Advances in Virus Research, Volume 74

Advances in VIRUS RESEARCH VOLUME 74 ADVISORY BOARD DAVID BALTIMORE ROBERT M. CHANOCK PETER C. DOHERTY H. J. GROSS B...

Author: Karl Maramorosch | Aaron J. Shatkin | Frederick A. Murphy

28 downloads 1015 Views 2MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

Advances in

VIRUS RESEARCH VOLUME

74

ADVISORY BOARD DAVID BALTIMORE ROBERT M. CHANOCK PETER C. DOHERTY H. J. GROSS B. D. HARRISON BERNARD MOSS ERLING NORRBY J. J. SKEHEL M. H. V. VAN REGENMORTEL

Advances in

VIRUS RESEARCH VOLUME

74 Edited by

KARL MARAMOROSCH Rutgers University, New Jersey, USA

AARON J. SHATKIN Center for Advanced Biotechnology and Medicine, New Jersey, USA

FREDERICK A. MURPHY University of Texas Medical Branch, Texas, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London, NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2009 Copyright # 2009 Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (þ44) (0) 1865 843830, fax: (þ44) (0) 1865 853333; e-mail: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-378587-9 ISSN: 0065-3527 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 09 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTENTS

1. Regulation of HIV-1 Alternative RNA Splicing and Its Role in Virus Replication

1

C. Martin Stoltzfus I. Introduction II. HIV-1 Splicing Regulatory Elements III. Evidence for the Functional Importance of HIV-1

2 3

Splicing Regulatory Elements in Virus Replication

21 30 32 33 33

IV. Strategies to Target HIV-1 Splicing with Antiviral Drugs V. Conclusions and Perspectives

Acknowledgments References

2. New Insights into Flavivirus Nonstructural Protein 5

41

Andrew D. Davidson Introduction The Methyltransferase Domain The RNA-Dependent RNA Polymerase Domain NS5 Interactions NS5 Phosphorylation NS5 Localization Emerging Roles for NS5 in Viral Pathogenesis Conclusions and Future Perspectives Acknowledgments References I. II. III. IV. V. VI. VII. VIII.

3. Replication of the Hepatitis Delta Virus RNA Genome

42 44 61 75 79 81 85 89 92 92

103

John M. Taylor I. II. III. IV. V. VI. VII.

Background Polymerase(s) Promoters and Priming Pausing and Switching Replication in the Nucleus Role(s) of the Delta Antigen Host Factors

104 106 108 110 112 113 115

v

vi

Contents

VIII. Viroid Analogy IX. Conclusions and Outlook

Acknowledgments References

4. Recent Epidemiology of Tick-Borne Encephalitis: An Effect of Climate Change?

116 116 117 117

123

E. I. Korenberg Introduction Major Debatable Issues The Ranges of Main Tick Vectors: Are They Really Expanding? Tick Abundance and TBE Virus Prevalence: Have They Changed? Tick Expansion to the Cities: Is It Related to Climate Change? What Is Known About Newly Formed TBE foci? Anthropurgic TBE foci: What Are the Principles of Their Formation? Since When Has TBE Morbidity Increased in the Cities? What Are the Main Causes of Changes in Parameters of TBE Morbidity? Conclusions Acknowledgment References

I. II. III. IV. V. VI. VII. VIII. IX. X.

Index Color plate section at the end of the book

124 127 128 129 131 133 134 135 136 137 138 138 145

CHAPTER

1 Regulation of HIV-1 Alternative RNA Splicing and Its Role in Virus Replication C. Martin Stoltzfus

Contents

I. Introduction II. HIV-1 Splicing Regulatory Elements A. Cis-regulatory elements that activate and repress metazoan mRNA splicing B. General strategy of HIV-1 RNA splicing C. Intrinsic efficiency of HIV-1 splice sites D. HIV-1 splice sites and their regulatory elements E. Alternative roles of the major splice donor sites 50 ss D1 and D4 in HIV-1 replication F. Effects of inclusion of small exons 2 and 3 on HIV-1 mRNA expression and stability G. Effects of RNA secondary structure on HIV-1 splicing H. Possible roles of HIV-1 Rev, Tat, and Vpr proteins in regulation of viral RNA splicing III. Evidence for the Functional Importance of HIV-1 Splicing Regulatory Elements in Virus Replication A. Sequence comparison of HIV-1 splice sites and regulatory elements B. Mutations of HIV-1 regulatory elements inhibit virus replication C. Overexpression and siRNA inhibition of cellular splicing factors affect HIV-1 splicing and inhibit virus replication

2 3 3 5 6 8 15 17 17 18

21 21 23

28

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74001-1

#

2009 Elsevier Inc. All rights reserved.

1

2

C. Martin Stoltzfus

D. Changes in expression of cellular splicing factors during HIV-1 infection IV. Strategies to Target HIV-1 Splicing with Antiviral Drugs V. Conclusions and Perspectives Acknowledgments References

Abstract

29 30 32 33 33

Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNA species, both completely and incompletely spliced, are produced by alternative splicing of the primary viral RNA transcript. In addition, about half of the viral RNA remains unspliced and is transported to the cytoplasm where it is used both as mRNA and as genomic RNA. In general, the identities of the completely and incompletely spliced HIV-1 mRNA species are determined by the proximity of the open reading frames to the 50 -end of the mRNAs. The relative abundance of the mRNAs encoding the HIV-1 gene products is determined by the frequency of splicing at the different alternative 30 -splice sites. This chapter will highlight studies showing how HIV-1 uses exon definition to control the level of splicing at each of its 30 -splice sites through a combination of positively acting exonic splicing enhancer (ESE) elements, negatively acting exonic and intronic splicing silencer elements (ESS and ISS elements, respectively), and the 50 -splice sites of the regulated exons. Each of these splicing elements represent binding sites for cellular factors whose levels in the infected cell can determine the dominance of the positive or negative elements on HIV-1 alternative splicing. Both mutations of HIV-1 splicing elements and overexpression or inhibition of cellular splicing factors that bind to these elements have been used to show that disruption of regulated splicing inhibits HIV-1 replication. These studies have provided strong rationale for the investigation and development of antiviral drugs that specifically inhibit HIV-1 RNA splicing.

I. INTRODUCTION HIV-1 is the etiologic agent of acquired immunodeficiency disease syndrome (AIDS) and currently over 30 million people worldwide are living with HIV-1 infection. Because of its importance in human disease HIV-1 has been the subject of intense study since its discovery in the early 1980s. The knowledge of the basic biology of HIV-1 has led to the development of a number of antiviral drugs that have been targeted to different steps of the virus life cycle. Through the use of a cocktail of several drugs, referred to as highly active antiretroviral therapy (HAART), the treatment

HIV-1 Splicing Regulation and Virus Replication

3

of AIDS has been revolutionized and this therapeutic approach has transformed the disease into a manageable chronic illness. However, one major problem with the antiviral drugs is the high frequency with which HIV-1 mutates to drug resistance. Thus, there is a pressing need to further investigate all steps of the virus life cycle in order to develop new antiviral drugs. One of the steps of the HIV-1 life cycle that has received relatively little attention as a target for antiviral drugs is the process of alternative RNA splicing. This is a complex process by which HIV-1 generates over 40 different spliced mRNAs from the single full-length unspliced RNA which is transcribed from the integrated viral provirus by RNA polymerase II. It is crucial for HIV-1 to maintain appropriate cytoplasmic levels of both spliced mRNAs for viral protein synthesis and unspliced viral RNA for use both as genome RNA and as an mRNA. In addition, the intracellular levels of the different viral mRNA species vary widely reflecting the efficiencies by which splicing occurs at the multiple alternative splice sites in the viral genome. Over the past 15 years, considerable progress has been made in understanding the mechanisms by which HIV-1 regulates its RNA splicing. As described below, this regulation is complex and involves the cooperative action of multiple positive and negative elements acting on the relatively weak core splice sites that characterize the HIV-1 genome. It also involves interaction of these cis-elements with a number of different cellular splicing factors. The purpose of this chapter is first, to briefly review the current knowledge of how HIV-1 cis-splicing elements and the trans-acting cellular and viral factors interacting with these elements regulate HIV-1 splicing. We will then discuss the extent of sequence homology of HIV-1 splice sites and splicing regulatory elements among different HIV-1 strains. This will be followed by a review of genetic evidence supporting the hypothesis that regulation of HIV-1 splicing is essential for efficient virus replication. We will also discuss how overproduction and inhibition of host splicing factors in infected cells affect HIV-1 replication. Finally, we will discuss several antiviral strategies that are being used to target HIV-1 splicing.

