cells. Aviles et al. (1977), using a trypsin-Giemsa banding technique for analysis of metaphase chromosomes, was able to identify hybrid clones in each of 14 mouse L cell sarcomas growing in C3H mice. About 90% of the hybrids were tumorigenic when injected back into host mice. In the first study to demonstrate a relationship between fusion and metastasis, Goldenberg et al. (1971, 1974) injected cells of a female human astrocytic glioma into the cheek pouch of male golden hamsters. Lethal metastases rapidly developed that were found upon chromosomal analyses to be composed of human × hamster hybrids. Wiener et al. (1972, 1974a) demonstrated that a highly tumorigenic mouse

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A9 sarcoma, when injected s.c. into mice regularly formed hybrids with host cells that were likely of hemopoietic origin. The hybrids were highly aggressive in vivo, in this case similar to the parental tumor cells. Kerbel et al. (1983) showed that methylcholanthrene-induced mouse sarcoma cells became highly metastatic after fusion in vivo with host cells of bone marrow origin. Larizza et al. (1984a, b), Larizza and Schirrmacher (1984) provided evidence that a highly metastatic variant of a low metastatic T-cell lymphoma was derived from spontaneous fusion with a host macrophage. The metastatic variant expressed the macrophage-specific antigen Mac-1 (CD18) that was not found on cells of the original tumor or any other of the tumors assayed except for a macrophage tumor line. They further showed that hybrids artificially generated with polyethylene glycol in vitro between the Eb T-cell lymphoma and bone marrow macrophages were highly metastatic and also expressed Mac-1. Fortuna et al. (1989, 1990) induced sarcomas in allophenic mice by treatment with methylcholanthrene and demonstrated that almost 1% of the tumor cells were hybrid in nature, with hybrid clones displaying both forms of the enzyme glucose phosphate isomerase. Clones of hybrids showed significantly increased lung colonization abilities compared to non-hybridized tumor cells isolated from the same cultures, although these traits diminished upon further in vitro passages. There is circumstantial evidence that hybrid formation occurs within human tumors. Atkin (1979) in a case study of human bladder cancer observed premature chromosome condensation (PCC) in about 6% of 284 tumor metaphases examined. PCC is a property of multinucleate cells, and Atkin postulated that this, in conjunction with increased ploidy seen in these cells, was evidence for fusions occurring between normal and malignant cells of the bladder, and that the normal cells were likely to be plasma cells that were present in the tumor in high number. Similar observations of PCC in human tumors were reported by Kovacs (1985). In a study of a freshly excised human ovarian tumor, Kerschmann et al. (1995) reported the presence of sarcomatoid cells that expressed both KP-1, a macrophage antigen, and cytokeratin, characteristic of carcinomas. The majority of the cells in the excised nodules were positive for only one or the other of the two markers. The authors concluded that this was evidence for macrophage × tumor cell hybridizations in vivo, and suggested that the presence of cholesterol crystals within the tumor might have catalyzed the fusions. Ruff and Pert (1984) reported that freshly excised human lung tumor cells, particularly those diagnosed as small cell carcinomas, expressed a number of macrophage-specific antigens, leading the authors to propose that such tumors are of hemopoietic stem cell rather than lung origin. Three reports describe spontaneous melanoma-host hybrids in mice: one of B16 melanoma cells (Hu and Pasztor 1975) and two of Cloudman S91 melanoma cells (Halaban et al. 1980, Chakraborty et al. 2000). In all three cases, the hybrids were hypermelanotic, showed increased dendricity and showed increased DNA content compared to the parental melanoma cells. All these lines showed higher tumorigenicity (Hu and Pasztor 1975, Halaban et al. 1980) and/or metastatic potential (Hu and Pasztor 1975, Chakraborty et al. 2000). We tested two of these hybrids and found that they produced ‘coarse melanin’, autophagosomes with

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multiple melanosomes. Further, when we fused mouse or human macrophages with Cloudman S91 melanoma cells in vitro, more than half of the 75 individual hybrids were of increased metastatic potential and autophagic (Rachkovsky et al. 1998, Pawelek et al. 2000, Rupani et al. 2004). Autophagy as a signature trait of cancer cell hybrids and human malignant melanomas alike is discussed later in this chapter. The hypermelanotic phenotype of our panel of macrophage-melanoma hybrids was puzzling. Why should fusion of a non-pigmented macrophage with a weaklypigmented melanoma cell yield highly pigmented hybrids? The short answer is aberrant glycosylation in the form of β1,6-branched oligosaccharides. Analyses of melanosomal proteins tyrosinase, TYRP-1, TYRP-2, and LAMP-1 in macrophagemelanoma hybrids showed that they were heavily glycosylated compared to parental melanoma cells. That LAMP-1 was one of these proteins provided the first indication that β1,6-branched oligosaccharides might be involved (Pawelek et al. 2000). LAMP-1 is one of the most heavily glycosylated of all proteins and is the chief substrate for GnT-V, (N-acetylglucosaminyltransferase V) that is rate-limiting in the formation of β1,6-branched oligosaccharides. GnT-V activates several pathways in metastastic progression. High GnT-V expression is a macrophage trait and it thus seemed likely that GnT-V might be elevated in macrophage-melanoma hybrids due to genetic input from the parental macrophage. Indeed, GnT-V and β1,6-branched oligosaccharides were elevated in the high metastatic hybrids (Chakraborty et al. 2001b). Use of glycosylation inhibitors and selective GnT-V inactivation in hybrids revealed that GnT-V expression was the underlying cause for both chemotactic motility and hyperpigmentation (Chakraborty and Pawelek 2007). It is possible that GnT-V itself induced formation of coarse vesicles. GnT-V transfection into mink lung cells induced production of LPHA-positive multilamellar vesicles and the process was dependent on autophagy (Hariri et al. 2000, Handerson and Pawelek 2003). Likewise, in macrophage-melanoma hybrids with high GnT-V expression, β1,6-branched oligosaccharides co-localized with coarse melanin vesicles (detected with the lectin LPHA) (Rupani et al. 2004). An example of melanoma cell fusion in vivo is seen in the development of a spontaneous melanoma metastasis to the lungs in a Balb c nude mouse (Chakraborty et al. 2000) (Fig. 16.1). Balb c mice are albino due to a homozygous mutation in tyrosinase (c/c), the rate-limiting enzyme in melanogenesis. Although the melanoma clone implanted into these mice was genetically wild type for tyrosinase (C/C), the cells produced little or no melanin in culture and formed amelanotic tumors in mice. Metastases, though infrequent, were generally small, amelanotic tumors in the lung, and were well-tolerated by the mice (Chakraborty et al. 2000). However, in one experiment a mouse developed a melanin-producing in transit metastasis near the site of implantation in the tail dermis. Because of this the tail was amputated and the mouse was followed to see if distant metastases developed. After 5 weeks the mouse became moribund with a massive, highly pigmented pulmonary metastasis. DNA analyses showed that cells from the metastasis had a genotype of C/c, indicating they were hybrids formed from fusion of the implanted tumor cells (C/C) with host cells (c/c). Cells from the metastasis showed an average 30–40% increase in DNA content, increased chemotaxis in vitro, activation of the glycosyltransferase

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Fig. 16.1 Spontaneous in vivo fusion in melanoma (Chakraborty et al. 2000). Cells from a clone of the Cloudman S91 mouse melanoma were implanted sub-cutaneously in the tail of a Balbc nu/nu mouse. The mice were albino due to a homozygous mutation in tyrosinase (c/c), the rate-limiting enzyme in melanogenesis. Although the melanoma clone was genetically wild type for tyrosinase (C/C), the cells produced little or no melanin in culture and formed amelanotic tumors in mice. Metastases, though infrequent, were generally small, amelanotic tumors in the lung, and were well-tolerated by the mice. In one experiment (designed for other purposes) what appeared to be a melanin-producing in transit metastasis developed (Panel a, asterisk) near the site of implant (bracket). The tail was amputated and the implanted tumor was formalin fixed, embedded in paraffin, and sectioned serially. Small numbers of highly melanized, coarse melanin-producing cells were found within the implanted tumor that were not seen in cultures of the parental melanoma cells and had thus been generated in vivo (Panel b, arrows). Five weeks after removal of the tail the mouse became moribund with a massive, highly pigmented pulmonary metastasis (Panel c, asterisk). Cells from the metastasis were cloned in soft agar. DNA analyses

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GnT-V, and production of β1,6-branched oligosaccharides (see below). They also produced coarse melanin/autophagosomes. Small numbers of highly melanized, coarse melanin-producing cells were found within the original implanted tumor. These were not present in the cultured parental melanoma cells and were thus generated in vivo (Chakraborty et al. 2000). Morphologically identical cells were cultured from the metastasis and determined to be C/c hybrids with host cells, indicating that fusion and hybridization had occurred in the original implant. Histopathology studies of the original implant revealed that it was infiltrated with macrophages, supporting the possibility that macrophage-tumor fusion had occurred there.

16.3 Tumor Associated Macrophages as Candidates for Cancer Cell Fusion Partners Munzarova et al. (1992) noted that a number of macrophage-like traits are expressed by metastatic melanoma and other malignancies and proposed that metastatic melanoma cells might be macrophge-melanoma hybrids. For example, Pernick et al. (1999) showed that human melanomas are often immunoreactive for macrophage markers such as CD68, alpha-1-antitrypsin, HAM56, Mac387 and muramidase. In breast cancer, Shabo et al. (2008) showed that expression of CD163, a macrophage scavenger receptor, is related to early distant recurrence and reduced patient survival. Tumor associated macrophages (TAM’s) facilitate both cancer initiation and progression (Balkwill et al. 2005, Lin et al. 2001, Mantovani et al. 2008, Pollard 2004). Macrophages are attracted through chemotactic signals to tumors where they exert their abilities for matrix degradation, tissue remodeling, stroma deposition, tropism and neoangiogenesis. These are normally employed in functions such as wound healing, osteogenesis, and embryogenesis (Pollard 2004). Since similar microenvironments exist within tumors, it is thought that macrophages become recruited to these “wounds that never heal” (Balkwill et al. 2005); or “tissues that never cease to develop” (Pollard 2004). Indeed macrophages are recruited to existing tumors by inflammatory cytokines and growth factors normally produced following wounding or infection (e.g. chemotactic chemokine CCL2; colony stimulating factor, CSF-1; vascular endothelial growth factor, VEGF-A)

Fig. 16.1 (continued) revealed that 12 of 12 randomly picked clones had a genotype of C/c, indicating they were hybrids formed from fusion of the implanted tumor cells (C/C) with host cells (c/c). Cells from the metastasis showed an average 30–40% increase in DNA content, increased chemotaxis in vitro, activation of the glycosyltransferase, GnT-V, and production of its enzymatic product, β1,6-branched oligosaccharides. Like the pigmented cells found in the primary implant (Panel b), they also produced “coarse melanin” – autophagosomes containing melanosomes and other organelles. Similar cells were cultured from the metastasis and were also seen in histolopathology sections of the pulmonary tumor. This indicated that the coarse melanin-containing cells originated in the primary implant through host-tumor cell fusion(s) (from Chakraborty et al. 2000)

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(Pollard 2004, Schioppa et al. 2003). Macrophages initiate neoplasia through release of reactive oxygen and nitrogen species that are mutagenic and carcinogenic (Lin et al. 2001). Tumor microenvironment cytokines – transforming growth factorβ1 (TGF-β1), interleukin-10 (IL-10), and macrophage-colony stimulating factor (M-CSF) – induce macrophages to differentiate from M1 to M2-type cells that produce tumor growth-promoting factors and stimulate angiogenesis (Balkwill et al. 2005). Macrophages accumulate in hypoxic regions of tumors through HIF-1-mediated upregulation of the chemokine receptor CXCR4 (Schioppa et al. 2003). The density of TAM’s has correlated with poor outcome in more than 80% of the human cancers studied, most notably in carcinomas of the breast, prostate, ovary, and cervix (Pollard 2004, Bingle et al. 2002). In these cancers potential benefits from TAM anticancer immune functions were apparently dominated by the TAM tumor-promoting abilities. This was demonstrated in a mouse mammary tumor model where mice carrying a null mutation for CSF-1 showed a marked reduction in TAM density. Mammary tumors developed normally in the null mutants but unlike those in wild type mice they tended not to metastasize (Lin et al. 2001). Thus, the presence of TAM’s was a key requirement for metastasis in this model. However, tumor progression is not completely explained by the presence of TAM’s. During transition to a metastatic phenotype, tumor cells notoriously co-opt leucocytic traits (Chambers et al. 2002, Pawelek 2005, Pawelek et al. 2006, Pollard 2004). Malignant cells are chemotactic, responding to chemokines and exhibiting their own matrix-degrading and angiogenic capabilities. Like migratory leucocytes, metastatic cells exhibit loss of homotypic adhesion, and the ability to transverse a basement membrane, migrate through the mesodermal matrix, intravasate into lymphatics or the blood circulatory system, extravasate from these vessels, and colonize lymph nodes and distant organs (Fidler 2003, Thiery 2002). But unlike normal leukocytes, cancer cells have deregulated mitotic cycles and their numbers continually increase, killing the host if left unchecked. During this process, invasive carcinomas and melanomas often lose differentiated traits such as E-cadherin expression, homotypic cell–cell adhesion, and cytokeratin or melanin production, while gaining mesodermal traits normally attributed to fibroblasts such as production of fibronectin and vimentin, loose adherence, mesenchymal motility mechanisms, and mesoderm-associated pathways such as the uPA/uPAR and HGF/cmet pathways (Friedl 2004, Kang and Massague 2004, Thiery 2003, Wang et al. 2004, Yang et al. 2004). This is known as the epithelialmesenchymal transition (EMT), and thought to be a process where cancer cells mimic the pathways through which the mesoderm is formed from the epithelium in early development (Friedl 2004, Kang and Massague 2004, Thiery 2003, Wang et al. 2004, Yang et al. 2004). A developmental connection to EMT in cancer was shown through analyses of transcription factors such as the Snail/Slug superfamily and Twist that control EMT in embryogenesis (Kang and Massague 2004). These factors regulate mesoderm formation during gastrulation, and were also associated with cancer progression (Friedl 2004, Kang and Massague 2004, Nieto 2002, Wang et al. 2004). It has thus been proposed that the complex processes in metastasis

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may be explained by the action of master regulatory genes normally associated with development (Chambers et al. 2002, Fidler 2003, Friedl 2004, Nieto 2002, Thiery 2002, Zhou et al. 2004). However, a uniform phenotype for EMT in cancer has not yet been described. Carcinomas and melanomas are notoriously heterogeneous, particularly as primary tumors (Clark et al. 1977, Ferlicot et al. 2004, Fidler and Hart 1982, Thiery 2002, Warner 1975). Many invasive and metastatic carcinomas and melanomas continue to produce cytokeratins or melanin, and not all invasive and/or metastatic carcinomas lose E-cadherin (Kerr 2004, Thiery 2003, Yang et al. 2004). Twist expression is not universal. In human breast carcinoma, Twist up-regulation is associated with invasive lobular carcinomas, but not with invasive ductal carcinomas, which make up 80% of breast cancers and which metastasize at a similar rate as the lobular (Kang and Massague 2004, Kerr 2004). If EMT defines tumor progression, why is it not expressed more uniformly? One explanation could be that EMT is transient: For example, metastases may regain differentiated traits in the process of colonizing lymph nodes or distant organs in a reversal process known as MET (mesenchymalepithelial transition) (Thiery 2003, Thiery and Morgan 2004, Wang et al. 2004, Yang et al. 2004) Another explanation could be that EMT is a consequence of tumor cell-myeloid cell fusion. Monocytes/macrophages and other myeloid cells are of mesenchymal origin, as shown in Drosophila, where double mutants in the mesoderm regulators Twist and Snail lack macrophages (Tepass et al. 1994). There is a growing list of myeloid-type traits that are shared by malignant cells. These include loss of homotypic adhesion, chemotactic motility, matrix degradation, immune signaling pathways, systemic migration, neoangiogenesis, and even multidrug resistance (Pawelek 2005, Pawelek et al. 2006). A key example is amoeboid motility, a characteristic of bone marrow-derived leukocytes, stem cells, and metastatic cancer cells alike. Amoeboid motility is required for migration through the stroma and dissemination via the circulatory system (Friedl and Wolf 2003, Wolf et al. 2003). In amoeboid motility, cells are highly deformable and because of their lack of stable focal adhesions can move at high velocities. The ability to undergo rapid shape-change allows for migration through tissue without the need to degrade matrix (Condeelis and Segall 2003, Friedl and Wolf 2003, Wolf et al. 2003). Moreover, monocytes/macrophages and malignant tumor cells both show high plasticity, such as the ability to differentiate into fibroblastic or endothelial-like cells and to exhibit vascular mimicry (Hendrix et al. 2003). This is mediated in part through FAK kinase, a monocyte/macrophageassociated enzyme whose expression is associated with both vascular mimicry and metastasis (Hess et al. 2005, McLean et al. 2005, Rovida et al. 2005). Similarly, neurotropins and neurotropin receptors are expressed by macrophages and are also associated with cancer anoikis-resistance and metastasis (Barouch et al. 2001, Geiger and Peeper 2005, Ricci et al. 2000). Melanoma and colon carcinoma cell lines express the macrophage-associated Toll Receptor-4 (TR-4) and are responsive to LPS (Molteni et al. 2006). The expression of Toll-like receptors could facilitate evasion of immune surveillance of metastatic cells (Huang et al. 2005, Molteni et al.

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Fig. 16.2 Malignant melanoma cells and tumor-associated macrophages (TAM’s) stained for three metastasis-associated markers: β1,6-branched oligosaccharides, GnT-V, and matriptase. Slides were bleached to decolorize melanin and stained by the immunoperoxidase reaction with the lectin LPHA for β1,6-branched oligosaccharides (A, tumor cells; B, TAM’s), with anti-GnT-V (C, tumor cells; D, TAM’s); or anti-matriptase (E, tumor cells; F, TAM’s). TAM’s were further verified by S100/azure blue staining (not shown). All fields were from the same tumor. (Handerson T and Pawelek J, unpublished)

2006). Cancer cells and macrophages both express multidrug-resistance proteins (ABC transporters) such as p-glycoprotein and other MDR proteins that confer chemotherapeutic resistance (Breier et al. 2005, Jorajuria et al. 2004, Michot et al. 2004).

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To illustrate marker co-expression, melanoma cells and TAM’s from the same histological section of a metastatic melanoma are each shown expressing GnT-V (E.C.2.4.1.155; N-acetylglucoseaminyltransferase V), β1,6-branched oligosaccharides, and matriptase, a GnT-V substrate (Fig. 16.2). These markers play key roles in both macrophage and cancer cell migration, and all three are prognostic indicators for metastasis and poor outcome in human cancers (Fernandes et al. 1991, Handerson et al. 2005, Handerson and Pawelek 2003, Ihara et al. 2004, Kang et al. 2003). Their high expression must have been acquired at some point during or following neoplastic transformation, since normal cutaneous melanocytes were negative.