II. HIV-1 SPLICING REGULATORY ELEMENTS A. Cis-regulatory elements that activate and repress metazoan mRNA splicing 1. Core splicing signals and exon definition For several recent reviews of RNA splicing and exon definition, the reader is referred to references (Black, 2003; Wang and Burge, 2008; Zheng, 2004). Core splicing signals include three sites which are present in most premRNA introns: the 50 -splice site (50 ss), the 30 -splice site (30 ss), and the

4

C. Martin Stoltzfus

branch point sequence (BPS). The 50 ss is a binding site for U1 snRNP. The 30 ss, which includes a polypyrimidine (Py) tract and a conserved AG sequence 30 -proximal of the Py tract, is a binding site for the heterodimeric cellular splicing factor U2AF. It is comprised of two subunits: U2AF65 binds specifically to the Py tract and U2AF35 binds to the AG sequence. The BPS has a loose consensus sequence YNCURAY (R is either A or G; Y is C or U; N is any nucleotide; underlined A indicates location of branch point). Early in spliceosome assembly BPS is bound by the branch pointbinding protein (SF1/mBBP) which is subsequently displaced by U2 snRNP as spliceosome formation proceeds. 50 -Splice sites are referred to as ‘‘strong’’ or ‘‘weak’’ depending on the extent of base pairing between U1 snRNA and the 50 ss, that is, the extent of homology to the consensus 50 ss. 30 -Splice sites are referred to ‘‘strong’’ or ‘‘weak’’ depending on the affinity of the splice site for U2AF and SF1/mBBP, that is, the extent of homology to the consensus 30 ss. In mammalian cells, 50 ss and 30 ss are initially recognized in pairs across exons. This interaction between factors at the 50 ss and 30 ss is referred to as ‘‘exon definition’’ or ‘‘exon bridging’’ (Hoffman and Grabowski, 1992; Robberson et al., 1990). Following this initial recognition step, spliceosomes can assemble to early splicing complex (E complex) that results in an irreversible commitment to the splicing reaction. Thus, exon definition is a key step in alternative splicing regulation.

2. Exonic and intronic splicing enhancers and silencers In addition to the core splicing signals, the RNA transcripts of many genes contain additional cis-elements that are necessary to facilitate or repress exon definition (for review, see Matlin et al., 2005; Wang and Burge, 2008). Such regulatory elements are particularly important in alternative splicing pathways. Exonic splicing enhancers (ESEs) are sequence elements within exons that preferentially bind to members of the serine–argininerich protein (SR protein) family. SR proteins have one or more N-terminal RRM domains that bind to ESE sequences and a C-terminal RS domain that acts to facilitate exon definition by interacting with the RS domains of other splicing factors. The serine residues in the RS domains are extensively phosphorylated by several types of protein kinases including SRPK1 and SRPK2, the Clk/Sty family, and DNA topoisomerase. A number of studies have shown that phosphorylation of SR proteins affect their functions in splicing (Graveley, 2000). Exonic splicing silencers (ESSs) are bound by splicing inhibitory proteins and they repress exon definition. ESS sequences are very diverse and most of the sequences are preferential binding sites for members of the cellular heterogeneous ribonuclear protein (hnRNP) families. Intronic splicing silencers and enhancers (ISS and ISE, respectively) have also been identified.


5

These elements facilitate or repress definition of exons that are surrounded by the intronic splicing elements.

3. Role of secondary structure The secondary structure of pre-mRNAs may also affect alternative splicing by exposing or sequestering core splicing signals and splicing regulatory elements (for review of the role of secondary structure in splicing, the reader is referred to Buratti and Baralle, 2004). A wellcharacterized example of how RNA secondary structure affects splicing is the inclusion of the alternative fibronectin EDA exon. In this case, a downstream sequence stabilizes an upstream ESE sequence within a loop of a stem-loop structure where it is accessible to SR proteins. This results in recognition of the EDA exon. Mutations in the downstream sequence cause a conformational shift such that the ESE is now present in the stem and is relatively inaccessible to SR proteins. This results in failure to recognize the EDA exon (Buratti et al., 2004). Another example is the alternatively spliced tau exon 10 in which the 50 ss is sequestered in a stem-loop element. Disruption of this stem-loop by mutations results in increased binding of U1 snRNP to the 50 ss and increased inclusion of exon 10 (Varani et al., 1999).

B. General strategy of HIV-1 RNA splicing The biogenesis of HIV-1 mRNAs requires the host cell RNA-splicing machinery to produce completely spliced mRNAs, which are transported from the nucleus to the cytoplasm by the endogenous cellular pathway. Splicing of viral RNA is inefficient and results in the accumulation of partially spliced and unspliced RNA whose transport from the nucleus to the cytoplasm is facilitated by the 18-kDa viral regulatory protein Rev. Rev serves as an adapter that targets the viral RNA to the Crm1dependent pathway for nuclear export. The role of Rev in transport of HIV-1 RNA has previously been the subject of a number of reviews and therefore this topic will not be addressed in detail in this chapter (Cullen, 2003; Pollard and Malim, 1998). Rev interacts with a highly structured region in the env gene, the Rev-responsive element (RRE). Early in infection only completely spliced 1.8-kb mRNAs, which encode the viral regulatory proteins Tat, Rev, and Nef, are transported and translated in the cytoplasm. As the infection progresses, sufficient Rev is produced to allow transport of the incompletely spliced 4-kb mRNAs and 9-kb unspliced viral mRNA (Kim et al., 1989; Klotman et al., 1991; Michael et al., 1991). The unspliced mRNA encodes the structural protein precursors Gag and Gag–Pol and also serves as genomic RNA. The incompletely spliced 4-kb mRNAs encode the Env protein as well as accessory proteins Vif, Vpr, and Vpu. Accumulation of incompletely spliced and unspliced

6

C. Martin Stoltzfus

mRNAs requires retention of the 30 -terminal intron between 50 ss D4 and 30 ss A7, which contains the RRE (Fig. 1B). In general, the identity of the individual HIV-1 mRNAs is determined by the proximity of the open reading frames to the 50 -end of the mRNAs (Fig. 1A). Translation of HIV-1 mRNAs follows the rules of ribosome scanning whereby protein synthesis is most often initiated at the first AUG. (This rule is broken in the case of the Env/Vpu reading frames in which both the 50 -proximal Vpu ORF and the downstream Env ORF are translated from the same set of mRNAs.) Each set of the HIV-1 mRNAs encoding a particular HIV-1 protein is spliced at the 30 ss immediately upstream of the protein open reading frame (Fig. 1B). The extent of splicing at each of the 30 ss is determined by the intrinsic strength of the splice site and by the positive and negative exonic and intronic ciselements that regulate this splice site. Some of the HIV-1 mRNAs are present in relatively high abundance (Env, Nef, Rev) and some are present in low abundance (Vif, Vpr, and Tat; Purcell and Martin, 1993). It has been generally assumed that these differences in mRNA abundance are primarily determined by the different efficiencies by which the splice sites are used.