16.4 BMDCs in Human Cancer and Stem Cell-Like Distribution Patterns The first confirmation of BMDC-tumor cell fusion in humans was reported where transcriptionally active malignant nuclei and normal nuclei were observed in tumorassociated osteoclasts from myeloma patients. In the osteoclast population, 30% of the nuclei were of malignant cell origin, indicating a remarkably high incidence of osteoclast-tumor cell fusion (Andersen et al. 2007, 2009). The potential relevance of this finding to myeloma pathobiology is not yet known. Other studies below have demonstrated the presence of donor genes in carcinoma cells of secondary malignancies arising after allogeneic bone marrow transplant, however for largely technical reasons, definitive proof for or against donor-host fusion was lacking in each. In the first reported case, a renal cell carcinoma (rcc) developed in a child following a bone marrow transplant from his cancer-free brother (Chakraborty et al. 2004). A lymph node metastasis of this tumor (the only tissue available) was analyzed by laser capture microscopy of tumor cells and PCR-based analyses for donor genes. Carcinoma cells throughout the tumor contained the donor-specific A allele of the ABO blood group indicating that BMDCs had in some manner become incorporated into the tumor. The patient history of radiation and immunosuppression prior to transplant increased the likelihood that the tumor arose de novo in the patient and that donor BMDCs became incorporated via fusion with preexising tumor cells. However, because a suitable patient-specific DNA sequence was unavailable, evidence for donor and patient genes in the same cells was lacking (Chakraborty et al. 2004). In the second such case (Yilmaz et al. 2005), tumor cells from a primary papillary rcc (prcc) arising after a male to female HSC transplant were found to exhibit a trisomy 17, a common abnormality in prcc and other cancers (Salama et al. 2003). About 1% of the trisomy 17-containing tumor cells also contained the donor Y chromosome in the same nucleus (Yilmaz et al. 2005). As above, this combined with the patient history suggested that fusion had occurred between tumor cells and donor HSC cells after development of the tumor (Yilmaz et al. 2005). However the possibility that the tumor was derived solely from a donor BMDCs, without fusion, followed by growth and widespread loss of the Y was not ruled out (Lau et al. 2007). Nonetheless Y-containing carcinoma cells were rarely

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Fig. 16.3 BMDC-engrafted renal carcinoma cells in pairs and clusters. H&E-stained renal carcinoma cells were co-localized to their Y chromosome-containing nuclei visualized by FISH (red). (∗ ) = trisomy 17-containing nuclei (green). Panels a and b show pairs of carcinoma cells containing one nucleus with a trisomy 17 and the other with a monosomy 17. Panel c shows a cluster of Y chromosome-containing carcinoma cells. In the H&E-stained sections, carcinoma cells were distinguished from normal cells by their large size, polygonal shape, abundant granular eosinophilic cytoplasm, and round or ovoid nuclei, usually located centrally, with fine chromatin. Verification of carcinoma cells was by pathologists. Asterisks (∗ ) denote nuclei containing a trisomy 17. Y chromosome: red; chromosome 17: green

found alone but in pairs resembling post-mitotic daughter cells and clusters suggesting a clonal origin of the cells (Fig. 16.3a–c). Should they have been daughter cells, it would also appear that the mitotic division had been asymmetric since many cases one of the Y-containing nuclei contained a trisomy 17 (∗ ) while the other contained a monosomy 17. Although it could not be proven that they were daughter cells or clonal clusters, the observations raise the possibility that they might have been mitocally-active cancer stem cells. (Bjerkvig et al. 2005, Dittmar et al. 2009, Guo and Lasky 2006) (Fig. 16.3). Also, Y-containing carcinoma cells were localized to a region covering only about 10% of the tumor, suggesting a clonal emergence of

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these cells. Supporting this, Y-containing carcinoma cells differed from the majority of carcinoma cells in this tumor through their high expression of β1,6-branched oligosaccharides, a risk factor in several cancers (below).

16.5 Cancer Cell Fusion and the Hybrid Phenotype Fusion-induced enhancement of metastasis and a differentiated trait such as melanin production is in contrast to previous studies where hybrids formed in vitro between normal epithelial cells or fibroblasts and tumorigenic cancer cells were generally suppressed in tumorigenicity compared to the parental cancer cells (Harris 1988, Herzog et al. 2007, Ramshaw et al. 1983, Sidebottom 1980, Stanbridge 1976, Weinberg 1991, Wiener et al. 1974b), with some exceptions (Levine 1995, Scaletta and Ephrussi 1965). These observations lead to the concept- and subsequent identification of a number of different tumor suppressor genes, that have been largely involved in control of progression through the cell cycle (Harris 1988, Weinberg 1991). Differentiated traits were also suppressed in such hybrids. For example, polyethylene glycol (PEG)- and Sendai virus-induced hybrids between fibroblasts and pigmented, tumorigenic melanoma cells were non-pigmented and non-tumorigenic (Davidson et al. 1966, Defendi et al. 1967, Gourdeau and Fournier 1990, Jonasson et al. 1977, Powers and Davidson 1996, Powers et al. 1994). The tendency of hybrids to lose chromosomes with successive cell divisions was exploited for chromosomal mapping of suppressor genes. However, when healthy leukocytes were used as fusion partners with cancer cells, co-activation of differentiated functions between parental genomes was seen, e.g. in leukocyte-hepatoma hybrids (Darlington et al. 1974, Malawista and Weiss 1974), leukocyte-myeloma hybrids (Giacomoni 1979), immunoglobulin-secreting hybridomas (Kohler and Milstein 1975), and macrophage-melanoma hybrids discussed herein. Thus, unlike tumor-suppressive fibroblasts and epithelial cells, hematopoietic cells enhanced malignancy and differentiation when hybridized with transformed cells. Expression of genes from both parental lineages in cancer cell hybrids could explain many properties of metastatic cells (Pawelek 2000, 2005, Pawelek and Chakraborty 2008a, b, Pawelek et al. 2006). For example, tropism to lymph nodes and organs and tissues such as bone marrow, brain, lung, and liver is a common trait of macrophages and metastatic cells alike. Likewise, the notorious multidrug resistance of malignant cells to chemotherapy due to high levels of p-glycoprotein (Gottesman and Ling 2006) could reflect that macrophages also express this phenotype (Lemaire et al. 2007).

16.6 Macrophage-Melanoma Fusion In Vitro Generates Altered Gene Expression and a Metastatic Phenotype In Vivo Tumor-BMDC fusions might explain how common gene expression patterns emerge for different tumor types. We, and others, have found that when BMDC-tumor cell hybrids were isolated in vitro with no selective pressure other than for

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Fig. 16.4 Metastatic potential of macrophage-melanoma hybrid cell lines compared to parental Cloudman S91 melanoma cells. Results are shown for in vitro-generated hybrids and one spontaneous in vivo hybrid (PADA). Melanin was estimated in pelleted cells from clones on their first passage in culture and before metastatic potential was determined. A minimum of 10–20 animals was tested for each clone. In addition representative clones were tested repeatedly during continuous passage in culture for up to 4 years where 30–90 animals were tested for each clone with similar results as above. Statistical analyses of metastatic potential revealed that p values for significance vs parental melanoma cells were < 0.0001 (∗ ); < 0.01 (dagger); < 0.05 (square)

growth in drug-containing media, remarkably high numbers of them exhibited a metastatic phenotype in mice. Further, the most metastatic clones tended to be highly melanized compared to parental melanoma cells or weakly metastatic hybrids (described below) (Fig. 16.4). In two separate rounds of isolation, a total of 75 clones of PEG-fused macrophage-melanoma hybrids were isolated in vitro. About half showed increased chemotaxis in vitro and metastasis in mice (Rachkovsky et al. 1998, Rachkovsky and Pawelek 1999, Pawelek et al. 2000). Similar results were obtained in T-cell hybridomas from fusion of healthy T-lymphocytes with T-lymphoma cells (Roos et al. 1985) and in hybrids between mouse T-cell

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lymphoma cells and bone marrow-derived macrophages or spleen lymphocytes, (Larizza et al. 1984a, b). High frequency emergence of a common metastatic phenotype in vitro without host selective pressure was surprising, particularly in view of the apparently chaotic nature of aneuploidy. In fact little is known of the regulation of gene expression in hybrids at the molecular genetic level. Evidence that BMDC-tumor hybrids express many of the same genes associated with invasive and metastatic cancers and that these genes are also expressed by macrophages and other migratory BMDCs is summarized below (also reviewed in (Pawelek and Chakraborty 2008a, b)).

16.6.1 SPARC SPARC (secreted protein acidic and rich in cysteine; osteonectin; BM40) is a modulator of cell-matrix interactions during development and is a key component of wound healing, tissue repair and hard tissue formation (Bradshaw and Sage 2001, Lane and Sage 1994). SPARC modulates cellular shape and as such is a counteradhesive factor (Bradshaw and Sage 2001). SPARC binds to several proteins of the extracellular matrix and is also a chaperone aiding proper folding of collagen in the endoplasmic reticulum (Martinek et al. 2007). In development, SPARC is expressed in late gastrulation during differentiation of invaginated epithelial cells into mesoderm (Damjanovski et al. 1998). Interestingly, SPARC is important in osteoclast formation (Fugita et al. 2002, Mansergh et al. 2007). In tissue macrophages SPARC is expressed in regions of neovascularization, for example in wound repair (Reed et al. 1993) and degenerative aortic stenosis (Charest et al. 2006). High SPARC expression is associated with tumor progression and poor outcome in melanoma and a number of carcinomas including breast, colorectal, ovarian and lung (Robert et al. 2006). SPARC acts as a regulator of melanoma EMT by downregulating melanoma E-cadherin with loss of homotypic adhesion, stimulates motility, and increases expression of mesenchymal markers such as matrix metalloproteinase MMP-9 (Alonso et al. 2007). The actions of SPARC are mediated through Snail, a transcription factor in the initiation of EMT during normal development and cancer (Barrallo-Gimeno and Nieto 2005). The SPARC gene provides an example of gene regulation in BMDC-tumor fusion. In fusions between mouse macrophages or human blood monocytes and weakly metastatic mouse Cloudman S91 melanoma cells, unfused melanoma cells, macrophages and monocytes all expressed SPARC mRNA, however the levels were threefold to fourfold higher per μg total RNA in hybrids (Kerbel et al. 1983, Chakraborty and Yamaga 2003). SPARC mRNA levels were highest in hybrids of high metastatic potential and lowest in weakly metastatic hybrids and parental melanoma cells. Moreover, hybrids between human monocytes and mouse melanoma cells expressed both human and mouse SPARC mRNA (Chakraborty et al. 2001a). This indicated that genomes from cells of the two different developmental lineages were both activated. Thus, for SPARC: gene expression was enhanced by hybridization of tumor cells with macrophages; high expression was

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correlated with high metastatic potential; and SPARC mRNA was produced in hybrids from the genomes of both parental fusion partners. That elevated SPARC expression was a characteristic of macrophage-melanoma hybrids provides a possible explanation for elevated SPARC and SPARC-mediated pathways in human melanoma and other cancers. It is not known whether other regulators of EMT and development in addition to SPARC were expressed in macrophage-tumor cell fusion hybrids (transcription factors Twist, Snail, and others (Gupta et al. 2005, Ma et al. 2007). However at least one, Twist, is activated in macrophages and regulates inflammatory cytokine production (Sharif et al. 2006, Sosi et al. 2003). By analogy to SPARC, this opens the possibility that Twist expression in some invasive carcinomas reflects expression of macrophage-lineage genes following macrophage-tumor cell fusion.

16.6.2 MCR1 and c-Met The melanocortin-1 (MC1, melanocyte stimulating hormone) receptor (MC1R) is activated by MC1 in healthy melanocytes and melanoma cells where, through cyclic AMP-dependent mechanisms, it activates melanogenesis and regulates proliferation along with several other actions (Carlson et al. 2007, Kanetsky et al. 2006). MC1R appears to play a role in melanoma progression at least in part through its activation of the proto-oncogene cMet, whose signalling pathway is a key regulator of metastasis in melanoma and many other cancers (Beuret et al. 2007, Boccaccio and Comoglio 2006, McGill et al. 2006). As with SPARC, gene expression for both MC1R and c-Met was increased in high metastatic macrophage-melanoma hybrids (Chakraborty et al. 1999, 2003). Moreover, each was involved in the induction of chemotactic motility in hybrids (Chakraborty et al. 1999, Rachkovsky and Pawelek 1999). Up-regulated MC1R mRNA expression in hybrids was associated with increased cellular binding of its ligand MC1, and amplified responsiveness to MC1 as shown by increased chemotactic motility, dendricity, and melanization (Pawelek et al. 2000, Rachkovsky and Pawelek 1999). Exposure of hybrids to MC1 also increased both the production of c-Met mRNA and responsiveness to HGF as a chemoattractant (Chakraborty et al. 2003). Thus the MC1/MC1R and HGF/c-Met pathways appeared to act coordinately in a positive autocrine loop to control chemotaxis and other functions in hybrid cells. This same relationship appears to be operative in malignant melanoma (Beuret et al. 2007). In melanoma, c-Met and MC1R are each regulated through the master transcription factor MITF (McGill et al. 2006) which itself is associated with tumor progression (Levy et al. 2006). Although it was not determined whether MITF was upregulated in experimental macrophage-melanoma hybrids, this appears to have been the case since mRNA’s for both c-Met and MC1R were elevated, an expected consequence of increased MITF (Bronisz et al. 2006, Garraway et al. 2005, McGill et al. 2006). High expression of MITF (Beilmann et al. 1997), c-Met (Gaasch et al. 2006, Lam et al. 2005), and MC1R (Lam et al. 2006, Manna et al. 2006, McGill et al. 2006, Taylor 2005) are all characteristics of monocytes/macrophages and other BMDCs.

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16.6.3 GnT-V and β1,6-Branched Oligosaccharides N-acetylglucosaminyltransferase V (GnT-V; Mgat5; E.C.2.4.1.155) is a Golgi complex enzyme that is highly expressed in myeloid cells and metastatic cancer cells. GnT-V and its enzymatic products, β1,6-branched oligosaccharides conjugated to N-glycoproteins, are associated with poor outcome in melanoma (Handerson et al. 2007) and carcinomas of the breast (Fernandes et al. 1991, Handerson et al. 2005), colon (Fernandes et al. 1991, Murata et al. 2000, Seelentag et al. 1998) lung (Dosaka-Akita et al. 2004) and endometrium (Yamamoto et al. 2007). β1,6-branched oligosaccharides were first purified from granulocytes (Fukuda et al. 1984). From structural analyses they are composed of poly-N-acetyllactose amines that are carriers of sialyl lewisx antigen (sialyl lex) and therein used by both leukocytes and metastatic cancer cells for binding to E-selectin and/or galectin-3 on endothelial cells during systemic migration (Sarafian et al. 1998, Sawada et al. 1993). GnT-V mRNA, protein, and/or enzymatic activity were elevated in high metastatic macrophage-melanoma hybrids formed in vitro (Chakraborty et al. 2001b), and following spontaneous host-tumor fusions in both lymphomas and melanomas in mice (Chakraborty et al. 2001b, Dennis et al. 1984, Kerbel et al. 1983). Multiple pathways in invasion and metastasis that are regulated by GnT-V were elevated in macrophage-melanoma hybrids – as seen below with motility-associated integrin subunits, cell surface expression of LAMP-1, and autophagy.

16.6.4 Motility-Associated Integrins The integrin subunits α2, α3, α5, α6, αv, β1, and β3 are all involved with migration of leucocytes and cancer cells. These same integrin subunits were significantly upregulated at the protein level in metastatic macrophage-melanoma hybrids compared to weakly metastatic hybrids and parental melanoma cells (Chakraborty et al. 2001b, Chakraborty and Pawelek 2010). Following stimulation with melanocortin-1, protein levels were further increased in high metastatic hybrids. These results correlated with findings that metastatic hybrids had acquired an MC1-inducible chemotactic phenotype that was directed toward fibronectin through the action of integrin α5β1 (Rachkovsky and Pawelek 1999). Of great interest, all the above subunits have been identified as substrates for GnT-V and their actions are strongly affected by their glycosylation status with β1,6-branched oligosaccharides (Chammas et al. 1993, Demetriou et al. 1995, Dennis et al. 1999, Guo et al. 2002, Jasiulionis et al. 1996, Leppa et al. 1995, Ochwat et al. 2004, Poche et al. 2003, Saitoh et al. 1992, Yamamoto et al. 2000, Zheng et al. 1994). For example, addition of β1-6 branched oligosaccharides onto the β1 integrin subunit by GnT-V reduced α5β1 integrin clustering and stimulated cell migration (188). Further, the above integrin subunits are each involved in metastasis. The α3β1 integrin is elevated and associated with increased migration and invasion in several types of metastatic cancers (Giannelli et al. 2002). α5β1 is a well-characterized receptor

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for fibronectin that is over-expressed in metastasis (Danen et al. 1994, Galbraith et al. 2007, Natali et al. 1995). Up-regulation of αvβ3, a vitronectin receptor, was described in various cancers including malignant melanoma and glioblastoma (Danen et al. 1994, Gladson and Cheresh 1991, Natali et al. 1997, Wong et al. 1998). Expression of the β1-integrin subunit is a key component of melanoma metastasis (Juliano 1993). The above integrins and integrin subunits are also highly expressed in macrophages where they are involved with many functions, including cell adhesion and migration, signal transduction, cell–cell recognition and phagocytosis (Ammon et al. 2000, Aplin et al. 1998, Elsegood et al. 2006, Kurita-Taniguchi et al. 2002, Shinji et al. 2007).

16.6.5 Cell Surface Expression of Lysosome Associated Protein-1 (LAMP-1) LAMP-1 is a preferred substrate for GnT-V and a major carrier of sialyl lex and poly-N-acetyl-lactose amines that bind to E-selectins and galectins (Chang et al. 2004). Cell surface LAMP-1 thus mediates binding to endothelial cells by both leukocytes and cancer cells (Chang et al. 2004, Sarafian et al. 1998, Sawada et al. 1993). Macrophge-melanoma hybrids showed elevated expression of cell surface LAMP-1 (Chakraborty et al. 2001b). This was seen in high metastatic macrophagemelanoma hybrids as well as peritoneal macrophages compared to that in parental melanoma cells and low metastatic hybrids.

16.6.6 Autophagy and Coarse Melanin As mentioned, the spontaneous mouse melanoma-host hybrid shown in Fig. 16.1 showed a high level of autophagy/coarse melanin (Chakraborty et al. 2000). This was also a characteristic of another spontaneous melanoma-host hybrid described previously (“PADA”) (Pawelek et al. 2000) and of macrophage-melanoma hybrids fused in vitro (Chakraborty et al. 2000, Pawelek et al. 2000, Rachkovsky et al. 1998). EM studies revealed that melanin was localized largely to heavily melanized melanosomes packaged in autophagosomes. Autophagosomes were verified by the presence of double limiting membranes and heterogeneous morphologies. They were also strongly positive for β1,6-branched oligosaccharides, implicating a role for GnT-V in their formation. These were surprising findings because healthy melanocytes do not appear to employ GnT-V in melanogenesis and the melanosomes are not packaged in autophagosomes but exist singly in the cytoplasm. That several independently-isolated melanoma hybrids all showed high levels of autophagy/coarse melanin raised the question as to whether this trait may be a signature of BMDC-melanoma fusion in human melanoma. While coarse melanin in melanoma had been known to pathologists for more than a century, and was shown to be due to autophagy (reviewed in Handerson and Pawelek 2003), its

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frequency in human cancers had not been evaluated (Clark et al. 1977). Analyses of several hundred cases have revealed that it is a common trait, expressed by 85% or more of melanomas (Handerson and Pawelek 2003, Handerson et al. 2007). It was further determined that coarse melanin-producing melanoma cells and melanophages (macrophages with autophagolysosomal vesicles containing undigested melanin) account for the well-known hypermelanotic regions of cutaneous malignant melanoma used in clinical diagnosis. As in macrophage-melanoma hybrids, coarse melanin vesicles in human melanomas contained β1,6-branched oligosaccharides (Handerson and Pawelek 2003, Handerson et al. 2007). In cutaneous malignant melanoma, β1,6-branched oligosaccharide-positive, coarse melanin-producing melanoma cells emerge clonally as “nests” within the in situ tumor and have the capacity for invasion into the dermis (Handerson and Pawelek 2003, Handerson et al. 2007). This suggests that BMDC-tumor cell fusion could be an explanation for the appearance of these cells. These findings prompted us to explore the levels of autophagy in human malignancies. Summarized below are our first observations of autophagy in a panel of cutaneous malignant melanomas.

16.6.7 Autophagy in Cutaneous Malignant Melanoma Twelve cases of superficial spreading melanoma (SSM) with both MIS and invasive component, and one case (#13) of acral lentigenous melanoma (ALM) with residual MIS but no invasive component were selected for the study. Patients ranged in age from 22 to 84 years with a median of 50 years. Eight patients were men and five were women. The lesions were located on the back (n = 4), leg (n = 5), chest (n = 2), abdomen (n = 1), and upper arm (n = 1). The depth of the melanomas ranged from 0.4 to 1.3mm with a median of 0.7mm. Eight melanomas were Clark’s level IV, four were Clark’s level III, and one was Clark’s level I. While all 12 cases of SSM had both MIS and invasive component, only four of them, cases 1, 7, 8, and 11, could be fully evaluated comparing the staining of normal epidermis, early MIS, and florid MIS within the same section. The remaining cases showed only florid MIS with little or no normal epidermis. Case #1 was selected as a representative case to illustrate the staining patterns. Sections stained with H&E from case #1 are shown in Fig. 16.5. Normal epidermis, peripheral to the area containing melanoma in situ, displayed a normal number and distribution of melanocytes in the basal layer (Fig. 16.5a). Adjacent to the normal epidermis was an area containing an irregular proliferation of single pleomorphic melanocytes in the basal layer and above the dermal epidermal junction (DEJ), representing early MIS (Fig. 16.5b). Contiguous with that focus and more towards the center of the lesion was florid MIS comprised of irregular nests of melanocytes in the epidermis, which varied in size and shape. An underlying invasive component of melanoma in the dermis displayed large atypical melanocytes in a nested pattern (Fig. 16.5c) Normal epidermis adjacent to MIS: In areas of the epidermis adjacent to a region of MIS, normal keratinocytes and melanocytes (asterisk) could readily be

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Fig. 16.5 Views from an H&E-stained sections of malignant melanoma in case #1. (a) Normal epidermis containing only a few melanocytes in the basal layer (∗ ). (b) Focus of early MIS showing an increased number of slightly pleomorphic single melanocytes at the dermal epidermal junction (DEJ) and above it. (c) Florid MIS in the epidermis with nests of melanocytes displaying irregular shapes, as well as single melanocytes, many of which are seen above the DEJ. Invasive melanoma, comprised of nests of atypical melanocytes, is present in the dermis

distinguished on H&E staining (Fig. 16.6a). Melanin was not apparent in normal melanocytes; however, it was evident that melanogenesis had occurred since adjacent keratinocytes contained brown melanin in their cytoplasm, presumably transferred from melanocytes. Normal melanocytes and keratinocytes did not stain for LC3B (Fig. 16.6b). Staining with the anti-Golgi 58k protein revealed large, globular, perinuclear Golgi complexes in both keratinocytes and melanocytes (Fig. 16.6c). There was no staining with LPHA in normal keratinocytes and melanocytes, confirming previous findings that these cells do not appear to produce β1,6-branched oligosaccharides (Fig. 16.6d) (Handerson and Pawelek 2003). Early MIS. Early MIS presents as a subtle proliferation of atypical melanocytes (melanoma cells) disposed as single units as well as in a few small nests at the

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Fig. 16.6 Medium and high power views of the staining patterns for H&E, LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides in sequential sections of normal epidermis from malignant melanoma case #1. Melanocytes are denoted with an asterisk (∗ ). Before staining, sections b–d were first bleached to decolorize melanin. Sections were subsequently stained for the specific marker and counterstained with hematoxylin. (a) An H&E stained section showing a normal number and distribution of melanocytes in the basal epidermal layer. (b) A section stained with anti-LC3B displaying negative staining with this marker. (c) A section stained with anti-Golgi 58k protein. In the higher power photomicrograph the brown globules represent the positively stained Golgi complex, sometimes overlapping and obscuring the nucleus. (d) A section stained with the lectin LPHA for β1,6-branched oligosaccharides showing negative staining

dermal epidermal junction and above it. Similar to normal melanocytes, melanoma cells in regions of early MIS did not show prominent melanin in their cytoplasm as seen with H&E staining (Fig. 16.7a). Cells of early MIS did not stain or stained only weakly for LC3B (Fig. 16.7b) and for β1,6-branched oligosaccharides (Fig. 16.7d). However, similar to normal melanocytes they did stain for the Golgi 58k protein in a globular perinuclear pattern, (Fig. 16.7c). Florid MIS. In florid MIS there is an irregular, asymmetric, and poorly circumscribed proliferation of melanoma cells. There are nests of melanoma cells that vary markedly in size and shape, which are not equidistant from one another. Single melanoma cells predominate over nests in some high power fields and there are individual melanoma cells as well as melanocytic nests above the DEJ. Melanoma cells are also seen down adnexal structures. In our study melanoma cells in florid MIS of all 13 cases produced coarse melanin to at least some extent (Fig. 16.8a). In addition, in all cases most if not all of the cells of florid MIS stained for the autophagosome marker LC3B with a heterogeneous vesicular pattern in the cytoplasm, indicating the presence of autophagosomes (Fig. 16.8b). Surprisingly, the Golgi 58k protein was distributed not in a globular perinuclear pattern, characteristic of normal cells and early MIS (above), but in a heterogeneous vesicular pattern, similar to that of coarse melanin and LC3B (Fig. 16.8c). In previous studies such a vesicular pattern for Golgi staining has been described as Golgi “fragmentation” or “vesiculation” (Dagher et al. 2003, Graves et al. 2001, Kovacs et al. 2004, Razi et al. 2009). Unlike melanocytes in normal epidermis and melanoma cells in early MIS, the nested melanoma cells in florid MIS produced β1,6-branched oligosaccharides, which, like the LC3B and Golgi 58k protein, also stained in a heterogeneous

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Fig. 16.7 A region of early MIS from malignant melanoma case #1 showing crowded melanocytes (melanoma cells) near the dermal epidermal junction. (a) A section stained with H&E. (b) A section bleached to decolorize melanin, subsequently stained with anti-LC3B, and counterstained with hematoxylin, showing faint staining for LC3B. (c) A section bleached, subsequently stained with anti-Golgi 58k protein, and counterstained with azure blue with a positive perinuclear staining. (d) A section bleached, subsequently stained with biotinylated LPHA, and counterstained with hematoxylin showing negative staining

vesicular pattern (Fig. 16.8d) (Handerson and Pawelek 2003, Handerson et al. 2007). Compared to the melanoma cells in small nests of florid MIS (Fig. 16.8), melanoma cells in larger nests tended to produce more prominent coarse melanin and stain more intensely for LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides (Fig. 16.9a–d). Thus, in florid MIS most if not all of the melanoma cells had autophagosomes. The autophagosomes contained LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides. This was confirmed through co-localization studies with coarse melanin and through electron microscopy. Co-localization of the Golgi 58k protein and β1,6-branched oligosaccharides with coarse melanin. Fresh tissue containing epidermis and dermis from case #13 of acral lentiginous melanoma with residual MIS was obtained. Half of the specimen

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Fig. 16.8 Small nests of melanoma cells from florid MIS in malignant melanoma case #1. Sections b and d were bleached, stained with antibody or lectin, and counterstained with hematoxylin. Section c was not bleached, but directly stained with the antibody and counterstained with azure blue. Arrows denote coarse melanin (a) and vesicular staining pattern (b–d). (a) A section stained with H&E. (b) A section stained for LC3B. (c) A section stained for the Golgi 58k protein. (d) A section stained for β1,6-branched oligosaccharides with biotinylated LPHA

was processed for electron microscopy and the other half was fixed in formalin and processed in a standard fashion. Tissue sections were cut and stained with H&E for light microscopic examination and immuno- and lectin histochemistry was performed. Histologic examination of H&E stained sections revealed florid MIS comprised of nests of melanocytes and single melanoma cells containing coarse melanin in their cytoplasm (Fig. 16.10). As mentioned, there was no invasive melanoma component in this case. Co-localization studies in case #13 were performed by photographing coarse melanin in unstained sections and then staining the same slides for either LC3B, the Golgi 58k protein, or β1,6-branched oligosaccharides (Fig. 16.11). Sequential sections were also bleached to decolorize melanin before staining with the same results (not shown). Photographs of coarse melanin-containing melanocytes were compared to photographs of the same melanocytes after staining. Widespread co-localization was found between coarse melanin and LC3B

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Fig. 16.9 Medium and high power views of large nests of melanoma cells within florid MIS from case #1. Sections b and d were bleached, stained with antibody or lectin, and counterstained with hematoxylin. Section c was not bleached, but directly stained with the antibody, and counterstained with azure blue. Arrows denote coarse melanin (a) and vesicular staining pattern (b–d). (a) A section stained with H&E. (b) A section stained for LC3B. (c) A section stained for the Golgi 58k protein. (d) A section stained for β1,6-branched oligosaccharides with biotinylated LPHA

Fig. 16.10 A region of florid MIS with melanoma cells in the epidermis producing coarse melanin (arrow) and melanophages in the superficial dermis also containing granular melanin in their cytoplasm (Case #13; H&E)

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Fig. 16.11 Co-localization of LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides to coarse melanin. The columns labeled “melanin” represent unstained sections photographed to depict coarse melanin. The sections on the right represent the same areas after staining with antibody or lectin. (a–d) Co-localization of LC3B to coarse melanin. (e–h) Co-localization of the Golgi 58k protein to coarse melanin. (i–l) Co-localization of β1,6-branched oligosaccharides to coarse melanin

(Fig. 16.11a–d), the Golgi 58k protein (Fig. 16.11e–h), and β1,6-branched oligosaccharides (Fig. 16.11i–l). Detection of autophagosomes by electron microscopy. A region of florid MIS from case #13 was analyzed by electron microscopy. Numerous vesicles surrounded by double membrane and filled with heavily melanized melanosome-like structures and other debris were seen (Fig. 16.12). The melanosomes seemed to be partially digested and might have represented only residual, undigested melanin; however,

Fig. 16.12 Electron micrographs of coarse melanin vesicles in a region of florid MIS from Case #13 (Fig. 16.6). (a) Low power view showing vesicles with heavily melanized melanosome-like structures. b and c High power views of individual vesicles. The arrows show that the vesicles are bordered by double membranes and by definition represent autophagosomes

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that remains to be further elucidated. Such double-membraned vesicles containing cytoplasmic organelles are defined as autophagosomes (Klionsky et al. 2008). Thus, we conclude that coarse melanin vesicles are autophagosomes containing LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides (Fig. 16.11).

Fig. 16.13 Medium and high power views of the dermal invasive component from malignant melanoma case #1. Sections b and d were bleached, stained with antibody or lectin, and counterstained with hematoxylin. Section c was not bleached but directly stained and then counterstained with azure blue. Arrows denote coarse melanin (a) and vesicular staining pattern (b–d). (a) A section stained with H&E. (b) A section stained for LC3B. (c) A section stained for the Golgi 58k protein. (d) A section stained for β1,6-branched oligosaccharides with biotinylated LPHA

Fig. 16.14 LC3B staining in the invasive components of malignant melanoma cases #1–12. Case #2 was stained with anti-LC3B and counterstained with azure blue. The remaining cases were first bleached to decolorize any melanin, stained with anti-LC3B, and counterstained with hematoxylin

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Invasive melanoma cells. While coarse melanin in florid MIS was present in all cases to some extent, only 4 of the 12 cases showed coarse melanin in the invasive component (cases 4, 5, 7, and 11). The invasive components of the remaining cases were only lightly pigmented or amelanotic as shown for case #1 (Fig. 16.13a). Regardless of the presence or not of coarse melanin, the invasive melanoma cells in all cases revealed positive heterogeneous vesicular pattern of staining for LC3B (Fig. 16.13b), the Golgi 58k protein (Fig. 16.13c), and β1,6-branched oligosaccharides (Fig. 16.13d), seemingly identical to that seen within cells of florid MIS (cf Figs. 16.8 and 16.9). Therefore, it appeared that the vesicles produced in the invasive components were also autophagosomes but with reduced melanin content. The melanoma cells in the invasive component of all 12 cases showed positive staining, for the autophagosome marker LC3B (Fig. 16.14) and the Golgi 58k protein (Fig. 16.15). The staining for both markers displayed a heterogeneous vesicular pattern. The same pattern was also seen for β1,6-branched oligosaccharides in all 12 cases of invasive melanoma (not shown). (Handerson and Pawelek 2003). Melanophages. Melanophages are macrophages filled with melanized vesicles that appear similar under light microscopic examination to coarse melanin seen in melanoma cells. These vesicles are presumably generated from engulfment via phagocytosis of cells containing melanin, followed by transfer of the engulfed cellular debris into autophagosomes. Our study revealed that melanophages express an

Fig. 16.15 Anti-Golgi 58k protein staining in the invasive components of malignant melanoma cases #1–12. Cases 1, 2, 3, 4, 7, 11, and 12 were stained with the anti-Golgi 58k protein and counterstained with azure blue. Cases 5, 6, 8, 9, and 10 were first bleached to decolorize any melanin, subsequently stained with anti-Golgi 58k protein, and counterstained with hematoxylin

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Fig. 16.16 Co-localization of granular melanin with heavily melanized vesicles in dermal melanophages (case #13). Melanin in unstained sections is displayed in the left column and the corresponding stained fields are shown in the right column. Arrows denote examples of co-localized vesicles. (a) A section stained for LC3B. (b) A section stained for the Golgi 58k protein. (c) A section stained for β1,6-branched oligosaccharides with biotinylated LPHA

autophagic phenotype that is strikingly similar to that expressed by melanoma cells of florid MIS and invasive melanoma. Co-localization studies showed that LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides were all constituents of the melanized vesicles in melanophages (Fig. 16.16). Electron micrographs of dermal melanophages in case #13 confirmed that the vesicles were autophagosomes (Fig. 16.17). These autophagosomes in the melanophages are limited by double membrane and, similar to autophagosomes

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Fig. 16.17 Electron micrographs of a dermal melanophage (Case #13). (a) Low power view showing numerous melanin-containing vesicles within the cytoplasm of a melanophage. Collagen bundles in the adjacent dermis are labeled by “c”. (b) High power view of a vesicle, which is an autophagosome, enveloped by a double membrane (arrow), and containing partially digested, heavily melanized melanosomes

in melanoma cells, contain what appears to be partially digested, melanized melanosomes, and other cellular debris. The melanophages were surrounded by collagen bundles (labeled “c”) confirming their dermal location. In summary of our initial studies of cutaneous malignant melanomas we show that autophagic tumor cells were the major, if not sole, cell type for florid MIS and for invasive cells in the dermis. In this study all melanomas were of the superficial spreading type with the exception of one case of ALM (case #13) used in EM and co-localization studies. Therefore, we cannot draw conclusions and make generalizations about other types of malignant melanoma. Using coarse melanin as a marker for autophagosomes, the autophagosomal protein LC3B, the Golgi 58k protein, and β1,6-branched oligosaccharides, all co-localized to autophagosomes. The presence of autophagosomes was additionally confirmed by electron microscopy. Tumor-associated melanophages also exhibited an autophagic phenotype, which was remarkably similar to that seen in melanoma cells. The presence of widespread autophagy in malignant melanoma can be explained by, and is consistent with, findings that most melanomas are under ER stress, an inducer of autophagy (Hersey and Zhang 2008, Rutkowski and Kaufman 2007). The results of this study provide a new view of melanoma progression, in which the metabolic energy balance of invasive cells may be dependent on autophagy. These findings suggest new paradigms for therapy involving inhibition of autophagy in order to deprive melanoma cells of this energy source. Could autophagy in human cancer result from fusions between cancer cells and macrophages or other phagocytes? In fact, macrophages express active autophagy as a part of the pathway for digestion of phagocytosed microorganisms and cells (Amer and Swanson 2005, Amer et al. 2005). Autophagy in macrophages

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Fig. 16.18 A model for generation of a metastatic phenotype following fusion of a melanoma cell with a macrophage. (1) A macrophage is attracted to a non-migratory melanoma cell in situ. The epigenomes of the two cells reflect their myeloid and melanocytic lineages respectively. The melanoma cell produces “fine” or “dusty” melanin – individual melanosomes in the cytoplasm, generally with a golden-brown color. Melanoma-associated macrophages are known as melanophages because they are laden with autophagolysosomal vesicles containing melanin from injested melanoma cells, and thus at times difficult to distinguish from melanoma cells at the light microscope level. (2) The macrophage and melanoma plasma membranes form close appositional contacts, normally as a prelude to injestion and destruction of the melanoma cell. However in some cases rather than the macrophage digesting the melanoma cell, the two cells fuse. (3) Following fusion a heterokaryon is formed with the two nuclei separate in the cytoplasm. (4) Genomic hybridization occurs and a mononuclear macrophage/melanoma hybrid emerges. From studies of macrophage/melanoma hybrids generated experimentally in vitro and of melanoma/host hybrids generated spontaneously in mice, such hybrids have a deregulated cell cycle, are aneuploid and exhibit epigenomes of both parental lineages. Some exhibit the myeloid capability for chemotaxis in vitro and tropism in vivo, common characteristics of metastatic cells (from Pawelek and Chakraborty 2008a)

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is linked to phagocytosis, interestingly, another characteristic of metastatic cancers (Coopman et al. 1998, Damiani and Colombo 2003, Lugini et al. 2003, 2006, Montcourrier et al. 1994). Moreover, macrophage vesicles, like those in experimental macrophage-melanoma hybrids and cancer cells, are positive for β1,6-branched oligosaccharides (Fernandes et al. 1991, Handerson and Pawelek 2003, Handerson et al. 2007). Therefore, activation of phagocytic and autophagic pathways in human cancers could reflect expression of imprinted genes of myeloid lineage in macrophage-tumor cell fusion hybrids. We suggest that should cancer cell autophagy be linked to phagocytosis as it is in macrophages, nutrients could be continuously phagocytosed from external sources and digested through autophagy, rendering metastatic cells constitutively independent of a direct blood supply. A model for a cell fusion origin of autophagy in malignant melanoma is presented below (Fig. 16.18).

16.7 Conclusions Metastatic macrophage-melanoma hybrids show high expression of SPARC, c-Met, MC1R, integrin subunits α3, α5, α6, αv, β1, β3, cell surface LAMP-1, GnT-V, and autophagy. This is paralleled in melanoma, and in a number of other cancers in which these molecules are associated with a migratory phenotype, enhanced survival, metastasis and poor outcome. Central to the metastatic phenotype is GnT-V which, through addition of β1,6-branched oligosaccharides to several of the above proteins, causes multiple phenotypic changes including increased chemotaxis, melanogenesis, and possibly autophagy. Expression of MC1R, MITF, c-Met, motility-related integrins, cell surface LAMP-1, GnT-V and autophagy are also characteristic of monocytes/macrophages and other BMDCs. Thus, expression of these molecules in cancer could be a result of fusion of cancer cells with migratory BMDCs and co-expression of imprinted genes from both parental fusion partners. While these molecules and traits are of course not the only factors involved in tumor progression, their high expression in BMDC-tumor hybrids provides a framework for understanding how fusion can explain metastasis. While the possibility of BMDC-tumor cell fusion has yet to be tested in human melanoma, it is consistent with the known properties of this highly aggressive malignancy.

16.8 Considerations for Studying Fusion In Vivo To prove fusion and genomic hybridization requires identification of genes or chromosomes from both of the putative fusion partners in the same cell or cells. Hence fusion has been well-documented in tumor xenografts in animals where hybrids were identified by the presence of both tumor and host genes. Little is yet known of the extent of cancer cell fusion in humans. While a few human cases have recently been reported (Andersen et al. 2007, 2009, Chakraborty et al. 2004, Yilmaz et al.