C. Intrinsic efficiency of HIV-1 splice sites Several approaches have been used to compare the intrinsic strengths of the HIV-1 50 ss and 30 ss compared to the strength of efficient splice sites. One approach was to substitute individual HIV-1 splice sites into a two exon–one intron human b-globin construct. These results indicated that 50 ss D1 and D4 were used as efficiently as the b-globin 50 ss. 50 ss D2 and D3, on the other hand, were used two to three times less efficiently. The efficiency of splicing at the different HIV-1 50 ss was directly related to the relative strengths of the 50 ss predicted by the homologies to the consensus metazoan 50 ss sequence. The HIV-1 30 ss were all shown to be used significantly less efficiently than the b-globin 30 ss (O’Reilly et al., 1995). Since the completion of this study, the roles of additional exonic regulatory elements in the HIV-1 genome have been defined. To determine the effects of these elements, the strengths of the HIV 30 ss were tested with or without their downstream exonic sequences in a one-intron HIV-based env reporter construct. This study indicated that, when the downstream 30 -exonic sequences were absent, 30 ss A1, A4c, A4a, A4b, A5, and A7 were all very inefficient in comparison to an optimized 30 ss. In contrast, 30 ss A2 and A3 were approximately 40% as efficient as the optimized 30 ss. When the exonic sequences were placed downstream of the 30 ss, the efficiencies of 30 ss A1, A4c,a,b, A5, and A7 were greatly increased. On the other hand, the efficiencies of 30 ss A2 and A3 were decreased approximately four- and twofold, respectively. These results emphasized

7


A

Vpr Nef

Vpu

5⬘ LTR

Vif

Gag Pro

Env

Pol

3⬘ LTR

Tat Rev

B 9-kb

5⬘ss D1

Genomic/ unspliced mRNA

D1a

D2 D3 D4

(D5)

RRE

Gag, pol A1 A2 A3 A5 (A6)

A1a

3⬘ss

A7

A4c,a,b Vif Vpr Tat exon 1 Env/vpu Env/vpu Env/vpu Env/vpu

4-kb mRNA

1

Noncoding exons

Tat exon 1,2 Rev Rev Rev Nef

1.8-kb mRNA

ESEM ESSV ESS2p/ESE2/ ESS2 3 4

ESE-Vif 1

1a 2

4cab G4

2

3

2

3

1.[2].[3].4.7 1.[2].[3].4c.7 1.[2].[3].4a.7 1.[2].[3].4b.7 1.[2].[3].5.7

1

Noncoding exons

C

1.2-I 1.[2].3-I 1.[2].[3].4-I 1.[2].[3].4c-I 1.[2].[3].4a-I 1.[2].[3].4b-I 1.[2].[3].5-I

ISS 6D

(ESE/ESS)

7

ESE2/ESS3

5 GAR

FIGURE 1 Diagrams showing the locations of splice sites, exons, and splicing elements in the HIV-1 genome. (A) Schematic diagram of HIV-1 genome. The open rectangles indicate open reading frames. The long terminal repeats (LTRs) are shown with the three regions comprising the LTRs shown as rectangles: U3-shaded; R-black; U5-open. Full-length RNA transcripts begin at the 50 -end of the R region of the 50 -LTR and poly(A) addition begins at the 30 -end of the R region in the 30 -LTR. Splice sites A6 and D5, which are present only in HXB2 and a few other B clade HIV-1 strains, are shown in parentheses. (B) Locations of 50 and 30 ss in the HIV-1 genome. The location of the RRE is also shown. The exons present in the incompletely spliced 4- and 1.8-kb mRNA species corresponding to the HIV-1 genes are shown as open rectangles. Noncoding exon 1 is present in all spliced HIV-1 mRNA species. Either both or one of the small noncoding exons 2 and 3 shown in black rectangles are included in a fraction of the mRNA species corresponding to the HIV-1 genes. The exon compositions of the RNA species are also shown. Species designated by an ‘‘I’’ are incompletely spliced mRNA species. Brackets indicate that mRNA isoforms containing neither exon 2 or 3, only exon 2 or 3, or both exons 2 and 3 are produced. (C) Locations of known splicing regulatory elements in the HIV-1. The exons are numbered according to the nomenclature shown in (B). Splicing enhancers are designated by white dotted rectangles and splicing silencers are designated by black rectangles.

8

C. Martin Stoltzfus

the importance of positive exonic sequences for splicing at 30 ss A1, A4c,a,b, A5, and A7 and negative downstream exonic sequences for splicing at 30 ss A2 and A3 (Kammler et al., 2006). One possible reason for the relatively low intrinsic splicing efficiencies of HIV-1 3’ss is the use of nonconsensus branch points at nucleotides other than A residues (Damier et al., 1997; Dyhr-Mikkelsen and Kjems, 1995). Splice sites 30 ss A4c,a,b and A5 have relatively short Py tracts with interspersed purines which could account for their relative low intrinsic efficiency.

D. HIV-1 splice sites and their regulatory elements The mRNAs encoding Vif, Vpr, and Tat are present at low abundance in infected cells or cells transfected with infectious plasmid DNA indicating that 30 ss A1, A2, and A3 are used relatively infrequently. In contrast, the mRNAs for Rev, Env/Vpu, and Nef are present at high abundance indicating that 30 ss A4c,b,a and A5 are used with relatively high frequency. Since approximately half the total spliced mRNA is completely spliced, 30 ss A7 is also used with relatively high efficiency. It has been found that each of the HIV-1 30 ss is regulated by a characteristic set of positive and negative cis-elements that act combinatorially to determine the efficiency by which this splice site is used. Most of these regulatory elements are present in the exonic and intronic sequences immediately downstream of the regulated 30 ss. In most cases, the splicing elements have been shown to be binding sites for cellular RNA-binding proteins. In this section, we summarize our current understanding of how the efficiency of splicing is regulated at each of the HIV-1 30 ss and highlight results indicating that exon definition is the key regulatory step. For a more detailed discussion of HIV-1 ESS, ISS, and ESE, the reader is referred to a previous review (Stoltzfus and Madsen, 2006).

1. Vif mRNA splice site: 30 ss A1 Vif mRNA is an incompletely spliced low abundance mRNA (approximately 1% of the incompletely spliced mRNA in infected cells) which is formed by splicing 50 ss D1 to 30 ss A1 (Fig. 1B). In a fraction of the completely spliced and incompletely spliced vpr, tat, and env/vpu mRNAs, the 50-nt exon defined by 30 ss A1 and 50 ss D2 (exon 2) is included (Purcell and Martin, 1993). Exon 2 does not contain an AUG and becomes part of the 50 -leader region of the mRNAs into which it is included. To test the role of 50 ss D2 on the splicing efficiency at 30 ss A1, mutants in the context of the infectious proviral plasmid pNL4-3 were created in which the predicted affinity of the relatively weak 50 ss D2 for U1 snRNP was increased or decreased relative to the wild-type sequence. These studies indicated that D2-up mutations, with increased affinity to U1 snRNP,


9

caused greatly increased inclusion of exon 2 as well as increased levels of spliced vif mRNA. This increase in level of vif mRNA was correlated with a corresponding increase in Vif protein levels. D2-down mutants, on the other hand, exhibited decreased levels of vif mRNA and no detectable inclusion of exon 2 into viral mRNAs. In general, the effects on levels of vif mRNA and Vif protein were correlated with the predicted affinity of U1 binding to 50 ss D2. Thus, 50 ss D2 acts as one of the enhancers of splicing at 30 ss A1 and this positive effect on exon definition does not require splicing at 50 ss D2 (Exline et al., 2008). Consistent with this hypothesis, the levels of vif mRNA and Vif protein were restored to wild-type levels by supplying a mutant U1 snRNA in trans whose 50 -end base pairs with the mutated 50 ss D2. The mutant U1 snRNA facilitates wild-type splicing at 30 ss A1 but is incapable of supporting splicing at 50 ss D2 (Mandal et al., 2009). In addition to the downstream 50 ss D2, the definition of exon 2 is facilitated by several other positive elements (Fig. 1B). The first of these elements is localized within the proximal 18 nt of exon 2 and has the properties of an ESE. Mutations within this sequence resulted in a decrease in exon 2 inclusion and greatly reduced levels of vif mRNA when tested in the context of infectious HIV-1 proviral plasmid pNL4-3. In HeLa cell nuclear extracts, this ESE (ESE-Vif) was bound selectively by the SR protein SRp75 (Exline et al., 2008). Two additional elements with the sequence UGGAAAG (M1 and M2) were detected in exon 2 downstream of ESE-Vif. Mutations within either the M1 or M2 sequence reduced exon 2 inclusion when tested using a three exon–two intron construct. Exon 2 sequences bind to the SR protein SF2/ASF; this binding was abrogated by mutations in either M1 or M2. These results were consistent with the hypothesis that M1 and M2 are SF2/ASF-dependent ESE and that both motifs are required for ESE activity. Surprisingly, in the context of pNL4-3, a mutation of M1 alone inhibited exon 2 inclusion into viral mRNAs but did not appear to significantly affect the level of incompletely spliced vif mRNA. This result suggested that the production of incompletely spliced vif mRNA is less dependent on the bipartite M1/M2 ESE than is exon 2 inclusion (Kammler et al., 2006). However, in these experiments, the effect of mutations of M2 alone on vif mRNA splicing were not tested. Mutations within a GGGG motif, which is immediately 30 -proximal of 0 5 ss D2, resulted in an increase in splicing at 30 ss A1, an increase in exon 2 inclusion, and increased vif mRNA and Vif protein when tested in the context of pNL4-3 (Exline et al., 2008). This suggests that the GGGG sequence acts negatively on exon 2 definition. Such 50 ss-proximal GGGG splicing silencers have been recognized in cellular genes and have been hypothesized to operate in concert with hnRNP A1-dependent ESS elements to provide a combinatorial code for splicing silencing in cells. The binding protein responsible for the negative effect on splicing of

10

C. Martin Stoltzfus

the GGGG motif has not yet been identified. The responsible protein appears not to be hnRNP F or H which do bind to the 50 ss-proximal GGGG motifs but which have been shown to have a positive rather than negative effect on splicing when bound at these sites (Han et al., 2005).

2. Vpr mRNA 30 -splice site: 30 ss A2 Vpr mRNAs are incompletely spliced low abundance mRNAs (approximately 2% of the incompletely spliced mRNA) which are formed primarily by splicing 50 ss D1 to 30 ss A2. There are also minor levels of vpr mRNA in which the 74-nt exon 3, which is defined by 30 ss A2 and 50 ss D3, is included (Fig. 1B). Exon 3 is also included in a fraction of tat, rev, env/vpu, and nef mRNAs (Purcell and Martin, 1993). Exon 3, like exon 2, does not contain an AUG and becomes part of the 50 -leader of these mRNAs. The dominant ciselement regulating splicing at 30 ss A2 is an ESS within exon 3 termed ESSV (Fig. 1C). ESSV is a member of the family of splicing elements with UAGcontaining motifs that preferentially bind to members of the cellular hnRNP A1 protein family (hnRNP A/B proteins; Bilodeau et al., 2001; Caputi et al., 1999; DelGatto-Konczak et al., 1999). Most of the ESSV activity is localized to a 16-nt element containing three (Py/A)UAG motifs (Madsen and Stoltzfus, 2005). Studies in HeLa cell nuclear extracts have indicated that binding of hnRNP A/B proteins to ESSV results in inhibition of binding of U2AF65 to the Py tract of the upstream 30 ss (Domsic et al., 2003). In the context of the infectious proviral plasmid pNL4-3, mutants with lesions in ESSV exhibit greatly increased inclusion of exon 3, an increase in incompletely spliced vpr mRNA, and an increase in Vpr protein (Madsen and Stoltzfus, 2005). In the presence of ESSV, up and down mutations within 30 ss D3 have relatively small effects on splicing at 30 ss A2, which indicates the dominance of the ESS. However when ESSV was inactivated, the 30 ss D3-down mutants dramatically decreased splicing at 30 ss A2 and the level of incompletely spliced vpr mRNA. The extent of this decrease correlated with the reduced predicted binding affinity of 30 ss D3 to U1 snRNA. As has been shown for 50 ss D2, 50 ss D3 acts as an enhancer of splicing and facilitates the production of vpr mRNA. This positive effect on exon 3 definition does not require splicing at 50 ss D3 but is dependent on the strength of U1 snRNP binding to 50 ss D3 ( J. Madsen and C. M. Stoltzfus, unpublished data). Further mutagenesis studies have revealed the presence of an additional positive element or elements within exon 3 downstream of ESSV. Mutations within this element(s) in the context of wild-type or ESSV mutants in subgenomic viral constructs (H. Schaal et al., unpublished data) and in the context of the viral genome (C. M. Stoltzfus et al., unpublished data) cause a decrease in exon 3 inclusion into completely and incompletely spliced mRNAs. The cellular binding protein or proteins interacting with this putative downstream ESE have not yet been identified.


3. Tat mRNA 30 -splice site: 30 ss A3

11

The tat mRNAs, formed by splicing 50 ss D1, 50 ss D2, or 50 ss D3 to 30 ss A3, are either completely spliced and encode two-exon Tat or incompletely spliced and encode one-exon Tat. The tat mRNAs are relatively low abundance, representing approximately 9% of completely spliced and 5% of incompletely spliced mRNAs (Purcell and Martin, 1993). Tat mRNAs spliced from 50 ss D2 to 30 ss A3 include exon 2 and those spliced from 50 ss D3 to 30 ss A3 include exon 3 or both exons 2 and 3 (Fig. 1B). As shown in Fig. 1C, splicing at 30 ss A3 is repressed by several ESS elements within the first tat coding exon (exon 4). The dominant ESS, an hnRNP A/B-dependent ESS, termed ESS2, is present approximately 70-nt downstream from 30 ss A3 (Amendt et al., 1994). ESS2 was mapped to a 10-nt core sequence containing two PyUAG motifs (Si et al., 1997). A second ESS, termed ESS2p, is present within the 50 -proximal 8-nt region of exon 4 (Jacquenet et al., 2001). ESS2p binds selectively to hnRNP H suggesting that it acts similarly to an hnRNP H-dependent ESS in one of the alternatively spliced exons of the rat b-tropomyosin gene (Chen et al., 1999). Mutagenic inactivation of either ESS2 or ESS2p in the context of the viral genome has indicated that ESS2p is a substantially weaker negative element than ESS2 (P. S. Bilodeau and C. M. Stoltzfus, unpublished data). Further mutagenesis of the 10-nt sequence immediately upstream of ESS2 revealed the presence of an additional cis-element regulating tat mRNA splicing. This region was shown to contain a binding site for the cellular SR protein SC35 which functioned as an ESE in in vitro splicing assays. The element was termed ESE2 (Zahler et al., 2004). Two different groups have shown that the binding sites for SC35 in ESE2 overlap with the hnRNP A1-binding sites in ESS2 and that SC35 and hnRNP A1 compete for these overlapping binding sites (Hallay et al., 2006; Zahler et al., 2004). Based on the data, two alternative models have been proposed to explain how the juxtaposed ESS and ESE elements act to regulate splicing at 30 ss A3. The first model proposes that inhibition of ESS2 activity by depletion of hnRNP A1 or mutations of ESS2 allow binding of SC35 to ESE2. This results in activation of splicing at 30 ss A3 by bridging through the SC35 RS domain to essential splicing factors U2AF and U1 snRNP (Zahler et al., 2004). The second model proposes that SC35 through its RNA-binding domain competes for the overlapping hnRNP A1 sites and blocks cooperative binding of additional hnRNP A1 molecules to exon 4, thus relieving splicing inhibition at 30 ss A3 (Hallay et al., 2006). This model is analogous to a proposed model, summarized below in Section II.D.5, to explain how SF2/ASF relieves hnRNP A/B repression of 30 ss A7.