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2005) only one of these involving macrophage-myeloma fusion in osteoclast formation definitively proved fusion (Andersen et al. 2007, 2009). The use of myeloma clone-specific immunoglobulin rearrangements as parental markers of myeloma cells can thus be used to further investigate questions of fusion in myeloma. Other studies have suggested that incorporation of BMDCs into tumor cells can occur through differentiation or neoplastic transformation without fusion (Avital et al. 2007, Cogle et al. 2007, Houghton et al. 2004). It is possible that both mechanisms are operative in cancer as well as in healthy tissue regeneration and repair, and this remains to be resolved. The use of allogeneic HSC transplants in medicine followed by the unfortunate development of secondary malignancies provides a potential source of pathology material for study (Chakraborty et al. 2004, Yilmaz et al. 2005). However, such cases are in limited supply and it will take some time to determine the extent of fusion in human cancer by this technique alone. Further, while this technique can verify fusion of BMDCs and cancer cells, the cellular nature of the BMDC cannot at this point be determined. Another problem is that the frequency of cancer cell fusion may be quite low, as it is in culture (roughly 1 in 105–107 non-fused cells) making fusion events difficult if not impossible to follow in vivo (Rachkovsky et al. 1998). Also, depending on the time when a particular tumor is analyzed, the number of hybrid cells could range from none, should hybridization not have occurred, to 100% if hybrids had overgrown a preexisting tumor or initiated a new tumor, e.g. a metastasis. Further, hybrid cells in a tumor could result from a single progenitor hybrid or from multiple hybrids formed from separate fusions. It is thus difficult to study the molecular mechanisms of cancer cell fusion in vivo, or to estimate its frequency. Until more progress is made in these and other areas, the impact of BMDC incorporation into human tumors, whether by fusion or other mechanisms, remains to be determined.

16.9 Implications Two of the hybrid-associated features described above, enhanced migration and autophagy, could together have important implications for the initiation of metastasis. Remarkably, both features may be activated through GnT-V-mediated addition of β1,6-branched oligosaccharides. For the primary carcinoma or melanoma cell, a migratory phenotype would imply loss of adhesion to adjoining cells in the epidermis, activation of matrix proteinases, induction of chemotaxis and tropism, and major cytoskeletal changes. But cancer cells often exist under hypoxic conditions. In such situations, autophagy could play a survival role by providing a nutrient source independent of the vasculature. Interestingly, the hypoxic regions of tumors have long been thought to select for more aggressive cancer cells that can survive under limited nutrient and oxygen supply (Graeber et al. 1996). Should cancer cell autophagy be linked to phagocytosis as it is in macrophages (see below), nutrients could be continuously phagocytosed from external sources (cellular debris, matrix fragments, etc.) and digested in autophagolysosomes, rendering metastatic cells constitutively independent of a direct blood supply.

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Tumor cell-BMDC fusion as a source of metastatic cells would imply that prevention of fusion, or of early, rate-limiting post-fusion events might prevent metastasis (e.g. Parris 2008). With better understanding should come better strategies for targeting vulnerable steps in fusion and the generation of hybrids. Postfusion events and hybrid formation could present other fruitful areas of focus, for example molecular steps governing the integration of parental fusion partner genes into hybrid genomes, or those involved with activation of master regulatory genes that are rate-limiting in development of a migratory phenotype. Early post-fusion cells are also likely to express unique antigenic profiles making them susceptible to immunotherapy. The cancer cell-BMDC fusion theory presents a unifying explanation for tumor progression. It seems that this theory is not only possible but likely to be correct to at least some degree, with the remaining question being how extensively does it contribute to progression of human cancers? There are many areas to consider regarding the therapeutic implications of fusion itself. Should it be determined that fusion indeed underlies metastasis, or at least some aspects of it, then new therapeutic paradigms would surely emerge, for example in prevention of fusion itself or destruction of fused cells based on unique molecular signatures. Based on the information gathered by several laboratories to date, we would urge more scientists to enter this most important and interesting area of cancer research. Acknowledgments We gratefully acknowledge the many and invaluable contributions of David Bermudes, Jean Bolognia, Douglas Brash, Dennis Cooper, Lynn Margulis, Josh Pawelek, James Platt, Michael Rachkovsky, Stefano Sodi, and Yesim Yilmaz. Supported in part by a gift from Amway, Inc.

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Chapter 17

Cell–Cell Fusions and Human Endogenous Retroviruses in Cancer Reiner Strick, Matthias W. Beckmann, and Pamela L. Strissel

Abstract The overall focus of this review is the characterization and functional role of cell–cell fusions in connection with human endogenous retroviruses (HERV) in cancer. Examples of multinucleated cells presented include placental syncytiotrophoblasts, muscle myotubes, bone osteoclasts involved in normal human development and cell–cell fusions detected in tumors. Examples of multinucleated cells in various cancers include germ cell tumors, glioblastoma, melanoma, lung, breast, ovarian and endometrial carcinomas. The role of different HERVenvelope proteins mediating fusion or regulation of cells in tumors is highlighted. Although multinucleated cells are detected in various tumors, their origin, functional role and overall cellular fate are ambiguous. The effect of multiple cancer cells fusing and in contrast cancer cells fusing with somatic cells is also discussed. Understanding tumorigenesis has to ultimately link the knowledge between the function and action of multinucleated cells, cell fusion, HERVs, retroviruses and cell signalling pathways. Keywords Cancer · cell-cell fusions · HERV · multinucleated cells · polyploidy · retrovirus · syncytin · virus Abbreviations ASCT BMD BMDC CD eff-1 EM env

Alanine, serine and cysteine selective transporters Bone marrow derived Bone marrow derived cells Cluster of differentiation Epithelial fusion failure 1 Electron microscopy Envelope

R. Strick (B) Department of Gynaecology and Obstetrics, Laboratory for Molecular Medicine, University-Clinic Erlangen, D-91054 Erlangen, Germany e-mail: [email protected]


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Group-specific antigen Hemagglutinin Hemolysis elevated liver enzymes low platelet count Human endogenous retrovirus Human immune deficiency virus Human T-cell lymphotropic virus Immunofluorescence Immunohistochemistry Intrauterine growth restriction Major facilitator superfamily domain containing 2 Multiple sclerosis retrovirus Pre-eclampsia Polymerase Radioimmunoassay RNA interference Surface unit Transforming growth factor Transmembrane unit

Contents 17.1 Development and Polyploidy . . . . . . . . . . . . . . . . 17.1.1 Short History of Cell–Cell Fusions . . . . . . . . . . 17.1.2 Cell–Cell Fusions in Development, Differentiation and Viral-Induced . . . . . . . . . . . . . . . . . 17.1.3 Cell–Cell Fusions During Tumorigenesis . . . . . . . 17.2 Human Endogenous Retroviruses (HERVs) . . . . . . . . . . 17.2.1 HERV Expression in Human Cancers . . . . . . . . . 17.2.2 HERVs in Cancer Cell–Cell Fusions: Driver or Passenger 17.3 Cell–Cell Fusions in Cancer: Functional Role or Dead-End . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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17.1 Development and Polyploidy One text book fact imperative for human somatic cells, which constitute the entire organism, is diploidy, where genetic information on chromosomes consists of two homologous copies of each autosome and two sex chromosomes. In the normal process of mitosis the total number of chromosomes is a diploid set with 2n members. One exception to the diploid rule mentioned in every textbook are germ cells, like eggs and sperms, which are generated by meiosis and have a haploid chromosome number (n). The phenomenon of somatic polyploidy, meaning the existence of extra copies of all chromosomes is mostly found in the world of plants, invertebrates and protista and in some vital human developmental processes like muscle, bone and placenta formation. In addition, the existence of polyploid somatic cells

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or aneuploidy (a loss or gain of certain chromosomes) in human malignancies is long-established. Although polyploid cells found in human tissues exist for many scientists, they mostly represent an exotic or odd research field and in vitro studies are mostly dismissed as artefactual. Therefore, this review will describe first, examples of polyploid human cells and show important differences in order to understand the different types of polyploidy; and second, depict the occurrence of cell–cell fusions as one cause for polyploidy in carcinoma; and third, describe human endogenous retroviruses (HERV) involved in cell–cell fusions and tumorigenesis. In order to understand polyploidy and the cellular causes for this phenomenon a short excurse into cell division and the prevention of it has to be made. Normal cell division is characterized by nuclear division (karyogenesis) and cytosolic separation (cytokinesis). Incomplete cytokinesis can be a programmed step in normal development producing tetraploid progeny (Glotzer 2001), or it can be part of cancer progression where tetraploid cells occur in tumors and further create genomic instability (Ganem et al. 2007). Following tetraploidization of cells subsequent divisions result in aneuploidy. Especially in the absence of functional p53 these cells are associated with a predisposition of tumorigenesis (for review: Margolis 2005). The suppression or slowing down of the spindle apparatus relative to the chromosomes usually leads to the formation of endopolyploid nuclei, where many copies of the same chromosomes are found in one nucleus (Fig. 17.1). The most common mechanism giving rise to endopolyploidy is endoreduplication, which means that two or more DNA replications take place without an intervening mitosis. If a cell divides after endoreduplication, the chromosomes appear as diplochromosomes or as bundles of multiple chromatids. In endomitosis, which is rarer than endoreduplication, chromosomes condense as in normal prophase, but the

Fig. 17.1 Three main possibilities of a cell (2n) leading to polyploidy are detected in different mammalian tissues: endoreduplication, endomitosis and cell–cell fusions. These polyploidies take place with a fusion of nuclei (karyogamy) or without (multinucleation). Multinucleated cells with nuclei from different origins are only found after cell–cell fusions

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nuclear membrane does not disintegrate. In endo-metaphase the sister chromosomes lie side-by-side giving the nuclei their characteristic appearance. After the sister chromosomes separate in endo-anaphase, they revert through endo-telophase into interphase. Polytenization is a modification of endoreduplication. The chromosomes are extended and the paired strands replicate again and again. The tendency to somatic pairing in diptera aligns the homologous chromomeres which form the bands in the polytene chromosomes. In other organisms, pairing of the homologous strands is more or less loose; consequently, banding is much less clear or even absent. Restitution, mostly from metaphase or anaphase, implies that the daughter chromosomes are included in the same nucleus and are products of defect spindle mechanisms or spindle poisons such as colchicine (C-mitosis). In cancer cells where mitosis similar to C-mitosis occasionally takes place spontaneously, is mostly the result of the formation of micronuclei and not polyploidy. Some well known examples describing the above mechanisms are megakaryocytes which produce platelets in mammals and represent normal polyploid cells due to a failure of karyogenesis and cytokinesis (Ravid et al. 2002, Fig. 1). Newer experiments further showed that the endomitotic process of megakaryocytes was a late failure of the cytokinesis due to a defect in the Rho/Rock-pathway (Lordier et al. 2008). Cardiomyocytes also have an incomplete cytokinesis leading to binucleated cells (Engel et al. 2006). Hepatocytes show high percentages of tetraploidy and octoploidy with mono- and binucleation in normal cells especially after liver damage and hepatitis due to incomplete cytokinesis, but not during carcinoma (Gupta 2000, Margall-Ducos et al. 2007). In normal adult human liver 30–40% represent polyploid cells (Kudryavtsev et al. 1993). Interestingly, tetraploid cells were able to proliferate resulting in mononucleated cells (Guidotti et al. 2003). In contrast to human trophoblasts, differentiation of mouse trophoblast stem cells into trophoblast giant cells is due to endoreduplication and suppression of cyclin-dependent kinase 1 by p57 (Ullah et al. 2008).

17.1.1 Short History of Cell–Cell Fusions After the establishment of the “cell theory” by Schleiden (1838) and Schwann (1839) that the basic structural element of all plants and animals is the cell, one of the first descriptions of multinucleated giant cells and the hypothesis of cell–cell fusion events was 1868 by Langhans in tuberculoid lesions. Today these specific multinucleated giant cells are morphologically classified as Langhans’ giant cells commonly found in immune granulomas and are characterized with less than 20 nuclei per cell arranged in a kind of a circle (Roger et al. 1972). Multinucleated cells were since then not only found in tuberculoid granulomas but also in other types of granulomas, like sarcoidosis (Okamoto et al. 2003), schistosomiasis (Boros 1989), sarcoid-like type granuloma e.g. colitis or rectal carcinoma (Williams and Williams 1967) and foreign body fusion events, like biodegradation of polymeric medical devices like sutures (Anderson 2000). The foreign body giant cells have in contrast to Langhans’ giant cells more than 20 nuclei per cell and are irregularly

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arranged in the cell. All of these multinucleated giant cells are most likely due to cell fusions of macrophages recruited to the site of inflammation (Helming and Gordon 2007). Other early descriptions of multiple mitosis and giant nuclei in tumors of a larynx were done by Hansemann (1890). Also Winge (1927) described giant cells with up to 40–50 nuclei, but smaller in size than normal nuclei, in human tumors, but also in tumors of mice induced with tar. Interestingly, early detections of multinucleated cells in normal tonsils were described, but especially induced after a measles infection (Finkeldey 1931). The direct connection of cell fusions after viral infections was later shown for e.g. fusion of glioblastoma cells after cytomegalovirus infections (Navarro et al. 1993) and astrocytoma cells following measles virus infection (Duprex et al. 1999). It is important to note that viral induced cell fusions do not need a virus infection per se since the Mason Pfizer monkey virus envelope gene was enough to induce cell fusions. These fused cells failed to proliferate and divide, except in case of inactivated p53 (Duelli et al. 2005). The link between virus-carcinoma and also virus-carcinoma-multinucleated cells will be important for understanding the etiology of virus induced malignancy. One example of virus-carcinoma-multinucleated cells is the human papilloma viruses induced cervix carcinoma and associated giant cells (Therman et al. 1983). Many novel virus-carcinoma connections were found and will be found, e.g. the recent connection that over 80% of Merkel cell carcinomas were associated with a newly discovered polyoma virus (Feng et al. 2008), as well as the connection of virus induced carcinoma with multinucleated cells. A connection between cell–cell fusion and tumorigenesis was already proposed 100 years ago. Aichel (1911) suggested that fusion (in the sense of fusion of gametes) of two somatic cells produces benign tumors, whereas fusion of a resident somatic cell with a leukocyte could produce a malignant tumor. The difference between these fusions and the different outcomes according to Aichel was due to the correct or incorrect (re)distribution of chromosomes, where a fusion between a resident somatic cell and a leukocyte would result in aneuploidy and evolve into malignancy. He did not support that cells acquire attributes, but that attributes of single (fusing) cells led to malignant hybrids. Since then, a plethora of publications described higher ploidies and aneuploidy in malignant cells. The tumor progression is mostly associated with chromosome or gene amplification, demonstrating that extragenetic dosage is advantageous. This is possible due to chromosome non-disjunction, endoreduplication and cell–cell fusions (for review: Larizza and Schirrmacher 1984, Hanahan and Weinberg 2000, Margolis 2005, Ganem et al. 2007, Larsson et al. 2008).

17.1.2 Cell–Cell Fusions in Development, Differentiation and Viral-Induced Cell–cell fusions can be transient, as in the case of the sperm and egg heterokaryon, which lead to a fusion of nuclei (synkaryon) and subsequent cell divisions, or they can be permanent leading to syncytia and multinuclear cells (Fig. 17.1).

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These syncytia have essential functions in human somatic tissues, as in the case of placenta, muscle and bone formation. Generally membrane fusions are energetically unfavourable and are therefore mediated by specific proteins to overcome repulsive hydration forces and promote hydrophobic attractive forces (Helm et al. 1992). Many proteins mediating the molecular mechanisms involved in cell–cell fusion during development of different organisms are known as further discussed in section 2 (for review: Oren-Suissa and Podbilewicz 2007, Larsson et al. 2008). Membrane fusions can also be associated with other diseases, like giant cells during inflammations, entry of enveloped viruses as well as virus induced cell–cell fusions. Also a lack of normal developmental cell–cell fusions can be associated with incorrect ossification like osteopetrosis (Everts et al. 2009), minute myotubes leading to centronuclear myopathy (Wöckel et al. 1998) and significantly reduced expression levels of the HERV-W envelope gene, Syncytin-1, found associated with pregnancy disorders like PE/IUGR and HELLP/IUGR (Langbein et al. 2008). Several organisms are used as cell–cell fusion models, like Caenorhabditis elegans (C. elegans), where during embryonic and post-embryonic development nearly one third of all cells fuse and ultimately generate the syncytial hypodermis, vulva, excretory gland, male tail, anchor cell, uterus and pharynx (Gattegno et al. 2007). Several genes were identified as promoting and inhibiting cell–cell fusion. For example, the membrane protein epithelial fusion failure 1 (eff-1) was identified as essential for cell–cell fusions in C. elegans and may act as a viral fusogen (Mohler et al. 2002). Eff-1 mutants demonstrated that cell–cell fusion in C. elegans limited cell migration, restricted cell fate determination and was essential for organogenesis and morphogenesis. 17.1.2.1 Cytotrophoblasts-Syncytiotrophoblasts During early gestation two trophoblast populations exist: (1) extravillous trophoblasts invade the maternal decidua basalis in order to attach the placenta to the uterus and to adapt maternal arteries for blood supply and (2) villous trophoblasts which differentiate and fuse with each other into a multinuclear syncytiotrophoblast. After the short period of forming the syncytiothrophoblast, maintenance of this multinuclear state is achieved through fusion of villous cytotrophoblasts into the existing syncytiotrophoblast. Interestingly, experiments showed that the syncytiotrophoblast does not replicate and is relatively transcriptionally inactive (for review: Huppertz 2009). Although syncytiotrophoblast maintenance is ongoing through cell fusion with villous cytotrophoblasts, apoptotic material from the syncytiotrophoblast is deposited in so called syncytial knots and released into the maternal blood (Huppertz 2009). 17.1.2.2 Myoblasts-Myotubes Mononuclear myoblasts fuse with each other to form bi- or trinucleated nascent myotubes. Additional rounds of cell fusion between myoblasts and nascent myotubes result in the formation of large, mature myotubes with hundreds or

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thousands of nuclei (Horsley and Pavlath 2004 and for review: Chen et al. 2007). Interestingly, muscle founder cells attract fusogenic myoblasts which migrate to each other, adhere over filopodia and fuse (Kim et al. 2007). 17.1.2.3 Osteoclasts Mononuclear pre-osteoclasts derived from haematopoietic cells, migrate to the bone surface and fuse to multinuclear osteoclasts which acquire after attachment and polarization bone resorbing (osteolytic) activities. Up to eight nuclei are normally in human osteoclasts, although osteoclasts of some diseases, like Paget disease, have over 100 nuclei (Roodman and Windle 2005). Many of the molecules involved in regulation of haematopoietic cells to osteoclasts (osteoclastogenesis) are known and all nuclei of osteoclasts are transcriptionally active (Bar-Shavit 2007). In addition to the migratory activity of osteoclasts on the bone, it was shown that osteoclasts were also able to migrate through cell layers (transmigration) (Saltel et al. 2006). 17.1.2.4 Unique Cell–Cell Fusions A unique situation is the vertebrate lens, where evidences suggest that epithelial fiber cells fuse during terminal differentiation. However, the fusion of the lens syncytium is only partial, where the cytoplasm of fused fiber cells remains partly portioned by a membrane (Shestopalov and Bassnett 2000). The function of the lens syncytium is physiological in order to combine metabolically active cells at the surface with quiescent cells at the centre of the lens and on the other hand physically by enhancing the transparency and correction of spherical aberrations (Shi et al. 2009). In male germ line development, the committed spermatid precursors fail to complete cytokinesis and form syncytia until the end of differentiation (Guo and Zheng 2004), but multinucleated spermatogonia due to true cell–cell fusions are found in elderly males (Miething 1995). 17.1.2.5 Experimental Stem Cell Fusions Stem cell fusions with somatic cells lead to pluripotent cells with stem cell character. Evidences for this statement were from cell fusions of mouse embryonic stem cells with adult mouse spleen cells, which resulted in hybrid cells with retained high pluripotency (Matveeva et al. 1998). Using cell hybridization of adult thymocytes with embryonic stem cells showed pluripotency in vivo and epigenetic changes similar to the stem cell (Tada et al. 2001). Similar fusion experiments were done with human embryonic stem cells and human fibroblasts which produced pluripotent hybrids with embryonic programming (Cowan et al. 2005). Considering the differentiation of fused stem cells Terada et al. (2002) demonstrated that mouse bone marrow cells spontaneously fused with embryonic stem cells in vitro and these cells differentiated to the phenotype of the recipient cells. In view of brain cells fusing with stem cells, Ying et al. (2002) detected cell fusions between mouse brain cells

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and pluripotent embryonic stem cells, which resulted in brain-embryonic stem cell hybrids with an altered phenotype, but with a full pluripotent character. In addition, introducing stem cells into traumatic rat brains led to cell–cell fusions of grafted stem cells with brain cells (Horváth et al. 2006). 17.1.2.6 Virus Induced Cell Fusions Well characterized are the fusion proteins of enveloped viruses, which are mostly sufficient to facilitate fusion in cell culture. Most viral fusion proteins are integral membrane glycoproteins which form trimers or tetramers (like influenza HAtrimers). Essential of viral fusion proteins are the fusion peptides or fusogens, which are hydrophobic amino acids within a membrane-anchored transmembrane subunit. Differentiated are class I fusogens, like HA of influenza virus with coiled-coil helical domains and class II fusogens, like the E-glycoprotein of the Tick Borne Encephalitis virus with a ß-barrel domain, which undergoes structural changes (Harris and Watkins 1965, Skehel and Wiley 1998, Lescar et al. 2001). Interestingly, like in viruses, fusion peptides were also found in cell–cell fusion proteins, like PH-30 of sperms (Blobel et al. 1992). The presence of multinucleated giant cells are even used today for differential diagnosis of chickenpox (varicella), herpes and herpes zoster from small pox, where small pox does not form multinucleated cells (Koranda 2004). In the case of HIV1, infected cells expressing the env of HIV-1 on their surface can initiate cell–cell fusion between infected and uninfected cells. Detection of syncytia seems to be symptomatic for the late HIV-1 pathogensis (Blaak et al. 2000) and are found esp. in the brain and adenoid (Budka 1986, Frankel et al. 1996). One other example of virus induced cell–cell fusions associated with cancer was the detection of virus-like particles and syncytia found in ascites of an HIV-1-positive patient with ovarian carcinoma (Rakowicz-Szulczynska et al. 1999). Lastly, a case report showed syncytia with virus-like inclusions in giant tumor of bone (De Chiara et al. 1998). 17.1.2.7 Bone Marrow Derived Cells (BMDC) Earlier experiments of mice with specific liver enzyme mutations transplanted with wild-type mice bone marrow cells resulted in wild-type liver cells due to cell–cell fusion and a change of the liver cell expression profile to those of bone marrow cells (Vassilopoulos et al. 2003). In addition, similar experiments showed that cell–cell fusions occurred between two diploid cells and diploid and tetraploid cells (Wang et al. 2003 and Fig. 17.2). However, as mentioned throughout the literature the phenotypic and functional changes of BMDC are the basis of a dispute: if these cells are changed due to differentiation based upon cell–cell fusion or solely transdifferentiation without fusion (for review: Pawelek 2005). BMDCs can be reprogrammed at new locations, by transdifferentiation and function as new cells (Krause et al. 2001); on the other hand BMDCs can fuse with cells and result in nuclei reprogramming where mRNA and pre-exisiting proteins in the cytoplasm directly change the outcome of cell fusion. Importantly, the explanation is probably not exclusively

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Fig. 17.2 Different possible outcomes of normal diploid cells (2n) after cell–cell fusion. Shown are preserved multinucleation with and without cell cycle arrest and division, as well as later fusions of nuclei and subsequent divisions leading to aneuploidy or apoptosis

transdifferention or cell–cell fusion, the existence of one does not exclude the other (Kodama et al. 2003, Ianus et al. 2003). Transdifferentiation was shown by several researchers. For example, Tran et al. (2003) demonstrated that BMDCs could differentiate into buccal epithelial cells; Ianus et al. (2003), showed in mice that BMDCs switched to pancreatic endocrine cells and Kodama et al. (2003), showed that splenocytes could regenerate pancreatic cells in mice without fusion. Although initially, some reviews focused on transdifferentiation of BMDCs as examples of plasticity of adult stem cells, presently with more evidences in the literature, the tone now represents cell–cell fusion of BMDCs as the main cause for switches of cell lineages (Rodi´c et al. 2004). In 2003 and 2004 heterotypic cell–cell fusion between BMDCs and resident somatic cells, such as hepatocytes, cardiomyocytes and skeletal muscle cells was demonstrated in several studies (Alvarez-Dolado et al. 2003, Nygren et al. 2004). Specifically BMD myeloid cells were shown to fuse with muscle fibers (Camargo et al. 2003). One explanation, for the observed cell type switching could be due to specific cell types. As noted above, although BMDC transdifferentiation to buccal epithelial cells or pancreatic cells occurs; these cell types do not fuse during development. On the other hand, cells undergoing BMDC-cell fusions represented cell types with a capacity to fuse during development, like liver cells, cardiomyocytes and skeletal muscle cells. Interestingly, in mice cell fusions of cardiac and skeletal muscle cells (cardiomyocytes with myoblasts) were also demonstrated (Reinecke et al. 2004).

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BMD macrophages generate syncytia under physiological conditions and reside everywhere, like Kupffer cells in the liver and microglia in the brain. More specific myelomonocytic cells were found to spontaneously fuse with hepatocytes (Camargo et al. 2004). Further evidences for cell–cell fusions were recently shown by irradiation experiments, which induced frequent heterotypic cell fusions between myeloid and lymphoid cells and hepatocytes (Nygren et al. 2008). In addition, chronic inflammation led to increased cell fusions of BMDCs with Purkinje cells (binucleated cells) in the cerebellum (Johansson et al. 2008). The technique of cre-lox recombination and lethally irradiated mice grafted with BMDC from mice expressing specific detectable proteins (green fluorescent protein and -Galactosidase) demonstrated after cre-lox recombination that these mice expressed these marker proteins in the brain, heart and liver, but not in skeletal muscle, gut, kidney or lung (Alvarez-Dolado et al. 2003). These experiments showed that fused cells developed into normal liver cells and in the cerebellum where binucleated cells were detected but with morphologically different nuclei (Alvarez-Dolado et al. 2003). Especially, reports of BMDCs fusing with brain cells, like Purkinje cells are known (Weimann et al. 2003, Johansson et al. 2008). A recent report even showed that cell fusion of BMDCs contributed to pericytes after induced strokes in mice (Piquer-Gil et al. 2009). On the other hand, maybe cell–cell fusion also serves as homeostasis in special organs like brain, heart and liver, like clearing of these cells and replenishing them with BMDC. Brain and liver cells also have a very slow turnover and could make them targets for cell fusions.

17.1.3 Cell–Cell Fusions During Tumorigenesis Numerous publications have described fusions of tumor cells with other tumor cells or with somatic cells. Unfortunately, many times a precise description of these so called fused cells was not given, so that the definition of true cell–cell fusion or endomitosis cannot be given, e.g. patient case reports. Additionally, the status of multinucleation or fused nuclei (karyogamy) could not be given either. When cell characterizations were stated they were given as examples of known cell–cell fusions like osteoclast-like or trophoblast-like cells. Experiments involving grafting human cancer cells into rodents produced tumors that partially or completely consisted of host-human hybrids and host tumor cells that appeared to be derived by spontaneous fusion (Goldenberg et al. 1974, Pathak et al. 1997). The role of fused cells explaining metastasis, esp. of BMDC or tumor associated macrophages and cancer cells was supported by spontaneous fusion of tumor cells with normal cells in mice followed by chromosome segregation resulting in tumor progression and metastasis (Kerbel et al. 1983, Larizza et al. 1984, Busund et al. 2003). Table 17.1 describes a variety of tumors where cell–cell fusions have been described. Below are some examples of cell–cell fusions in different tumors expanded on from the literature. Breast cancer: Cell–cell fusions between breast cancer cells were described by Miller et al. (1988). Further it was shown that human breast cancer cell lines can

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Table 17.1 Selected examples of multinucleated cells found in human tumors Tumors

Multinucleated cells described as

References

Breast carcinoma Breast carcinoma Breast carcinoma Breast carcinoma (DCIS) Ovary

Osteoclast-like Stromal-like Osteoclast-like Giant Giant cell

Uterus/leiomyosarcoma

Osteoclast-like; fusion of spindle-cells Syncytiotrophoblast-like Giant, trophoblast-like

Athanasou et al. (1989) Factor et al. (1977) Krishnan and Longacre (2006) Coyne (2007) Veliath et al. (1975), Yasunaga et al. (2008) Watanabe et al. (1996)

Uterus Uterus Germ cell tumors Seminoma Testicular teratoma Liver Liver Gallbladder Lung Lung Pancreas

Syncytiotrophoblast-like Syncytiotrophoblast-like, giant

Bone Soft tissue (PEComa) Melanoma Thyroid

Epithelial syncytial giant Osteoclast-like Osteoclast-like Giant Osteoclast-like Giant, pleomorphic, osteoclast-like Osteoclast-like Giant Monster cells, pleomorphic Giant

Hodgkin lymphoma Glioblastoma

Endomitotic Giant

Strick et al. (2007) Jones et al. (1991), Pesce et al. (1991) Ulbright (2005) Miettinen et al. (1985) Ulbright (2005) Atra et al. (2007) Munoz et al. (1980) Akatsu et al. (2006) Laforga (1999) Nakahashi (1987) Deckard-Janatpour et al. (1998); Robinson et al. (1977) Werner (2006) Folpe et al. (2005) Boyd et al. (2005) Albores-Saavedra and Wu (2006) Drexler et al. (1989) Homma et al. (2006)

fuse with endothelial cells in vitro and in vivo (mice) (Mortensen et al. 2004) and fusion could be inhibited with an inhibitory peptide against the HERV-W envelope protein, Syncytin-1 (Bjerregaard et al. 2006). Cell–cell fusion of breast cancer cell lines expressing Syncytin-1 was repressed with antisense RNA against Syncytin-1 (Bjerregaard et al. 2006). Syncytin-1 was found in 38% of breast tumors by immunohistochemistry and showed a positive prognostic factor for breast cancer (Larsson et al. 2007). The question of bone metastasis and osteolytic activity of breast cancer could be answered with the findings that tumor associated macrophages from breast cancer patients were capable of differentiation into multinucleated osteoclasts in the presence of breast cancer fibroblasts and tumor cells (Lau et al. 2007). Similar results were found with metastatic melanomas (Lau et al. 2006). Grafting human cells from pleural effusion from a patient with breast cancer into mice resulted in cell hybrids with human and mice chromosomes due to spontaneous cell fusion. These human cancer-mouse stromal cells were tumorigenic (Jacobsen et al. 2006). Recent results showed that bone metastatic breast cancer

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cells induce migration of multinucleated osteoclasts in vitro (Saltel et al. 2006), indicating the stimulative activity of cancer cells. Germ cell tumors: Germ cell tumors are a very heterogeneous class and have for male and female very different incidences. For example, 95% of ovarian germ cell tumors are teratomas, but only 4% of testicular germ cell tumors. On the other hand seminomas count for 50% of testicular germ cell tumors and dysgerminoma for 2% of ovarian germ cell tumors (for review: Ulbright 2005). Choriocarcinomas are one of the rarest germ cell tumors. Seminomas are found with multinucleated cells (Miettinen et al. 1985) as well as testicular teratomas (Ulbright 2005). Giant neoplastic cells were often found in pure seminomas, but giant mononuclear cells less frequent in spermatocytic seminomas (von Hochstetter et al. 1985). Some germinomas and esp. choriocarcinomas were found with distinct syncytiotrophoblast cells (Mostofi and Sesterhenn 1985). Especially, choriocarcinoma cell lines are widely used as ideal test cells for cell–cell fusion studies. Fusion of teratocarcinoma cells with adult mice thymocytes produced different tissue types and carcinoma in mice indicating pluripotency (Miller and Ruddle 1976). Melanoma: Tumor cells with giant nuclei, so called monster cells, were previously described in basal cell carcinomas, dermatofibromas, pleomorphic fibromas and cutaneous melanomas. In the latter multinucleated cells were also detected and statistical association with ulceration and depth of invasion found (Boyd et al. 2005). Glioblastoma: One example of the difficulty to find the cause for multinucleation of giant cells is shown for giant cell glioblastoma: Giant cell glioblastomas are rare among glioblastomas, and are characterized by many multinucleated giant cells. A molecular analysis of the cell cycle kinase cdc2 and Rho-kinases showed inactivity in the giant cell glioblastoma with the conclusion that these cells entered into early mitosis but not through late mitosis demonstrating nuclear division but no cytokinesis and therefore endomitosis (Maeda et al. 2003). In another report, giant cell glioblastomas were found with little mitotic potential and p53 mutations concluding some cell fusions (Takeuchi et al. 2006). Finally, the existence of CD98 positive giant cell glioblastomas with low proliferative potential speaks for the development of these cells by cell fusion and syncytial formation (Takeuchi et al. 2008). Endometrial carcinoma: In contrast to endometroid endometrial carcinomas, which count for over 80% of the total endometrial carcinomas, non-endometrioid endometrial carcinomas are not derived from benign lesions. Non-endometrioid endometrial carcinomas show a high array of different histological subtypes, like serous, mucinous, clear cell, squamous and poorly differentiated tumors with trophoblastic elements and giant cells (for review: Clement and Young 2004). These trophoblastic and giant cell carcinomas have areas of multinucleated cells, which are described as syncytiotrophoblast-like and are considered highly aggressive (Jones et al. 1991, Pesce et al. 1991). However, multinucleated cells were also identified in primary endometrioid endometrial carcinomas (Strick et al. 2007). Lung carcinoma: Large cell lung carcinomas, which count for 10–16% of lung cancers, also include so called giant cell carcinomas (Yesner 1985, Laforga 1999).

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Interestingly, giant cell carcinomas of the lung are specified as the most malignant lung carcinoma and include multinucleated and spindle form cells, which have phagocytotic activities (Razzuk et al. 1976). Hodgkin lymphoma: Hodgkin lymphoma shows several interesting findings in the course of the disease (for review: Küppers 2009). One phenomenon is the finding of multinucleated Hodgkin-Reed-Sternberg cells, which are derived from mononuclear Hodgkin cells probably by endomitosis (Jin and Woodgett 2005). Earlier experiments with a cell line from Hodgkin disease showed spontaneous formations of multinucleated cells, which had the same surface antigens as the monocleated cells and were still proliferative with mitosis, however without telophase (Drexler et al. 1989). BMDC fusions in cancer: Several examples are shown that BMDC-resident somatic cell fusions also play a role in multiple tumorigenesis (Liu et al. 2006). For example, transplanted BMDCs from mice showed cell fusion with normal and transformed intestinal cells, esp. with intestinal progenitor or stem cells, demonstrating a role of BMDCs in intestinal cell regeneration and/or tumorigenesis (Rizvi et al. 2006). Induction of inflammation and epithelial proliferation in mice induced cell fusions of BMDC with intestinal cells, proposing that increased inflammation and proliferation could lead to cell fusions and intestinal diseases and cancer (Davies et al. 2009). On the other hand, a connection between bone marrow stem cells and gastric cancer was shown, however the mechanism was unclear (Houghton et al. 2004). The latter example explained that the inflammatory environment probably favored the development of cancer been linked to homing and engraftment in peripheral tissue by BMDCs (e.g. Helicobacter). Human cancer cell fusions following transplantations: Only a few reports of cancer cell–cell fusions in humans are documented: Fusion of myeloma cells and osteoclasts (Andersen et al. 2007) and cell–cell fusions in two cancer patients occurring after organ transplantations (Chakraborty et al. 2004, Yilmaz et al. 2005). Additional evidence of solid cancers after allogenic bone marrow transplantation demonstrated the presence of donor-derived malignant cells (Avital et al. 2007). Signal transduction pathways and cell fusions: Several genes were identified as promoting or inhibiting cell–cell fusion, which were classified as transcription factors regulated by e.g. the Ras- and Wnt-pathways but also developmental genes, like the Homeo box-genes (for review: Shemer and Podbilewicz 2003). To date other signalling pathways have been implicated in the regulation of cell–cell fusion, for example protein kinase B or AKT2. Interestingly, AKT2 over expression has been found in many cancer types (Cheng et al. 1996). Introducing and activating a conditional AKT2 gene in human epithelial kidney cells resulted in multinucleated cells, caused by both endomitosis and cell–cell fusion (Jin and Woodgett 2005). Over expressing either the dual-specificity-tyrosine-phosphorylation-regulated kinase 1A (MNB/DYRK1A) or AURORA-A in Hela-cells resulted in multinucleated cells (Funakoshi et al. 2003, Meraldi et al. 2002). Interestingly, AURORA-A elevated protein levels is often found in cancers (Zhou et al. 1998), esp. in breast cancer (Miyoshi et al. 2001).

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17.2 Human Endogenous Retroviruses (HERVs) HERVs are derived from exogenous retroviral infected germ cells which integrated into the genome. Calculations showed that infection of the germline by the HERV precursors could have been more than 40 million years ago like for HERV-FRD or less than 200,000 years ago, like for HERV-K113 (Blaise et al. 2003, Turner et al. 2001). More than 8% of the human genome is considered retroviral origin, like retrotransposons, HERVs and elements with HERV origin (Bannert and Kurth 2004). More than 200 families of LTR-containing retroelements are defined by Repbase (Jurka 2000). HERVs are grouped into class I, II or III according to the sequence similarity to exogenous retroviruses: Class I HERVs, like HERV-W, -H, -R, -P, -T and -FRD are related to gammaretroviruses such as the murine leukaemia virus; class II HERVs, like HERV-K are related to betaretroviruses such as the mouse mammary tumor virus and class III HERVs, like HERV-L are related to spumaretroviruses such as the human foamy virus (for review: Nelson et al. 2003 and Tristem 2000). HERVs like exogenous retroviruses code for at least three genes: gag (group-specific antigen) encoding the structural protein of the core, pol (polymerase) encoding the viral enzymes like reverse transcriptase and env for the viral envelope. Many HERV sequences integrated through evolution adjacent to gene promoters and regulate transcription of cellular genes with functional consequences for normal tissues and malignancies (for review: Bannert and Kurth 2004). In contrast to pure retrotransposons, HERVs contain the env gene, coding for a viral membrane protein. HERV families are further distinguished due to the t-RNA codon for amino acids used for initiation of transcription. As an example, the primer binding site for a specific tRNA used to initiate reverse transcription is the tRNA(Trp) for HERV-W (Tristem 2000). Like exogenous retroviruses, HERVs consist of a provirus with the following assembly: LTR-gag-pol-env-LTR. Most of the HERV genes are nonfunctional due to recombinations, mutations and deletions; however 17 gag, 13 pol and 18 (29) env from all different HERVs encoded full-length open reading frames and could produce functional proteins (de Parseval et al. 2003, Villesen et al. 2004, Blaise et al. 2005). A few families, esp. the HERV-K family have been shown to form viral but non-infectious particles (Löwer et al. 1984, Bieda et al. 2001, Büscher et al. 2005). However, using the env protein from HERV-K108 infectivity to simian immunodeficiency virus pseudotypes was shown, demonstrating a full functional env gene (Dewannieux et al. 2005). Especially env genes of different HERVs were examined for expression. The env gene of HERV-W, called Syncytin-1 was found expressed in placentas and plays an essential role in cell–cell fusion of cytotrophoblasts to multinuclear syncytiotrophoblasts (Mi et al. 2000, Blond et al. 1999). So far, three env genes from different HERVs were found fusogenic in vitro assays: Syncytin-1, the env of HERV-FRD, also called Syncytin-2 and the env of HERVP(b). The regulation of Syncytin-1 for cell specific expression was found to be dependent on the methylation in the U3 part of the 5’LTR (although no CpG-islands were found) (Matousková et al. 2006). Syncytin-1 on chromosome 7q21.2 belongs to the class I fusion genes like other exogenous virus (paramyxovirus, influenza,