12

C. Martin Stoltzfus

4. Rev and env/nef 30 -splice sites: 30 ss A4c, A4a, A4b, and A5

These four 30 ss are contained within a 40-nt region near the middle of the HIV-1 genome (30 ss central cluster) and are used to produce completely spliced rev mRNAs (A4c,a,b), completely spliced nef mRNAs (A5), and incompletely spliced env/vpu mRNAs (Fig. 1B). The mRNAs created by splicing in this region are relatively abundant and represent approximately 90% of the completely spliced mRNAs and 92% of the incompletely spliced mRNAs. Within the incompletely and completely spliced mRNA species arising from splicing in this region are isoforms that include exon 2, exon 3, or both exons 2 and 3. The frequency of splicing at 30 ss A5 is greatest, followed by 30 ss A4a and A4b. The frequency of splicing at 30 ss A4c is very low (Purcell and Martin, 1993). Because the intrinsic strengths of 30 ss A4c, A4a, A4b, and A5 are all very weak, it suggested that activation of these splice sites requires splicing enhancers, the downstream strong 50 ss D4, or both of these positive splicing elements. Indeed, a guanosine–adenosine-rich ESE (GAR ESE) was discovered within exon 5 and downstream of 30 ss A5. The GAR ESE also was shown to activate splicing at the downstream 50 ss D4 and thus is a bidirectional splicing enhancer. The GAR ESE contains two predicted SF2/ASF-binding sites [SF2(1) and SF2(2)] as well as a predicted SRp40binding site. Selective binding of SF2/ASF and SRp40 to GAR ESE was confirmed by experiments to test binding of purified SR proteins (Caputi et al., 2004). In the context of a three-exon, two-intron subgenomic env expression construct, mutations of the SRp40 binding had only a slight effect on splicing within the 30 ss central cluster whereas mutations of both SF2/ASF-binding sites greatly reduced activation of all 30 ss within the 30 ss central cluster. Mutation of only the proximal SF2/ASF-binding site SF2 (1) specifically decreased splicing at 30 ss A5 compared to splicing at 30 ss A4c,a,b; mutation of only the distal site SF2(2) had a smaller effect than mutations of SF2(1) and inhibited splicing at all the 30 ss in the central cluster. A third SF2/ASF-binding site SF2(3), which also contributes to the selective usage of 30 ss A5, was also detected in the region overlapping 30 ss A5. In addition, the region of exon 5 downstream of the GAR, referred to as E42, was shown to be necessary for inclusion of exon 5 and GAR activation of the downstream 50 ss D4. The E42 region by itself does not facilitate U1 snRNP binding in the absence of the GAR enhancer and suggests that E42 may be necessary to recruit additional unidentified factors that mediate interactions between SR proteins bound to GAR and U1 snRNP bound to 50 ss D4 (Asang et al., 2008). In addition to the GAR ESE, splicing at all the 30 ss within the 30 ss cluster was enhanced by the strength of the downstream 50 ss D4, which is used to define exons 4c, 4a, 4b, and exon 5. Mutations with 50 ss D4 that were predicted to decrease affinity for U1 snRNP inhibited splicing at 50 ss D4 and reduced the usage of all 30 ss within the 30 ss cluster. Splicing could be


13

restored upon addition of a compensatory U1 snRNA that re-established complementarity to the mutated 50 ss (Asang et al., 2008). These experiments also showed that, analogous to the results discussed above for 50 ss D2 and D3, exon definition but not splicing at 50 ss D4 is required in order for U1 snRNP to activate the upstream 30 ss splice sites within the 30 ss cluster and enhance the production of incompletely spliced env/vpu mRNAs. Interestingly, in contrast to the effect on exon 5 inclusion, either SF2(1) or SF2(2) alone were not sufficient to activate production of vpu/env mRNA. In addition to the role of cis-acting splicing elements, splicing in the central cluster is affected by overlap of the core splicing signals of the 30 ss. Splicing in the 40-nt region containing 30 ss A4c,a,b and 30 ss A5 involves the usage of different sets of branch points for splicing at each of the four alternative AGs. Two of the branch points used for splicing at 30 ss A5 overlap the AGs used for splicing at 30 ss A4a and A4b (22- and 16-nt upstream of 30 ss A5, respectively; Swanson and Stoltzfus, 1998). Mutations of the 30 ss A4b AG have previously been shown in vitro and in vivo in the context of the viral genome to dramatically increase splicing at 30 ss A5 (Purcell and Martin, 1993; Riggs et al., 1994; Swanson and Stoltzfus, 1998). A possible model for this phenomenon is that factors bound at or near the AG of the A4b splice site may interfere with the formation of spliceosomes at 30 ss A5 by blocking access to the BPS.

5. Nef and tat, rev exon 2 splice site: 30 ss A7

This 30 ss in combination with 50 ss D4 is used to remove the 30 -terminal RRE-containing intron of HIV-1 RNA and generate completely spliced 1.8kb mRNAs for two-exon Tat and Rev as well as Nef (Fig. 1B). Regulation of this splice site is complex and includes several hnRNP A/B-dependent ESS elements, ESE elements, and an intronic splicing silencer (ISS) which was shown to bind hnRNA A1. The ESS was first named ESS3 was mapped to the region 75- to 90-nt downstream from 30 ss A7 (Amendt et al., 1995; Staffa and Cochrane, 1995). Subsequent experiments showed that ESS3 is bipartite and that each of the subelements AGAUC (ESS3a) and UUAG (ESS3b) can inhibit splicing independently (Si et al., 1998). A region upstream of ESS3a with the sequence GAAGAAGAA (GAA3) corresponds to a known ESE element responsive to SF2/ASF and deletion of this element greatly reduced splicing at A7 in vitro and in transfection experiments using a subgenomic construct (Amendt et al., 1995; Staffa and Cochrane, 1995). This suggested its role as an enhancer which was termed ESE3. Subsequently, it was shown that there are additional ESE elements that bind SF2/ASF and SC35 upstream of the (GAA)3 in exon 7 (Mayeda et al., 1999; Tange and Kjems, 2001). Some mutations of the (GAA)3 element increased splicing at A7 rather than decreased splicing as expected for inactivation of an splicing enhancer. These results suggested that the