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filovirus, ebola virus, HTLV, lentivirus, HIV-1) and is cellularly processed and proteolytically cleaved into a surface unit (SU) and transmembrane unit (TM). The 538 amino acid long Syncytin-1 is glycosylated and makes homotrimers (Cheynet et al. 2005). Syncytin-1 and Syncytin-2 were found up-regulated after cAMP-stimulation, either with Forskolin, which induces the adenylate cyclase or directly with e.g. SPcAMP indicating a protein kinase A regulation (Prudhomme et al. 2004, Strick et al. 2007, Chen et al. 2008). Furthermore, when Syncytin-1 was intrinsically or transiently activated by cAMP cell–cell fusion increased. Multiple cells and cell lines (human and non-human) demonstrated the fusogenicity of Syncytin-1 and on the other hand RNAi and inhibitory antibody experiments identified Syncytin-1 as the cause for cell–cell fusion in vitro. In addition, the receptors for Syncytin-1 were identified as ASCT-1 and -2, which bind to the N-terminal SU-domain of Syncytin-1 (Cheynet et al. 2006). Additionally, the C-terminal, called the cytoplasmatic region was identified as negatively regulative for cell–cell fusion (Chang et al. 2004, Drewlo et al. 2006). Analyses of genomic polymorphisms of Syncytin-1 and Syncytin-2 identified multiple changes and even non-synonymous changes, but none of them impaired cell–cell fusion capabilities in vitro, leading to the assumption that Syncytin-1 and -2 have an essential role for humans, especially in human reproduction (Mallet et al. 2004, de Parseval et al. 2005). In addition, Syncytin-1 can function as a normal env protein and also with other retroviruses, pseudotyping HIV-1 (An et al. 2001, Lavillette et al. 2002). Although, Syncytin-1 expression was proven essential for mediating cytotrophoblast fusion to the syncytiotrophoblast in placenta it was also detected in other normal and tumorigenic tissues and cell lines, however HERV-W gag and pol expression was only detected in selected tissues and cancer cells (Yi et al. 2004 and Table 17.2). In addition to cell–cell function, many HERV env proteins also contribute to immunomodulatory actions, like autoimmunity caused by superantigens or immunosuppression (for review: Balada et al. 2009). Several neuropathological diseases, like schizophrenia, rheumatoid arthritis and multiple sclerosis have been attributed to the role of Syncytin-1 and another env gene of the HERV-W family member called the multiple sclerosis retrovirus (MSRV) (Antony et al. 2007, Antony et al. 2004, Karlsson et al. 2001). The other fusogenic env protein is Syncytin-2 of HERV-FRD on chromosome 6p24.1, with MFSD2, a putative carbohydrate transporter as the receptor (Esnault et al. 2008, Blaise et al. 2003). In contrast to Syncytin-1, Syncytin-2 expression is more restricted to placental cells like villous cytotrophoblasts (Malassiné et al. 2007). The third env protein, which was shown to be fusogenic in an ex vivo assay was HERV-P(b) on chromosome 14q32.12. To date, not much is known about the expression of this env in cancer, but it was shown to be widely expressed in human tissues (Blaise et al. 2005). Interestingly, not much is known about the regulation of HERV expression. However, one early regulation shown for HERV was steroid hormones. The production of HERV-K virus particles and provirus in the breast cancer cell line T47D was estradiol and progesterone-dependent (Keydar et al. 1984, Ono et al. 1987). T47D cells also induced HERV-K gag, pol and env expression after estradiol and progesterone stimulation, and may be important for breast carcinogenesis. Golan

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R. Strick et al. Table 17.2 Selection of HERV expression found in different human cancers

HERV

Gene

Method

Tissue or cells

References

HERV-W HERV-W

Syncytin-1 Syncytin-1

PCR RT-PCR

Yi et al. (2001) Yi et al. (2004)

HERV-W

gag

RT-PCR

HERV-W

pol

RT-PCR

HERV-W

Provirus

EST mapping

Ovar carcinoma cell lines All tissues and cancer cell lines Brain, testis, placenta, spleen All tissues, except heart and uterus Choriocarcinoma

HERV-W

Syncytin-1

Breast cancer and cells

HERV-W

Syncytin-1

HERV-W

Syncytin-1

Immunoblot + IHC Immunoblot + RT-PCR IHC

HERV-W

pol

Microarray, PCR

Control and breast cancer

HERV-W

Syncytin-1

IHC

Colo-rectal carcinoma

HERV-S HERV-T

pol pol

RT-PCR PCR

HERV-R

ERV-3, env

Northern

HERV-R

ERV-3, env

HERV-R

ERV-3, env

cDNA hybridization Northern

Cancer cell lines Carcinoma, cancer cell lines Breast cancer, glioma, rhabdo-myosarcoma and cell lines Osteosarcoma cell line

HERV-R

ERV-3, env

HERV-R

ERV-3, env

HERV-R/ ERV9 HERV-R

pol

RT-PCR, northern Tissue microarray Microarray, PCR

env

RT-PCR

HERV-P HERV-P

gag, pol, env env

RT-PCR RT-PCR

HERV-K

Virus particles

EM

HERV-K

Virus particles

Centrifugations, RIA

HERV-K

ERV-3, env

HERV-K

gag, virus particles

cDNA hybridization IF, EM

Endometrial carcinoma + cell lines Breast cancer

Yi et al. (2004) Yi et al. (2004) Stauffer et al. (2004) Bjerregard et al. (2006) Strick et al. (2007) Larsson et al. (2007) Frank et al. (2008) Larsen et al. (2009) Yi et al. (2004) Yi and Kim (2007) Cohen et al. (1988)

Leib-Mösch et al. (1990) Lung cancer Andersson et al. (1998) Prostate cancer Wang-Johanning et al. (2003) Ovarian cancer Wang-Johanning et al. (2007) Control and breast cancer Frank et al. (2008) Liver+ lung tumor Ahn and Kim (2009) Cancer cell lines Yi et al. (2007) Colon and liver tumors Ahn and Kim (2009) Teratocarcinoma cell line Bronson et al. (1979) Breast cancer line T47D Keydar et al. (1984), Ono et al. (1987) Breast/gastric/melanoma Leib-Mösch cancer cell lines et al. (1990) Teratocarcinoma cell lines Boller et al. (1993)

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Table 17.2 (continued) HERV

Gene

Method

Tissue or cells

References

HERV-K

gag, env

Germ cell tumors, choriocarcinomas

Herbst et al. (1996)

HERV-K

Virus particles

In situ hybridization, immunoblot EM

Teratocarcinoma cell lines

HERV-K

env

RT-PCR

Breast cancer

HERV-K

gag

RT-PCR, northern Leukaemia

HERV-K

Np9, gag

RT-PCR, immunoblot

Bieda et al. (2001) Wang-Johanning et al. (2001)+ (2003) Depil et al. (2002) Armbruester et al. (2002)

HERV-K

Provirus

EST mapping

HERV-K HERV-K

Virus particles, provirus env

RT-PCR, immunoblot, IF, Tissue microarray

HERV-K

mel

PCR

HERV-K

RT

IHC

HERV-K

pol

microarray, PCR

HERV-K

RT

HERV-K

PCR, Northern, IHC gag, env, PCR, immunoblot, virus particles EM

HERV-K

env

Tissue array

HERV-K

Virus particles

EM and RNA

HERV-K

env

RT-PCR

HERV-H

env

RT-PCR

HERV-H

Provirus

EST mapping

HERV-H

env

RT-PCR

HERV-H

gag

Signal sequencing

HERV-H

env

RT-PCR

Germ cell and mammary tumors, leukaemia and cell lines Cancer of testis, uterus, Stauffer et al. stomach, brain, skin and (2004) normal brain and skin Melanoma Büscher et al. (2005) Ovarian cancer Wang-Johanning et al. (2007) Pancreatic cancer SchmitzWinnenthal et al. (2007) Breast cancer and normal Golan et al. breast (2008) Normal and breast cancer Frank et al. (2008) Normal and breast tumors Golan et al. (2008) ContrerasPlasma of patients with Galindo et al. lymphoma and breast (2008) cancer Breast cancer Wang-Johanning et al (2008) Melanoma cell lines Serafino et al. (2009) Testicular cancer Ahn and Kim (2009) T-cell leukaemia cell lines Lindeskog and Blomberg (1997) Cancer of prostate, colon, Stauffer et al. testes, bone marrow, (2004) intestine, bladder, cervix Human tissues and cancer Yi et al. (2006) cell lines Colon carcinoma Alves et al. (2008) Cancer of liver, lung, testes Ahn and Kim (2009)

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R. Strick et al. Table 17.2 (continued)

HERV

Gene

Method

Tissue or cells

HERVFRD HERV-F

pol

Microarray, PCR

Control and breast cancer

HERV-F

pol

HERV-E

env

HERV-E

env

HERV-E

provirus

HERV-E

env

HERV-E

env

HERV-E

pol

HERVE/ER

gag, pol, env

pol

References

Frank et al. (2008) RT-PCR Cancer cell lines Yi and Kim (2004) Microarray, PCR Control and breast cancer Frank et al. (2008) immunoblot Endothelial, colon and Turbeville et al. prostate carcinoma (1997) RT-PCR, northern Prostate cancer Wang-Johanning et al. (2003) EST mapping Control and breast cancer Stauffer et al. (2004) Tissue microarray Ovarian cancer Wang-Johanning et al. (2007) RT-PCR Most tissues and all cancer Yi and Kim cell lines (2007) Microarray, PCR Control and breast cancer Frank et al. (2008) RT-PCR Leukaemia cell lines Prusty et al. (2008)

IHC = immunohistochemistry, IF = immunofluorescence, EM = electron microscopy, RIA = radioimmuno assay

et al. (2008) found that HERV-K RT was more induced in breast cancer biopsies than normal tissue and that HERV-K RT positive tumors showed both a shorter overall and metastasis-free survival. In addition to HERV-K, Syncytin-1 was also found steroid hormone inducible in endometrioid endometrial carcinoma cell lines due to an estrogen response element in the LTR of HERV-W, which bound specifically to estrogen receptor alpha (Strick et al. 2007). Endometrioid endometrial carcinoma represents a steroid hormone dependent tumor and the regulation between steroid hormone proliferation and cell–cell fusion was found to be TGF-β1/β3 dependent (Strick et al. 2007).

17.2.1 HERV Expression in Human Cancers Next to the detection of the fusogenic HERVs [HERV-W, -FRD and P(b)] in human cancers and cancer cell lines, multiple non-fusogenic HERVs were identified in different human cancers (Table 17.2). Especially, gag-pol- and env-gene expression from four HERV families was found associated with different cancers and cancer cell lines: HERV-E, HERV-H, HERV-R and esp. HERV-K (Table 17.2). In particular, not only gene expression of HERV-K families were found associated with different human cancers (cell lines), but also HERV-K virus particles were found. For example, HERV-K gene expression and particles were found associated with advanced

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state melanoma and related with the switch from adherent to non-adherent cell growth. RNAi-experiments specific for HERV-K prevented this transition (Serafino et al. 2009). To date, infectious HERV-K has never been detected even after co-cultivation with target cells. In addition, several reports showed that human patients exhibited serological response to HERVs, like HERV-K gag and env in melanoma, prostate and testicular cancer (Hahn et al. 2008, Ishida et al. 2008, Goedert et al. 1999). The fusogenic Syncytin-1 of HERV-W was found in breast, endometrial and colon cancer. Interestingly, in breast cancer Syncytin-1 was expressed in 38% of cases and correlated with a positive prognostic role, whereas in a retrospective analysis of colorectal cancers over-expressed Syncytin-1 was associated with a decreased overall survival of rectal but not of colon carcinomas (Larsson et al. 2007, Larsen et al. 2009). Taken together strong in vitro evidences point to a direct involvement of single HERV genes in cancer.

17.2.2 HERVs in Cancer Cell–Cell Fusions: Driver or Passenger HERV gene expression and over expression are detected in primary human tumors, but evidence to prove a direct connection of HERV env genes with cell–cell fusions is ongoing (Table 17.2). So far, as stated before only three HERV env genes, Syncytin-1 (HERV-W), Syncytin-2 (HERV-FRD) and the env of HERV-P(b) were shown to be involved in cell–cell fusions in vitro (Blaise et al. 2003, Blaise et al. 2005, Strick et al. 2007). The main candidate of fusogenic env gene involved in cancer cell fusions is Syncytin-1 in choriocarcinoma, breast cancer and endometrial carcinomas. Several choriocarcinoma cell lines showed Syncytin-1 and Syncytin-2 and their receptors ASCT-1/-2 and MFSD2 expression and some of them cell–cell fusions (Kudo and Boyd 2002, Borges et al. 2003, Esnault et al. 2008) For breast cancer cell lines expressing Syncytin-1 cell fusions occurred between the cancer cells but also with endothelial cells. These cell–cell fusions were inhibited by antisense RNA as well as with an inhibitory peptide against Syncytin-1 (Bjerregaard et al. 2006). Expression of Syncytin-1 and the receptor ASCT-1 in breast cancer tumors and endothelial cells were also detected proposing that cell–cell fusion of breast cancer cells and endothelial cells could also occur in patients. This is similar to nude mouse endothelial cells fusing with human breast cancer cell lines (Mortensen et al. 2004, Larsson et al. 2007). In another human tumor, endometrioid endometrial carcinoma, Syncytin-1 and ASCT-1 and -2 receptor expressions were found significantly higher than in control endometrium. Cell–cell fusion experiments with endometrial carcinoma cell lines were demonstrated as dependent upon Syncytin-1 expression using RNAi experiments. Multinucleated cells in endometrial carcinomas were found in paraffin sections proposing that Syncytin-1 expression could be connected with cell fusions in this carcinoma (Strick et al. 2007). Similar experiments were done in ovarian carcinomas and a direct connection of Syncytin-1 with cell–cell fusions was found in vitro (Strick et al. in preparation). Similar to e.g. Mason Pfizer virus env gene, which was enough to induce cell fusions (Duelli et al.

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2005) many experiments with Syncytin-1 demonstrated the sufficiency of the env (or even parts of env) to mediate cell fusions as well as C-terminal truncations. For example, truncations up to 55 or 58 C-terminal amino acids of the 538 amino acid Syncytin-1 increased fusogenicity in human cancer cells (Drewlo et al. 2006, Chang et al. 2004), but in contrast C-terminal truncations of 67 or 82 amino acids inhibited cell fusions (Cheynet et al. 2005, Chang et al. 2004).

17.3 Cell–Cell Fusions in Cancer: Functional Role or Dead-End Polyploidy and multinucleation are hallmarks of many types of human malignancies, leading to genetic instability and aneuploidy and development of cancer (Hanahan and Weinberg 2000). In addition, highly malignant cells generally show higher ploidy levels than their less malignant progenitor tumor cells (review: Larizza and Schirrmacher 1984). Cancer cells may fuse spontaneously with several types of resident somatic cells, esp. with BMDC and these hybrids are proposed to play an important role in metastasis (Pawelek and Chakraborty 2008, Liu et al. 2006, Jacobsen et al. 2006, Vignery 2005). Several consequences of fused cancer cells in tumorigenesis are possible: (1) Cancer cell fusions result in karyogamy, cellular divisions and new tumorigenic functions; (2) Cancer cell fusions stay multinuclear and progress into cell cycle arrest or divide; or (3) the cancer cell hybrids apoptose (Fig. 17.3). In favour for the first possibility are the examples of detected tetraploid cells in tumors and their functional role in the progress of tumorigenesis (Ganem et al. 2007, Margolis 2005). On the other hand, many examples of tumors are known of the existence of multinuclear cells in tumors, however if they are permanent or transient is not known (Table 17.1). Then again many evidences are in favour of the third possibility, but with modifications. According to some references are cell fusions due to intact viruses doomed to either apoptose or cell cycle arrest (Zhivotovsky and Kroemer 2004). Another possibility is the phenomenon of mitotic catastrophy, which was defined as a type of cell death occurring during mitosis. Besides e.g. microtubule poisons and over-duplication of centrosomes, mitotic catastrophy can also be induced by fusion of mitotic cells with interphase cells, but also be the destiny of common multinucleated cells, which undergo karyogamy and metaphase and result into apoptosis or division with an asymmetric cell division (Castedo et al. 2004 and Fig. 17.3). One checkpoint regulator for a direct apoptosis after karyogamy of multinucleated cells was Chk2 (Castedo et al. 2004). Although many sources speak for apoptosis or cell cycle halt of fused cancer cells, the existence of multinucleated cells in tumors, like giant cell glioblastoma etc. are established and not just a rare accident and play even a role for prognosis. The fate of cancer cell-somatic cell hybrids could depend upon the existing cellular RNA and proteins, which are brought into the hybrid and could result to: (1) a gain of functional tumor suppressor genes with the consequence of reduced malignancy as shown with fusions after somatic cell hybridizations or inactivated viruses (Harris et al. 1969, Harris 1988, Stanbridge 1976, Wiener et al. 1974a), or (2) a loss of tumor suppressors (for review: Anderson and Stanbridge 1993). It was shown that malignant and normal human cell fusions resulted into a suppression of tumor

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Fig. 17.3 Possible consequences of cell–cell fusions between normal somatic (2n) or normal stem cells (2n) with tumor cells. Two main outcomes are distinguished: with and without fusion of nuclei (karyogamy versus multinucleation). Different reports showed that karyogamy after cell– cell fusion and repeated cell divisions resulted in loss or gain of specific chromosomal genes (aneuploidy). Several reports also proposed the possibility of novel so called cancer stem cells after cell–cell fusions and karyogamy, which can be self renewing or lead to new tumor cells. This scenario could also be conceivable with multinucleated cells

formation, whereas cancer cell–cell fusions kept their malignancy (Stanbridge 1976, Wiener et al. 1974b). On the other hand cell–cell fusions between cancer cells and BMDCs, esp. macrophages have an important impact on tumorigenesis, like metastasis and proliferation. For example, the detection of rectal cancer cells expressing macrophage specific proteins (CD163) probably due to cancer cell fusions with macrophages had very high metastatic activities (Shabo et al. 2009), or cancer-macrophage fusion hybrids caused higher proliferation and vascularisation of tumors (Busund et al. 2002). Important is the possibility of even partial reprogramming of the somatic cell or even of the cancer cell after fusion. This was found esp. for BMDC-somatic cell hybrids after cell fusions, because tissue-specific genes normally not found in haematopoietic cells were found in skeletal muscle cells (Camargo et al. 2003, Gussoni et al. 1999, Ferrari et al. 1998), brain cells (Weimann et al. 2003) and liver cells (Vassilopoulos et al. 2003, Lagasse et al. 2000); probably due to reprogramming. This kind of reprogramming after cell–cell fusion could be particularly important if one partner is a BMDC or a stem cell, cells which are highly plastic, and the other partner is a resident somatic cell. A cell–cell fusion could then generate new hybrid pluripotent cells, which could have the potential to differentiate tissue or regenerate damaged tissue. Conversely, if this reprogramming is deregulated, this would provide a mechanism for cancer development.