14

C. Martin Stoltzfus

(GAA)3 element can act as either an ESS or ESE in the context of exon 7 and for this reason was termed a Janus element (Marchand et al., 2002). Based on in vitro splicing assays and RNA footprinting, it has been proposed that hnRNP A/B proteins exert their negative effect by initiation of binding of hnRNP A/B proteins to ESS3 or (GAA)3 followed by cooperative binding of additional hnRNP A/B proteins to other lower affinity sites within exon 7 and to the ISS. Addition of SF2/ASF either with or lacking the RS domain prevents cooperative hnRNP A/B protein binding initiated at ESS3 or (GAA)3. This failure to initiate cooperative binding of hnRNP A/B proteins is thought to result in increased splicing at 30 ss A7 and binding of U2AF65 to the PPT (Damgaard et al., 2002; Marchand et al., 2002; Zhu et al., 2001). The in vitro splicing data have suggested that in HeLa cell nuclear extracts repression by ESS3 and the intronic ISS dominate activation by exonic ESE elements. In contrast, data obtained by transfection of HeLa cells with a subgenomic construct containing splice sites D4 and A7 and the env gene intron have shown that deletion of ESS3a or deletion of both the (GAA)3 element and ESS3a had little effect on splicing at 30 ss A7. On the other hand, deletion of the (GAA)3 element in the presence of wild-type ESS3a resulted in a dramatic decrease in splicing. These results suggested that one of the functions of the (GAA)3 element is to counteract the effect of ESS3a (Pongoski et al., 2002). It has also been shown that placement of the exon 7 region, which contains ESE3, ESE3a, and ESS3b, downstream of 30 ss A7 resulted in a dramatic increase in splicing at A7 suggesting the dominance of the positively acting ESE3 and other ESE in exon 7 over the negatively acting ESS3 elements (Kammler et al., 2006). These results suggest that there may be differences in the ratio of hnRNP A/B proteins to SF2/ASF in HeLa cell extracts where ESS3 appears to be dominant compared to the same ratio in the nucleus of living cells where ESE3 appears to be dominant.

6. HXB2 tev splice sites: 30 ss A6 and 50 ss D6

The HXB2 HIV-1 strain contains a novel 30 ss within the env gene (30 ss A6) that is not conserved in other HIV-1 strains and a 50 ss (50 ss D5) 170-nt downstream that is conserved in HXB2 and only a few other B clade HIV-1 strains (Fig. 1B). The usage of these two splice sites in HXB2 results in inclusion of exon 6D and the production of a low abundance spliced mRNA encoding a novel 28-kDa protein Tev whose amino acid sequence corresponds to the first tat coding exon, a portion of the env gene encoded by exon 6D, and the second rev exon. The hybrid Tev protein has functional Tat but not Rev activity (Benko et al., 1990; Salfeld et al., 1990). A naturally arising HXB2 point mutant within exon 6D exhibited a dramatic increase in inclusion of this exon. The mutation was localized to a U-to-C change within exon 6D (Wentz et al., 1997). Caputi and Zahler


15

showed that U-to-C, U-to-A, and U-to-G mutations all increase binding of SR proteins to exon 6D and that the mutant sequences functioned as an SC35-dependent ESE when tested in heterologous dsx in vitro splicing substrates. In addition, they showed that, in the context of the U-to-C mutation, hnRNP H family members bind with increased affinity to a GGGA sequence 3 nucleotides downstream from the mutation and this binding is essential for inclusion of exon 6D into HIV-1 mRNAs. A third element that affects exon 6D inclusion in the context of the U-to-C mutation is a polypurine element further downstream in exon 6D. Paradoxically, this element promotes SR protein binding in HeLa cell nuclear extracts but also serves as an ESS when present in a dsx in vitro splicing substrate (Caputi and Zahler, 2002).

7. Gag–pol splice sites: 30 ss A1A and 50 ss D1A

A novel 50 ss and a 30 ss (50 ss D1A and 30 ss A1A) within a highly conserved region of the pol reading frame were found to define a 190-nt exon (exon 1A) that is included into several HIV-1 mRNA species at a very low level (Fig. 1B; Lutzelberger et al., 2006). The sequence of 50 ss D1A is AG/GUAAGA and differs from consensus only at position þ 6 relative to the splice site. The sequence of 30 ss A1A, on the other hand, has a short Py tract and would be predicted on this basis to be relatively weak. The function of the 50 ss D1A in the context of the HIV-1 genome was tested by a mutation to decrease its affinity for U1 snRNA. This resulted in an approximately threefold decrease in the level of unspliced viral RNA. The wild-type level of unspliced RNA was restored in the 50 ss D1A mutant by expression of U1snRNP with a compensatory change in the U1 RNA sequence. These results suggested that 50 ss D1A may be necessary in the HIV-1 genome to prevent degradation of unspliced viral RNA. The authors speculate that one possible mechanism for this effect is that U1 snRNP bound to 50 ss D1A may recruit SR proteins to the viral RNA. These SR proteins may in turn stabilize binding of Rev and result in more efficient export of unspliced RNA.

E. Alternative roles of the major splice donor sites 50 ss D1 and D4 in HIV-1 replication In addition to the role of the major 50 ss D1 and D4 in defining exon 1 and exons 4, 4a, 4b, and 5, respectively, during HIV-1 RNA splicing there is evidence that these two strong splice sites may play additional roles in the expression of HIV-1 RNA. One function for which the strong HIV-1 50 ss have been implicated is in RNA stability. In the context of a single intron env expression vector, Lu et al. showed that mutations within 50 ss D4 resulted in a drastic inhibition of env mRNA accumulation and loss of Env expression either in the presence or absence of Rev. Env expression

16

C. Martin Stoltzfus

could be recovered by coexpression of a mutated U1 snRNA that restored wild-type U1 base pairing (Lu et al., 1990). These studies were confirmed and extended by Kammler et al. who showed that the affinity of U1 snRNP for 50 ss D4 was directly related to the level of Env expression. Kammler et al. also showed that the conserved dinucleotide GU in 50 ss D4 was required for splicing at 50 ss D4 but not for stabilizing env mRNA and Env expression (Kammler et al., 2001). A subsequent study by the same group showed that in a three-exon, two-intron env expression context, mutations of 50 ss D4 reduced splicing at the central 30 ss cluster but these mutations did not affect transcript stability. The authors speculated that the difference in the results obtained with the three and two exon constructs is the presence of 50 ss D1 in the two-intron construct which may provide the necessary stabilization function that D4 provides in the two exon construct (Asang et al., 2008). Experiments to test the effect of mutating 50 ss D1 in the context of pNL4-3 have shown that such mutations activate a cryptic 50 ss 4-nt downstream from 50 ss D1. All species of spliced mRNA and proteins accumulated at a reduced rate and the 50 ss D1 mutant displayed a delayed production of virus. Since the cryptic 50 ss is a less strong splice site than 50 ss D1, spliced mRNAs may accumulate at a lower rate than wild-type HIV-1 and this may explain the delayed phenotype (Purcell and Martin, 1993). A later study showed that if both 50 ss D1 and the cryptic 50 ss site were mutated in pNL4-3, only unspliced HIV-1 RNA accumulated in transfected cells. This result supported the authors’ hypothesis that downstream splicing of HIV-1 RNA from 50 ss D4 to 30 ss A7 is dependent on prior splicing of the upstream intron from 50 ss D1 to a downstream 30 ss (Bohne et al., 2005). Although not directly addressed by the authors, these results also suggest that 50 ss D1 is not absolutely required for stabilization of unspliced HIV-1 RNA. It is possible that in this mutant, the unspliced RNA stabilization function may supplied by 50 ss D1A (Lutzelberger et al., 2006). A second function of 50 ss D1 is to suppress one of the two 30 -cleavage and polyadenylation sites in the HIV-1 genome. In HIV-1 as in a number of other retroviruses, the poly(A) signals are duplicated within the R regions of the 50 - and 30 -LTRs of the provirus. This necessitates a mechanism whereby the upstream poly(A) site is suppressed and only the downstream poly(A) is used during the processing of the viral RNA. Ashe et al. found that substitution of the heterologous CMV promoter for the HIV-1 LTR promoter or the closeness of the initiation site for transcription to the poly(A) site did not affect the suppression of the upstream poly(A) site. However, mutations that decreased the affinity of U1 snRNP for 50 ss D1, which is 200-nt downstream of the 50 -LTR poly(A) site, activated the usage of this poly(A) site. Suppression was restored by targeting binding of U1 snRNP to a location near the mutated 50 ss A1 (Ashe et al., 1995, 1997). The suppression activity of U1 snRNP requires stem-loop 1 of U1 snRNA


17

and, since U1-70K binds to this region, it suggests that this protein may be responsible for the effect (Ashe et al., 2000).

F. Effects of inclusion of small exons 2 and 3 on HIV-1 mRNA expression and stability Since the discovery of HIV-1 mRNA isoforms containing one or both of the small noncoding exons 2 and 3, there have been a number of studies intended to define possible functions for these exons in mRNA metabolism or translation. This question has been addressed in several ways. One study created nef cDNA expression clones with exon 2, exon 3, or neither exon 2 or 3 upstream of the nef open reading frame and found that each of these expressed Nef equally well (Schwartz et al., 1990). Muesing et al. assayed several different tat expression constructs by their ability to transactivate the LTR promoter. These results showed that the constructs with either exon 2 or 3 in their 50 -leaders demonstrated small increases in transactivation but was not clear from the data whether these differences were statistically significant (Muesing et al., 1987). Krummheuer et al. investigated the effect in HeLa-T4þ cells of exon 2 or 3 both in the context of a single intron env expression vector and in an LTR CAT expression vector. These studies indicated that constructs with either exon 2 or exon 3 in the 50 -leader of the mRNAs resulted in significantly increased or decreased gene expression, respectively. The effects of exons 2 and 3 appeared to be posttranscriptional, affected RNA stability, and occurred in the nucleus (Krummheuer et al., 2001). Madsen and Stoltzfus (2006) found that the stability of total HIV-1 mRNAs in 293T cells transfected with pNL4-3 mutants overexpressing HIV-1 mRNAs containing either exon 2 or exon 3 did not differ significantly from each other or from wildtype viral mRNAs. Further data have indicated that 50 ss D2-down mutations, which completely prevent inclusion of exon 2, do not significantly affect virus replication in permissive T-cell lines under conditions where Vif is not required (Mandal et al., 2009). To explain these discordant results, it is possible that the differences in the effects of the small noncoding exons on RNA stability or function may be dependent either on the types of cells used in the assays or on the HIV-1 constructs that were used to determine the effects.

G. Effects of RNA secondary structure on HIV-1 splicing The secondary structures of the region surrounding three of the HIV-1 30 ss have been determined by chemical and enzymatic probes and the binding sites for cellular splicing factors have been mapped by RNA footprinting analysis. Based on these secondary structure data, models for protein binding to ESS and ESE elements have been proposed.

18

C. Martin Stoltzfus

This subject has recently been reviewed and thus will not be discussed in detail in this chapter (Saliou et al., 2009). The exon 3 region defined by 30 ss A2 and 50 ss D3 appears to be folded into a single stem-loop structure. In the structural model, the ESSV core sequence is exposed in the loop region and is accessible to binding of hnRNP A1 proteins (Saliou et al., 2009). The region containing the 50 -part of exon 4 and 30 ss A3 is folded into an extended stemloop structure. HnRNP A1 was shown to bind simultaneously to both ESS2 and ESE2, which is proposed to be the initiation site for cooperative binding of additional hnRNP A1 molecules (Hallay et al., 2006). In the model for the region surrounding 30 ss A7, the RNA appears to be folded into a structure with three stem-loops. Stem-loop 1 contains the ISS element, stem-loop 2 contains the 30 ss and ESE3, and stem-loop 3 contains ESS3. The initiation site for hnRNP A1 binding is proposed to be the ESE3 Janus element discussed above in Section II.D.5. Additional hnRNP A/B proteins are then proposed to bind to the other two stem-loop structures. The RNA–protein binding is thought to be stabilized by protein–protein interactions through the Cterminal glycine-rich domains of the hnRNP A/B proteins (Damgaard et al., 2002; Marchand et al., 2002). For each of the three regulated 30 ss, SR protein-binding sites were shown to overlap the sequence where hnRNP A/B protein binding is initiated. SF2/ASF was shown to bind selectively to the region downstream of 30 ss A2 and A7 whereas SC35 binds selectively to the region downstream of 30 ss A3 (Marchand et al., 2002; Saliou et al., 2009; Tange and Kjems, 2001). When overexpressed, the SR proteins are thought to displace the bound hnRNP A/B proteins and thus abrogate the negative effect of hnRNP A1 on splicing. The major HIV-1 50 ss D1 is predicted to be embedded in a relatively stable stem-loop RNA structure (SD stem-loop). When the SD stem was further stabilized by mutagenesis, virus replication was inhibited due to the failure of the viral RNA to be spliced efficiently. Several revertant viruses were isolated upon long-term passage of the mutated virus in which the SD stem-loop was destabilized. In addition, another type of second site mutation occurred upstream of 50 ss D1 within the RNA dimerlinkage structure (DLS). Further analysis indicated that this mutation created an alternative 50 ss which restored splicing and efficient virus replication (Abbink and Berkhout, 2008). Whether the SD stem-loop normally plays an important role in the regulation of HIV-1 splicing has not yet been established.

H. Possible roles of HIV-1 Rev, Tat, and Vpr proteins in regulation of viral RNA splicing Rev-mediated export of unspliced and incompletely spliced viral mRNA has been shown to compete with HIV-1 splicing and increasing the rate of transcript splicing by strengthening the 30 ss results in a decrease of