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A deregulation of reprogramming could lead to an up-regulation of genes involved in invasion and metastasis like fusion of migratory bone marrow cells and cancer cells (for review: Pawelek and Chakraborty 2008). On the other hand the new fused hybrid cells could also be capable of being a cancer stem cell as suggested by Bjerkvig et al. (2005). This is in particular interesting for the tumor development and metastasis. Cancer stem cells were already demonstrated in leukaemia, breast cancer (Al-Hajj and Clarke 2004) and brain cancer (Singh et al. 2004, Galli et al. 2004). More evidences speak also for a functional role of cell fusions with regard to tumor metastasis, esp. the sustainment of the different tropisms of the single cells after fusion. It was shown that spontaneous cell fusion between bone-tropic and lung-tropic cancer cells generated stable hybrids with dual metastasis tropism to both organs without nuclear reprogramming in vitro and in vivo (Lu and Kang 2009). In addition to the acquisition of metastasis properties after cell fusion, the latter report also demonstrated a high level of chromosomal and phenotypic stability in hybrids during long-term passage in vitro and in vivo. These results demonstrated that the cancer cell hybrids maintained the genomic, transcriptomic and phenotypic characteristics of the original mononuclear cells. Chromosome stability after cell fusion was also demonstrated with other cell systems, e.g. fusion beween HeLa and fibroblasts (Stanbridge et al. 1982), between mouse mammary tumor cells (Miller et al. 1988) or between embryonal stem cells and fibroblasts (Cowan et al. 2005). Considering the genomic maintenance, but also the equality of different cells, cell fusion of human and mouse cells demonstrated expression of both parental partners in the hybrid (Chakraborty et al. 2001). Therefore, cell fusion of cancer cells with different metastatic abilities or fusion of cancer cells with somatic cells could increase the metastatic spreading. Significant for the treatment of cancer could also be the treatment resistance of cancer cell hybrids, because survival in other organs could be possible due to new tropisms and outright acquired chemoresistance after cell fusion. Many fundamental cell–cell fusion questions about cell–cell fusions are not completely answered up to date, for example: • How are cell–cell fusions regulated? • Which specific proteins (ligands-receptors) drive the cell–cell fusion process and which roles do HERVs play? • Do fused cells have transcriptional and translational changes compared to the same but non-fused cells? • What determines the dominance of one nucleus after cell–cell fusion and why are in some cases nuclei equal after cell–cell fusions? • What role does the existing RNA and proteins of each fusing cytoplasma play? • What factors prevent and support karyogamy after cell–cell fusion? • Which multinucleated cells are in cell cycle arrest (with or without transcriptionally active) or divide? • The unravelling of the phenomenon of cell–cell fusions, in normal human development and in carcinogenesis will help to explain the aetiology of some cancers, metastasis, relapse, and possibly treatment resistances.

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Index

A Abi, 155 Acetylation, 130, 132 Acrosin, 188 Acrosomal exocytosis (AE), 187–189, 191, 195–196 Acrosomal matrix, 187–188, 196 Acrosomal shroud, 187–188, 196 Acrosome, 181, 187–189, 191, 195, 281 Actin, 2, 4, 54–55, 139–165, 190, 238, 241, 254, 257–258, 270, 272 Actin filaments, 154, 254 Actin foci/plugs, 152–153, 156, 161 Actin ring, 238, 241 ADAM, 5, 173, 193, 268, 283–286 ADAM12, 193, 207, 239 ADP ribosylation factor (Arf), 145, 152, 258 AKT2, 407 Allantois, 119 Allogeneic DC, 328–329, 334–336, 338 Allogeneic tumor cells, 329 Alpharetroviruses, 22, 25, 69 Amitosis, 287 Amoeboid motility, 361 Amphotericin B, 50, 52 Amphotropic, 15, 45, 48 Anchor-cell fusion failure-1 (AFF-1), 225 Aneuploidy, 6, 354, 367, 397, 399, 403, 414–415 Antigen cross-presentation, 320 Antigen presentation, 321, 330, 332 Antigen-presenting cells, 164, 178, 318 Antisense, 31, 88–89, 100, 122, 129, 206, 210–211, 270–271, 405, 413 Antisocial, 151 Ants, 151, 157 AP-1, 73, 98

Ap-2, 73, 98 Apoptosis, 6, 44, 99–100, 103, 128, 134, 193, 207–208, 224, 403, 414 Appressorium, 176 Arp2/3, 157, 161 ASCT1, 4, 29, 78, 126, 143, 207, 269–270, 409, 413 ASCT2, 29–30, 45, 78, 101, 123, 126, 133, 207, 269–270 Astrocytes, 102, 133–134 Autologous DC, 328–329 Autophagosomes, 356–357, 359, 370, 373–374, 377–381 Autophagy, 6, 357, 369–384 Avian leukosis virus (ALV), 23–25, 50, 69 B Basolateral membrane, 74 Betaretroviruses, 22–26, 69, 71, 408 BeWo cells, 87, 89–90, 98, 100, 121, 124, 127, 129–130, 207–211, 213–214 Blastocyst, 205, 291, 303, 307 Blood–brain barrier, 338 Blow, 144, 152–153, 155–157, 159, 162 Blown fuse, 152 BM40, 354, 367 Bone marrow-derived cells (BMDCs), 6, 280, 290–291, 293, 296–301, 303, 306, 354–355, 363–365, 367–368, 383–384, 402–404, 407, 415 Bone mass, 227, 241, 244 Bone resorption, 5, 222, 238–239, 241, 244 β1,6-Branched oligosaccharides, 357, 359, 362–363, 365, 369–381, 383–384 Breast cancer, 4, 80, 103, 129, 131–133, 269, 320, 329, 336–339, 359, 361, 404–405, 407, 409–413, 416

L.-I. Larsson (ed.), Cell Fusions, DOI 10.1007/978-90-481-9772-9, C Springer Science+Business Media B.V. 2011

427

428 C Ca2+ , 21, 100–101, 174, 188–189, 209, 222 CAAT box, 98 C2C12 cells, 141, 144–145, 160–161 Cadherin, 174, 268 Caenorhabditis elegans, 2, 400 Calcium, 2, 188–189, 238, 256, 259, 261 Calcium signaling, 238 Calpain, 209 CAMP, 3, 5, 98, 100, 103, 121, 129, 132–133, 208, 254, 256, 272, 409 Cancer cell fusion, 5–6, 269, 351–385, 407, 413–415 cells, 3–7, 103–104, 129, 131–133, 243, 269, 299–301, 320, 329–330, 351–385, 398, 404–407, 409–416 stem cells, 229, 299–302, 355, 364, 415–416 vaccines, 317, 340 Capacitation, 187, 195 Cardiomyocytes, 280, 298–299, 398, 403 Caspase, 5, 8, 100, 207, 209–211, 214 Cathepsin, 50–52, 241 CD151, 176, 190, 194, 284–285 CD200, 5, 223, 226–229, 240–242 CD200R, 5, 223, 226–229 CD4, 15–17, 44–45, 51, 53, 71, 77, 320–321, 328, 331, 340–341 CD4+ T cells, 71, 320, 328, 340–341 CD47, 5, 223, 226–229, 239 CD8+ T cells, 321, 328, 331, 341 CD81, 4–5, 176–177, 180, 190, 192, 194, 196, 239–240, 268, 284, 285 CD9, 4–5, 66–67, 171–181, 189–192, 194, 196, 239–240, 258, 268, 272, 284–285 CD9-EGFP, 178–179 C. elegans, 155, 225, 400 Cell adhesion molecule, 2, 4, 53–54, 141, 143, 148–153, 156–157, 160–161, 163, 165, 173–174, 191–192, 239, 256–258, 268, 283–284, 289, 298, 360, 370 Cell division control protein 42 homolog (Cdc42), 144, 155, 161, 258 Cellocytose, 227–228 Cellular protooncogene homologous to Rous sarcoma virus (Src), 238, 259–260 c-Fos, 237, 242 Chemotaxis, 354, 357–359, 366, 368, 382–384 Choriocarcinomas, 208, 213, 406, 410–411, 413 Chorion, 119 Chorionic epithelium, 119

Index Chorionic villi, 95, 119, 205–206 Chromosomal instability, 104 Class I and II fusion proteins, 67 Class III fusion proteins, 67 Clinical trials, 336–339 Clonal expansion, 222 cMet, 368 c-Myc, 305 Coarse melanin, 356–359, 370–371, 373–379, 381 Cofilin, 54, 154 Coiled coil helices, 19 Coiled coil structures, 18, 19–20, 67, 77 Colitis, 294, 398 Collagen folding, 367 Colorectal cancer, 131, 133, 336, 413 Colorectal tumors, 293 Confocal fusion analysis, 291, 295, 327 Connexin43, 239 Co-receptor, 15–16, 17, 25, 77, 92 Co-receptor blockers, 16–17, 25, 77, 92 Costimulatory ligands, 331 CpG methylation, 94, 99, 130 Creatine kinase, 251 CREB binding protein (CBP), 129, 130, 132 Cre recombinase, 161, 290, 292–293, 303 CRISP1, 195 Crk, 144, 152, 156–157, 160 Crkl, 144, 160 CX6 CC, 70, 72, 90, 123, 125 CXXC motif, 20–21, 24, 32, 70, 72–73 Cyclic AMP, 98, 368 Cytokine, 102, 133, 206–207, 236–237, 255, 259–260, 298, 330, 339, 368 Cytokinesis, 7, 152, 235, 293, 397–398, 401, 406 Cytoskeleton, 2, 4, 6, 52, 54–55, 141, 153, 162, 165, 189–190, 209–210, 257–258, 268, 272 Cytotoxic T lymphocytes (CTL), 319–320, 329, 330, 332, 339–341 Cytotrophoblast (CT), 94–97, 99–102, 119–120, 129, 205–206, 209–210, 212–213, 409 D Dedicator of cytokinesis (DOCK), 140 Delta, 142 Deltaretroviruses, 23–24, 69 Dendritic cell maturation, 330–331 Dendritic cells (DC), 7, 24, 177–178, 180, 221–222, 227, 237, 315–342 Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), 24–25

Index Dendritic cell-specific transmembrane protein (DC-STAMP), 5, 222–223, 226–227, 241–243 Disintegrins, 173, 187, 192–194, 207, 268, 283 Disulfide bond isomerization, 32 Disulfide isomerase motif, 125 Disulfide isomerization, 20, 23–24, 32 DNAX-activating protein 12 (DAP12), 222, 238–240, 242 Dock180, 144, 152, 155–156 DRAP27, 175 Drosophila, 4, 6, 67, 98, 139–165, 244, 257–258, 261, 287–288, 361 Duf/Kirre, 142–144, 150–153, 157, 160, 163 Dumbfounded/Kin of irre, 150 E E-cadherin, 239, 360–361, 367 Ecotropic, 15, 22, 43, 45–46, 48–51 Effector T cells, 338 Egg, 5–6, 17, 66, 154, 177–181, 187, 189–196, 205, 239, 244, 281–285, 399 Egg exosomes, 179–181 Electrofusion, 325–328, 332, 334–336, 338 Electron-dense plaques, 146–148, 157, 160 Embryogenesis, 142, 151, 154, 205, 250, 287, 359–360 Embryonic stem cells, 66, 176, 229, 279–280, 282, 286–287, 290, 296–297, 302–305, 307, 401–402 Endogenous class I processing pathway, 328 Endogenous JSRV (enJSRV), 30–31, 68, 83–84, 86–88, 90–92, 97, 122 Endogenous retroviruses (ERVs), 13–14, 23, 26–32, 68, 81–82, 86, 88, 97, 103, 120, 395–416 Endometrial carcinoma (EnCa), 103, 120, 133, 281, 334–335, 406, 410, 412–413 Endomitosis, 7, 397, 404, 406–407 Endoplasmic reticulum (ER), 15, 17, 44, 47, 69–72, 102, 134–135, 237, 367, 381 Endopolyploid, 397 Endoreduplication, 397–399 Endosomes, 18–19, 25, 50–51, 73–74, 78–79, 127, 175, 319 Endothelial cells, 3, 103–104, 133, 270, 355, 369–370, 405, 413 Enfuvirtide, 19 Enhanced green fluorescent protein (EGFP), 178–179 Enveloped viruses, 15, 43, 69, 75, 400, 402 Envelope (env), 3, 13–14, 16, 27–32, 68–83, 94–97, 120, 126, 211, 267–273, 408–414

429 Envelope protein, 3, 13, 15–17, 19, 21–26, 30–32, 67, 71, 81, 87, 89–90, 92, 97, 99, 101, 120–121, 124–127, 134, 211, 405 Epididymal glycoprotein DE, 195 Epigenetic regulation, 130–131 Epigenetic state, 308 Epithelial fusion failure (EFF-1), 165, 225, 268, 400 Epithelial-mesenchymal transition (EMT), 302, 354, 360–361, 367–368 Epsilonretroviruses, 22–23, 69 Equatorin, 194–195 ER stress, 134–135, 381 ERV-3, 89, 211, 410 ERVWE1 transcripts, 80, 82–83, 85–87, 97, 104 E-selectin, 369–370 Estradiol, 103, 409 Estrogen, 133, 412 Exosomes, 5, 75, 177–181, 191, 243 Experimental hybrids, 355 Extracellular degradation, 224 Extracellular lysosome, 224 Extracellular signal-regulated kinase1/2 (ERK), 207–208 Extravillous trophoblast, 205, 212, 400 Ezrin, 54 Ezrin-radixin-moesin (ERM), 4, 54, 190 F F-actin plugs/foci, 141, 151, 153, 156, 163 FF family of fusogens, 225 FBW2, 130, 132 Fertilin β, 193–194, 283 Fertilization, 5, 17, 44, 172–175, 178–179, 181, 187, 189–192, 194–196, 205, 239, 244, 281–285, 296 Filamentous (polymerized) actin (F-actin), 4, 141, 151–159, 161–164, 272 Filopodia, 3, 145–146, 150, 162, 192, 254–256, 271–272, 401 FK506, 242 Flippase, 100, 208 Fluorescence activated cell sorting (FACS), 327 Foamy viruses, 25–26, 69 α-Fodrin, 209–211 Follistatin, 259–261 Foreign body giant cell (FBGC), 239, 241, 302 Forskolin, 121, 127, 208, 213, 409 Founder cell (FC), 142–143, 145–146, 148–151, 157, 162, 164, 222, 321, 324, 328, 330, 332–336, 338–342

430 Fumarylacetoacetate hydrolase (FAH), 243, 297 Fungus, 176–177 Furin, 47, 70, 72, 90, 123–124 Fusion competent myoblast (FCM), 146–153, 155, 157–159, 162–164, 251, 255, 261, 287 Fusion efficiency, 280–281, 324–329, 332–335, 338, 342 Fusion index, 89, 161, 252, 270 Fusion inhibitors, 19, 25, 31, 91, 208 Fusion peptide (FP), 18–19, 24–25, 31, 46, 50, 67, 69–70, 72, 76–77, 79, 84, 89, 123, 125, 127, 225, 283, 402 Fusion pores, 18, 46, 67, 76–77, 79–80, 157–158, 161–163, 209, 258 Fusion proteins, 20–21, 25, 27, 30, 66–67, 77, 79, 90, 172, 209, 225, 268, 283, 285, 402 Fusion restricted myogenic adhesive structure (FuRMAS), 4, 139–165 Fusion theory, 353–354, 385 Fusion from within, 22 Fusion from without, 5, 22 Fusogen, 3, 162, 164, 211, 225, 229, 318, 321, 324–327, 400 Fusogenic membrane glycoproteins (FMG), 324, 326–327, 335 G β-Galactosidase, 252, 290–292, 295, 404 Galectin-3, 369 Gammaretroviruses, 13, 20–23, 26, 29, 32, 69, 71, 408 Ganglioside, 178 Gap junctions, 207, 239 GCM1, 3–4, 128–132, 134, 207–208 GCM1-knockout, 128–129 GCMa, 98, 272 Genomic instability, 6, 300–301, 397 Germ line infection, 26 Giant cells, 5, 43, 66, 88, 95–97, 212, 221–225, 235, 237, 242–243, 302, 398–400, 402, 405 Giant nuclei, 399, 406 Gleefull, 143 Glial cell missing (GCM), 98, 128, 132, 207–208 Glioblastoma, 370, 399, 405–406, 414 Glioma, 333–334, 336–337, 340, 355, 410 Glucocorticoid receptor, 98 Glucose transporter 1 (GLUT1), 24, 45 Glycosylation, 45–46, 49, 70–72, 84–90, 92, 124, 191, 195, 256, 357, 369

Index Glycosyltransferase, 357–359 Gp160, 127, 134 GPI-anchor, 93 Granulomas, 224, 398 Green fluorescent protein (GFP), 153, 158, 161, 288, 291–293, 295, 297–298, 302–303, 306, 326–327 Group-specific antigen (gag), 13–14, 26, 28, 49, 69, 73–75, 86, 97, 120, 408–413 Growth factor, 119, 206–207, 222, 250, 259–260, 268, 338, 359–360 Growth hormone, 259–260 GSK-3β, 130–132, 134 H Hairpins, 19, 46, 67, 77, 79–80, 161, 268 β-hCG, 213–214 Heart, 119, 279, 298–299, 303, 404, 410 HELLP syndrome, 102, 131 Hemagglutinating virus of Japan (HVJ), 326 Hemagglutinin (HA), 17–19, 50, 79, 127, 134, 402 Hemifusion, 18–19, 66–67, 76–77, 79–80, 147, 149, 282 Hemolysis, elevated liver enzymes and low platelets (HELLP), 102, 131, 400 Hepatocyte growth factor (HGF), 360, 368 Hepatocytes, 176, 243, 280, 291, 294, 297–298, 398, 403–404 Heptad repeats (HR), 18–20, 24–25, 31, 69–70, 72, 77, 124–125, 127 Herpes simplex virus, 102 HERV-FRD, 29, 82, 85, 87, 90, 121, 127, 211, 408–409, 413 HERV-H, 81, 411–412 HERV-K, 27, 30, 81–82, 408–413 HERV-P(b), 29, 409, 413 HERV-R, 89, 410, 412 HERV-W, 27, 29, 80, 82, 85–87, 91–94, 121, 211, 400, 405, 408–410, 412–413 Heterokaryons, 7, 66, 280, 301–302, 304–305, 307, 318–321, 332, 382, 399 Heterophilic cell-cell fusion, 243 Heterotypic fusions, 6–7, 66, 280 Hibris (Hbs), 143–144, 150, 152, 323 Histone acetylation, 130 HIV-1 co-receptors, 77 Hodgkin lymphoma, 405, 407 Homeostasis, 2, 221, 224, 295, 307, 404 Homotypic fusions, 7, 66, 225, 280 Homotypic interaction, 225 HSP70, 178 HSP90, 178–179 HSPC300, 155

Index Human endogenous retrovirus (HERV), 7, 14, 23, 26, 27–32, 44–45, 99, 101, 104, 121, 134, 269, 395–416 Human immunodeficiency virus (HIV), 5, 15–17, 19, 25, 43–45, 47–55, 69, 134, 225 Human immunodeficiency virus type 1 (HIV-1), 13, 15, 17, 19, 23–25, 53, 67, 71–74, 77–80, 94, 172, 177, 180, 402, 409 Human placenta, 87, 90, 95, 99, 119, 121, 178, 205–206, 208, 211–212 Human T-cell lymphotropic virus type 1 (HTLV-1), 23–25, 32, 73 Human Teratocarcinoma-Derived Virus, 81 Hyaluronidase 2 (HYAL2), 30–31, 68, 92–93, 97 Hybridomas, 7, 318, 324, 365–366 Hypoxia, 102, 131, 354 I IFN-γ, 101–102, 331, 340 IgSF8, 190, 192 IL-1β, 98, 133 IL-4, 101, 222, 236–237, 239, 241–242, 259–260, 268, 330 IL-10, 294, 306, 338, 360 IL-12, 329–330, 332–337, 339–340 IL-13, 259–260 Immunoglobulin superfamily (IgSF), 150, 190–192, 257 Immunological synapse, 164 Immunosuppressive, 24, 29–31, 70, 89, 102, 121–122, 134–135, 339 Imprinting, 98 Induced pluripotent stem, 302, 305 Inflammation, 229, 279, 291, 294, 306, 399, 404, 407 Inflammatory bowel disease, 294 Initiator caspase 8, 209 Inner acrosomal membrane (IAM), 188, 195 Insulin growth factor 1 (IGF-1), 259–260 β1-Integrin, 268, 283, 370 Integrin alpha3beta1, 176 Integrins, 53–54, 173, 175–176, 187, 192–194, 238, 254, 257–258, 268, 283–284, 340, 369–370, 383 Intercellular syncytial fusion, 212 Interference, 16–17, 21, 29, 67–69, 78, 84, 93, 99, 101, 126, 129, 133, 227, 271–272, 289 Intestinal epithelial stem cells, 291, 293 Intracytoplasmic sperm injection (ICSI), 181, 194

431 Invadopodia, 164 IZUMO, 5, 66, 174–175, 191–192, 196, 285 J Jaagsiekte sheep retrovirus (JSRV), 15, 23, 25, 30, 50, 72, 78, 83, 86, 92–93, 97 JAR cells, 130 JEG-3 cells, 129 JSRV receptor, 93 K Karyogamy, 397, 404, 414–416 Kette, 145, 153, 155–157, 159, 161 Ki67, 210, 294 Kirrel, 144, 288 Klf4, 305 Koala retrovirus (KoRV), 82 L Labyrinth, 68, 88, 96, 98, 119–120, 122, 128 Labyrinth zone, 96 LacZ, 252, 290 Lameduck (Lmd), 143 Lamellopodia, 254 LAMP-1, 357, 369–370, 383 Langhans’ giant cells, 398 Large extracellular loop (LEL), 175–176, 189–190 Lens, 401 Lentiviruses, 22–23, 25–26, 69, 72–73, 75, 82 Lentivirus lytic peptides (LLP), 73 Leukocyte-specific protein tyrosine kinase (Lck), 55 Leukocyte-tumor hybrids, 330, 360–361, 365, 369, 371, 399 LIN28, 305 Lineage commitment, 279, 290 Lipid rafts, 22, 52–53, 55, 74, 80, 187, 256 Lmd/Minc/Gfl, 143 Long Terminal Repeats (LTR), 4, 26, 69, 85–86, 94, 97–99, 120, 129–132, 408, 412 LoVo cells, 124 Lung cancer, 92, 406, 410 Lung carcinoma, 336, 406–407 Lymphocyte, 24–25, 44, 74, 81, 101, 178, 239, 319, 322, 332, 340, 366–367 M Macrophage activating factor (MAF), 236 Macrophage colony stimulating factor (M-CSF), 207, 220, 222–223, 236, 238, 242, 360 Macrophage fusion factor (MFF), 236

432 Macrophage fusion receptor (MFR), 5, 223, 226–228, 239 Macrophage giant cell (MGC), 235–237, 239, 240–244 Macrophage-melanoma hybrids, 357, 365–366, 368–371, 383 Macrophages, 5–7, 66–67, 74, 102, 180, 190, 206–207, 219–230, 233–244, 280, 297, 301–302, 306, 340, 354–357, 359–363, 365–383, 399, 404–405, 415 Major facilitator superfamily domain containing 2 (MFSD2), 30, 68, 93–94, 96, 123, 126–127, 129, 134, 269, 409, 413 Mammalian target of rapamycin (mTOR), 259, 261 Mannose receptor, 237, 239, 255, 259–260, 337 MARVEL domain, 162–163 Mason-Pfizer monkey virus (MPMV), 23–24, 48, 124 MCF-7 cells, 131, 329 Megakaryocytes, 398 Melanocortin-1 receptor (MC1R), 368, 383 Melanomas, 357, 359–361, 369, 371, 381, 405–406 Melanophages, 371, 376, 379–382 Membrane vesicles, 178–179, 187–189, 196, 213 Membrane vesiculation, 145–149, 151, 162–163, 187 Mesenchymal stem cells, 291 Metastasis, 103–104, 175, 229, 243–244, 308, 331, 333, 336, 338, 340–342, 353–355, 357–363, 365–366, 368–370, 383–385, 404–405, 412, 414–416 Metastatic phenotype, 353–354, 360, 365–383 Methylation, 3–4, 26, 94, 98–99, 104, 130, 173, 304, 408 Methyl-β-cyclodextrin, 52–53 MIC3, 175 Microdomains, 2–4, 52–53, 55, 75, 80, 190, 285 Microphthalmia-associated transcription factor (MITF), 222, 368, 383 Micropores, 325 Microvilli, 178–181, 195 Migration, 3, 6–7, 90, 120, 148, 155, 163, 191, 252–253, 255–256, 260, 289, 331, 354, 361, 363, 369–370, 384, 400–401, 406 Mind bomb 2 (Mib2), 143–144 Mitogen-activated protein kinases, 208 Mitotic catastrophy, 414 MMP9, 240–242

Index MN9, 5, 185, 194–195 Moesin, 4, 54, 190 Monoblasts, 221 Monocytes, 5, 194, 221, 243, 321, 331, 361, 367–368, 383 Mononuclear phagocyte system (MPS), 221 Monosialo ganglioside 3 (GM3), 178–179 Motility, 175, 190, 192, 195, 255, 259–260, 354, 357, 360–361, 367–370, 383 Motility-related protein 1 (MRP-1), 175 Mouse, 15, 23, 25, 27, 43, 45–46, 50, 67–69, 78, 81, 83, 86–89, 92, 94, 96–97, 119–120, 122, 128, 133–134, 144–145, 160, 176, 178–180, 188, 190–193, 195, 251–253, 255–257, 259, 283–284, 292–295, 297–298, 303–306, 327, 333, 355–358, 360, 366–367, 370, 401, 405, 408, 413, 416 placenta, 88, 96, 119, 122 Mucin 1 tumor-associated antigen (MUC1), 329–330, 332–333 Multinucleation, 221–229, 235–237, 239, 241–242, 244, 288, 397, 403, 404, 406, 414–415 Multiple sclerosis (MS), 80–81, 102–103, 133–134 Multiple Sclerosis associated RetroVirus (MSRV), 80–82, 101, 121, 409 Multivesicular bodies (MVB), 74–75, 177, 243 Murine leukemia viruse (MLV), 13, 15–17, 22–24, 31–32, 43, 45–52, 67, 69, 71, 73–75, 77, 80, 90–94, 124–125, 128 Muscle fibers, 6–7, 44, 49, 51, 55, 66, 205, 268, 286–287, 317, 403 Myelin, 102, 133 Myeloma, 6, 227, 318, 330, 333–334, 336, 340, 363, 365, 384, 407 Myoblast adhesion, 256 Myoblast alignment, 148, 254, 256 Myoblast city (Mbc), 144, 152, 155–157, 160–161 Myoblast elongation, 6, 254 Myoblast incompetent(Minc), 143 Myoblast migration, 155, 256, 260 Myoblast-myotube fusion, 259–261 Myoblast recognition, 157, 256 Myoblasts, 3, 6–7, 49, 66–67, 139–165, 209, 239, 249–273, 281, 286–289, 303, 400–401, 403 Myocardial-infarction, 298 Myoferilin, 261 Myofibers, 250, 253, 256, 259–261, 268

Index Myogenesis, 22, 49, 52, 161, 165, 254–256, 258, 261, 268 Myogenin, 250–251, 270 Myonuclear number, 252, 259–260 Myosin heavy chain, 251 Myostatin, 259–260 Myotubes, 6–7, 142–153, 155–160, 162–164, 208, 244, 251–252, 254–256, 258–261, 268, 270–272, 281, 289, 400–401 N N-acetylglucosaminyltransferase V (GnT-V), 357, 369 Nanog, 305 Nap1, 153, 155, 161, 258 Nascent myotube, 6–7, 159–160, 251, 255, 258–261, 289, 400 Neph1, 144, 160 Nephrin, 144, 160, 259, 261 N-ethylmaleimide-sensitive factor (NSF), 188–189 Neural stem cells, 279, 289–291, 304–305, 309 Neurotropin, 361 NF-κB, 98, 102, 222–223, 226, 329 Nitric oxide (NO), 102–103, 195 NO synthetase, 102 Notch, 142 Nuclear factor of activated T-cells (NFAT), 242, 259–260 Nuclear factor of activated T-cells c1 (NFATc1), 222, 242–243, 259 Nuclear factor of activated T-cells c2 (NFATc2), 259–260, 268 Nuclear reprogramming, 6, 290, 302–305 O Oct4, 302–303, 305 Old astrocytes specifically induced substance (OASIS), 102, 118, 134 Oligodendrocytes, 102, 133–134 Oligodeoxynucleotides containing CpG motif (CpG ODN), 326, 331, 335, 339–340 Oncoprotein, 92 Oocyte, 66, 175, 180, 187, 190–191, 193–196, 281, 283–286 OK-432, 331, 338, 340 Osteoblast, 236–237, 241, 244 Osteoclast differentiation factor (ODF), 237 Osteoclastogenesis, 224, 226–227, 237–238, 240, 242, 401 Osteoclastogenesis inhibitory factor (OCIF), 237 Osteoclasts, 3, 5, 221–223, 227, 236–239, 241–242, 244, 363, 367, 384, 404–405

433 Osteonectin, 354, 367 Osteopetrosis, 222, 224, 227, 238, 400 Osteoporosis, 5 Outer acrosomal membrane, 187–188, 196 P P21, 251 Parabiosis, 293 Parathyroid hormone, 237 Pathogen-associated molecular patterns (Pamps), 228 Perivitelline space (PVS), 178–179, 181, 191 Phagocytosis, 5, 78, 228–229, 237, 282, 327, 370, 379, 383–384 PH-dependent fusion, 78 PH-independent fusion, 78 Phosphatidylserine, 5–6, 100–101, 208–209 PI-3K/Akt signaling, 132 Placenta, 3, 5, 27, 29–31, 44–45, 66–68, 80, 83, 87–91, 94–104, 119–122, 127–132, 134–135, 165, 178, 190, 205–206, 208–209, 211–214, 269, 286, 296, 298, 307, 396, 400, 408–410 Placental protein, 213 Plasma membrane, 4–5, 15–16, 22, 25, 43, 51–52, 66, 69–70, 74–75, 89, 99–100, 143, 149, 155, 163–164, 177, 180, 187–196, 205, 208–211, 213, 224, 226–227, 229, 243, 256, 261, 281, 382 Podosomes, 164 Pol, 13–14, 27–28, 69, 86–87, 97, 120, 408–412 Polarisation of budding, 74 Polyethylene glycol, 318, 324, 332, 356, 365 Polyploidy, 291, 396–398, 414 Polytropic, 15–17, 45, 48 Pore model, 79 Preeclampsia (PE), 102, 131–132, 134– 135, 206 Progesterone, 68, 98, 133, 409 Progesterone receptor, 98, 133 Prognostic impact, 103, 133 Programmed cell death, 5 Proliferation, 49, 68, 89, 94, 103–104, 119, 129, 190, 210, 280, 289, 293–294, 306, 328, 368, 371–373, 407, 412, 415 Promoter, 69, 87, 94, 97–99, 129–132, 153, 237, 242, 292–293, 302–303, 327, 408 Proprotein convertase, 123 Prostaglandin F2α , 259–260 Proteasome, 129 Protein kinase A (PKA), 3, 5, 98, 121, 129, 132, 134, 207–208, 254, 272, 409

434 Protein scaffold, 4–5 Protein tyrosine kinase, 55 Provirus, 13, 22, 26, 69, 81, 85–86, 408–412 Pseudotyping, 91, 93–94, 409 PSG17, 190 PTGFRN, 192 PTP1B, 189 R Rab, 188 Rabbit, 3, 31, 83–87, 94, 96–97, 122, 134, 235–236, 269, 322 Rac, 55, 152, 155, 157, 160–161, 258 Radixin, 4, 54, 190 Rafts, 2, 4, 18, 22, 52–53, 55, 74, 80, 96, 100, 187, 256 RANK ligand (RANKL), 5, 221–222, 227, 237–238, 242 RD114 virus, 78, 82 Receptor activator of NFκ B (RANK), 5, 221–223, 229, 237–238 Receptor-binding domain (RBD), 24, 93, 126 Receptor blocking, 92 Receptor interference, 16, 21, 29, 68–69, 126 Recurrence-free survival, 103, 133 Redox mechanims, 102 Reduction division, 293, 301 Regenerating myofibers, 259 Regeneration, 66, 250, 253, 257, 259, 268, 279, 281, 289–291, 293–299, 303, 305–309, 384, 407 Regulatory T cells (Treg), 338–339 Renal cancer, 338 Reproduction, 3, 174, 409 Reprogramming, 6, 290, 292, 301–305, 402, 415–416 Retrotransposons, 82, 98, 103, 408 Retroviridae, 22–23, 69 Retrovirus, 2–3, 11–33, 43–51, 55, 67–69, 71–75, 77–83, 85–93, 97, 99, 101, 103–104, 120–122, 124, 126, 128, 131, 395–416 Retrovirus D-type receptor (RDR), 92–93, 99, 126 Reverse transcriptase (RT), 69, 80, 408 Rho, 52, 55, 398, 406 Rho-associated, coiled-coil containing protein kinase (ROCK), 55, 398 Rolling pebbles (Rols), 145, 151–152, 157, 162 Roughest/Irregular optic Chiasma (Rst/IrreC), 140, 143–144, 150–153 R-peptide, 21–22, 24, 29, 43, 48–50, 70, 75–76, 90–91, 124

Index S Satellite cells, 7, 165, 268 Scar/Wave, 154–157 Schizo/Loner, 145, 152, 155, 157 Schneider cells, 150 Secreted protein acidic and rich in cysteine, osteonectin, BM40 (SPARC), 354, 367–368, 383 SEL, 175, 189 Self recognition, 5, 228 Self-tolerance, 229 Serine proteases, 123, 255 SH2 domain containing phosphatase-2 (SHP-2), 259–260, 268 Sheep3, 15, 23, 25, 30–31, 50, 67–68, 83, 85–86, 88–89, 92, 94, 97, 103, 122 Signal regulatory protein-alpha (SIRP α), 5, 226–229 Simian retrovirus (SRV), 69, 92, 78, 101 Singles bar, 144, 162 Skeletal muscle, 3, 6–7, 44, 66, 93, 100, 159–160, 165, 205, 208–209, 250, 256, 259–260, 268–269, 281, 286–287, 297–298, 307, 403–404, 415 Snail, 354, 360–361, 367–368 SNAP receptors (SNARE), 4–5, 66–67, 187–189, 196 Soluble NSF attachment protein (SNAP), 188–189 Sox2, 305 Sp-1, 98 Sperm, 5–6, 17, 171–181, 187–196, 205, 239, 244, 281–286, 399 Spermatid precursors, 401 Spermatogonia, 401 Sperm-egg fusion, 5–6, 171–181, 190, 192–193, 195, 239, 282, 285 Sperm factor, 194 Sperm-oocyte adhesion, 283 Spontaneous hybrid formation, 7, 355 Spumavirus, 22–23, 25–26, 69 Sra-1/PIR121, 155 Stem cell, 6, 66, 94, 96, 119, 129, 165, 176, 212, 221, 223, 228–229, 243, 250, 277–309, 317, 355–356, 361, 363–365, 398, 401–403, 407, 415–416 Stem cell fusion, 279–282, 285–295, 297, 299–303, 305–307, 309 Sticks and stones (Sns), 143–144, 150–153, 156–157, 160, 163–164, 261 Sumoylation, 130 Superinfection, 16 Superinfection resistance, 16

Index Surface subunit, 15 Surface unit (SU), 15, 17–18, 20–21, 23–25, 32, 43–44, 46–47, 49–50, 69–73, 75–76, 79, 90, 93, 123–127, 132, 272, 409 Syk, 238, 240, 242 Synapse, 4, 74, 164 Synaptobrevin, 187, 189 Synaptotagmin I, 189 Syncytial plaques, 97 Syncytin, 3–5, 7, 29–30, 44–45, 48, 63–104, 121, 123–127, 129, 131, 133–135, 165, 178, 211–212, 214, 225, 267–273 Syncytin-1, 3–4, 6, 31–32, 68, 80, 82–104, 120–134, 211–212, 225, 268–272, 400, 405, 408–410, 412–414 Syncytin-1 transactivation, 129 Syncytin-2 receptor, 4, 27, 29–30, 83, 85–87, 89–93, 95–97, 99, 101–102, 121–122, 124–132, 134, 211, 408–409, 413 Syncytin-A, 3, 68, 86–88, 90, 93, 96–98, 122, 125, 129, 134, 269 Syncytin-B, 68, 87, 93, 96–97, 122, 129, 134 Syncytin-A and -B, 86, 90, 93, 122, 269 Syncytin-Ory1, 68, 83–84, 86–87, 90, 92, 96–97, 122, 269 Syncytiotrophoblats, 96 Syncytium, 21, 43–44, 47–49, 51–55, 89, 119, 159, 205–206, 208–210, 213, 289, 307, 401 Synkaryons, 7, 280, 399 T TATA box, 98 T-cell activation, 318, 331 T-cell tolerance, 330 Teratomas, 406 Testis, 87, 93, 97, 187, 191–193, 285, 410–411 Tetraploid, 296, 303, 397–398, 402, 414 Tetraspanins (Tspan), 4, 66, 173, 175–178, 180, 187, 189–194, 196, 239, 284–286 Three-cell cluster, 341 Thrombspondin-1(TSP-1), 227 Time-lapse microscopy, 150, 252, 254, 287, 291 Toll-like receptor agonists, 331 Toll-like receptors (TLR), 228, 331 Transdifferentiation, 279, 296, 298, 302, 402–403 Transforming growth factor-β (TGF- β), 103–104, 206–207, 222, 338–339, 352, 360, 412 Trans-Golgi network(TGN), 73

435 Translocase, 100, 208 Transmembrane domain (TM), 24, 31, 45, 66–67, 69, 71, 75, 79–80, 84, 91, 93, 123, 127, 175, 189, 284 Transmembrane subunit (TM), 15, 18, 20–21, 23–25, 29, 31–32, 43–44, 46–50, 69–76, 79–80, 84, 90, 92, 102, 123–125, 127, 134, 143, 272, 409 Transplantation, 279, 290–293, 296–297, 300, 306, 407 Trogocytosis, 178, 191 Trophoblast, 3, 5, 30–31, 68, 86, 88–90, 94, 96–98, 100, 120, 122, 128–129, 131, 203–214, 269–270, 286, 307, 398, 400, 404–405 Trophoblast binucleated cells, 97 Tryptophan, 27, 71, 79, 101 T-SNAREs, 4–5, 189 Tspan-29, 175 Tumor associated macrophages, 302, 359–363, 404–405 Tumor cell fusion, 300, 329, 353–355, 359, 363, 368, 371, 383 Tumor-host hybrids, 355 Tumor-initiating cells, 300 Tumor microenvironment, 338, 360 Tumor necrosis factor (TNF-α), 102, 206–207, 222, 237, 329, 340 Tumor stem cells, 229, 302 Tumor suppressor genes, 6, 133, 293, 365 Tumor suppressors, 414 Tumor x tumor hybrids, 355 Tunicamycin, 49, 124 Type D mammalian retrovirus receptor, 126 Type D receptor, 67, 78, 84, 92, 99, 124, 126, 212 Tyrosinase, 327, 357–358 Tyrosine kinase, 55, 221–222, 272 U Ubiquitin, 129–130, 132, 143 V Vacuolar adenosine triphosphatase (v-ATPase), 241 VCA, 54, 154–155 VCAM-1, 54, 298 Vesicle associated membrane protein (VAMP), 189 Vesicles, 5, 66, 69, 73–75, 146–148, 156–157, 159–165, 177–181, 187–189, 191, 196, 205, 210, 213, 243, 256–258, 284, 357, 371, 377–383

436 Vesicle-SNARE (v-SNAREs), 189 Vesicular stomatitis virus, 326 Villous trophoblast, 205–206, 208–213, 400 Viral FMG, 326 Virologic synapse, 74 Virus-like particles, 27, 80–81, 402 Virus receptor types, 92 Vitamin D, 237 Vrp1/sltr, 145, 155–158 W WASP family Verprolin-homologous protein (WAVE), 145, 154–157, 161, 258 WASP-interacting partner (Wip), 145, 154–160 Wheat germ agglutinin (WGA), 259

Index Wiscott-Aldrich syndrome protein, (WASP), 54, 145, 154–160, 258 Wnt, 294, 407 X XC cells, 22, 49–50, 85 Xenotropic, 15–16, 45, 48 Xenotropic/polytropic receptor, 45 Xist, 304 Y Y-chromosome, 291, 298 Z Z-discs, 152 Zebrafish, 4, 141, 144–145, 160, 288 Zona pellucida, 173, 178–179, 181, 187–188, 193, 196, 281

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