19

Rev function (Kammler et al., 2006). Rev interaction with the RRE is able to override the cellular functions that normally act to retain the unspliced and incompletely spliced mRNA in the nucleus. An important unanswered question is whether Rev interaction with the RRE, in addition to its role in HIV-1 RNA transport, plays a direct role in inhibiting HIV-1 splicing by interacting in some way with the cellular splicing machinery. A possible link between Rev and splicing factors was suggested by studies of HIV-1 replication in mouse cells. In infected mouse cells, HIV-1 replication is characterized by a decrease in unspliced and incompletely spliced mRNA, a decrease in Gag protein level, and a decrease in virus production. Expression of human p32 protein in several mouse cell lines was shown to reverse this excessive splicing phenotype. A single Gly to Asp mutation at position 35 of the human p32 sequence to change the protein to the corresponding mouse p32 sequence at this position resulted in the loss of rescue (Zheng et al., 2003). Several laboratories have reported that both murine and human p32 proteins bind to the basic domain of HIV-1 Rev (Luo et al., 1994; Tange et al., 1996). The human p32 protein also binds to SF2/ASF and has been shown to inhibit its phosphorylation and RNA-binding activity (Petersen-Mahrt et al., 1999). It has been proposed that p32 could serve as a bridge between Rev, which is bound to the RRE, and SF2/ASF which is bound to HIV-1 ESE elements in the HIV-1 genome. Such an interaction may inhibit the activity of SF2/ASF and cause an inhibition of HIV-1 splicing (Tange et al., 1996). The HIV-1 14-kDa Tat transactivator Tat binds to the TAR sequence at the 50 -end of the HIV-1 RNA and is necessary for facilitating elongation of viral RNA transcription by recruiting elongation factor P-TEFb to the viral promoter (Price, 2000). It has been proposed that, in addition to its wellcharacterized role in transcription, Tat may also have a role in regulation of HIV-1 splicing (Berro et al., 2006). As shown for HIV-1 Rev, Tat also binds to the p32 protein in vitro; this binding was shown to occur with increased affinity when Tat was acetylated at lysines at amino acids 50 and 51 of Tat. Consistent with a role for acetylated Tat in HIV-1 splicing, transfection of a HeLa cell line containing an integrated full-length Tat-minus HIV-1 provirus with a K50A, K51A-mutated Tat expression plasmid resulted in an approximately twofold reduction in the ratio of unspliced to spliced HIV-1 RNA compared to wild-type Tat. When both Tat and p32 were expressed they were shown to colocalize in the nucleus whereas p32 alone was localized in the cytoplasm (Berro et al., 2006). More recent data from this group have implicated a third component of the complex, the cellular kinase CDK13, which was shown to interact with both acetylated Tat and p32 (Berro et al., 2008). Using several assays for HIV-1 splicing, Berro et al. showed that overexpression of CDK13 resulted in an increase in the ratio of spliced to unspliced viral RNA and a decrease in virus replication. Knockdown of CDK13 using siRNA, on the other hand, increased virus

20

C. Martin Stoltzfus

production in cells transfected with pNL4-3. CDK13 was further shown to bind to SF2/ASF and, using several in vitro assays, shown to phosphorylate SF2/ASF; this is a possible mechanism by which CDK13 can increase splicing of viral RNA. These results led to a model suggesting that early in HIV-1 infection CDK13 may promote complete splicing of HIV-1 RNA through activation of SF2/ASF-dependent ESE. As acetylated Tat accumulates during infection it recruits binding partner p32 which in turn acts to inhibit phosphorylation of SF2/ASF by CDK13. The effect would be to decrease the activity of SF2/ASF and allow increased accumulation of unspliced and incompletely spliced HIV-1 mRNAs later in HIV-1 infection. It was recently shown that, in the context of a single intron HIV-1 env expression construct, splicing of identical RNA transcripts was increased approximately twofold when the RNA was transcribed under control of the CMV promoter compared to the HIV-1 LTR promoter transactivated by Tat (Bohne and Krausslich, 2004). The authors indicate that this effect was independent of acetylation of Tat since K50A Tat mutants did not behave significantly different from wild-type Tat in this assay but they did not test the double K50A, K51A mutant used by Berro et al. (2006). It was suggested by Bohne and Krausslich that the difference in splicing when RNA is produced from the two promoters may indicate that HIV-1 LTR promoters, as well as other retrovirus LTRs, have been selected to produce excessive amounts of unspliced RNA. It should be noted however that, in the context of full-length infectious plasmid pNL4-3 with mutations that prevented the expression of Tat, Chang and Zhang (1995) found no significant differences in the percentages of unspliced, incompletely spliced, and completely spliced HIV-1 mRNAs when the transcription was driven by either by the Tat-transactivated HIV-1 LTR promoter or the CMV promoter in the absence of Tat. It is not clear if these conflicting results are due to the use of different HIV-1 constructs or to differences in the construction of the hybrid promoters. The HIV-1 vpr gene encodes a 14-kDa protein which is incorporated into virions and which plays a role in infection of macrophages and other nondividing cells by facilitating the nuclear import of preintegration complexes. Vpr also acts to inhibit cells in the G2/M phase of the cell cycle. Vpr was also shown to be a general inhibitor of cellular mRNA splicing when tested by in vitro splicing assays but had significantly reduced effect on splicing in cells transiently transfected with Vpr (Kuramitsu et al., 2005). Subsequent experiments indicated that Vpr binds to a cellular splicing-associated protein SAP145 and prevents association of SAP145 and another splicing-associated protein SAP49. It was proposed that the Vpr cell cycle arrest may be caused, not by the inhibition of splicing, but by inhibiting the formation of the SAP145–SAP49 complex, which in turn induced G2 checkpoint activation (Terada and Yasuda, 2006). However, more recent experiments from a number of


21

laboratories have indicated that G2 checkpoint activation is caused by interaction of Vpr with a Cul4A-containing E3 ligase complex (for a recent review, see Dehart and Planelles, 2008). Thus, the possible significance for virus replication of the Vpr effect on splicing is not yet clear.

III. EVIDENCE FOR THE FUNCTIONAL IMPORTANCE OF HIV-1 SPLICING REGULATORY ELEMENTS IN VIRUS REPLICATION A. Sequence comparison of HIV-1 splice sites and regulatory elements Since its introduction into the human population, the major HIV-1 group, referred to as group M, which represents over 90% of all HIV-1 strains, has spread into many different areas of the world and has diverged into multiple subtypes or clades. Currently HIV-1 has been divided into nine different clades: A–D, F–H, J, and K. In addition, a number of intraclade recombinants have been isolated. Most HIV-1 strains in the United States and Europe belong to the B clade whereas in Africa, most strains belong to the A and C clades. In addition, there are two other more divergent HIV-1 groups, the outlier (group O) and new (group N). In contrast to the group M viruses, groups O and N viruses, which represent only a small number of HIV-1 isolates, have remained confined to a small region of West Africa. Groups M, N, and O are each thought to have originated from independent transmissions of an HIV-1 progenitor from primates to humans (Sharp et al., 2001). One criterion for the functional importance of HIV-1 splice sites and splicing elements is the extent of sequence homology among the different HIV-1 strains. Most of the results we have discussed so far in this chapter have been obtained using the HIV-1 B clade infectious proviral clone pNL4-3, which has served as a model system for numerous studies on HIV-1 replication (Adachi et al., 1986). Sequence comparisons of the pNL4-3 50 - and 30 -splice sites to those in the other group M clades and to groups N and O are shown in Fig. 2. These data show that most of the 50 -and 30 -splice sites are strongly conserved in all the group M clades as well as in groups N and O. An exception is the region containing pNL4-3 rev 30 -splice sites 4c, 4a, and 4b where there is extensive sequence variation among the different virus clades. This high sequence diversity is accompanied by alternative locations for the rev mRNA splice sites even among virus isolates from the same clade (Bilodeau et al., 1999). The sequences of the known HIV-1 splicing elements are compared in Fig. 3. In general, most of the ESE elements (ESE-Vif, ESE M1 and M2, ESE2, ESE3), ESS2p, and ISS are highly conserved among all of the viruses. The GAR ESE is also reasonably conserved among the group M viruses but

3⬘ss A2

3⬘ss A1A NL4-3

3⬘ss A4c

3⬘ss A3

A1

rc

g u

a

A2

a

g

a

B

c ac

u

D

c

u

F1

ac

F2

ac

G

a

u

H

a

J K

n*

c

uy g ac

n* g

c

uy

3⬘ss A5

3⬘ss A4b

g

u

ay

u u

u

a

y

c

cy*ng

c

y* g

c c

uc g ac

g

c

uu

ag

g

g

c

uy

cag

g *g

u

n* a

c

uu

ag

g *g

u

c

ag

g

c

rg

gg

u

a

ac

u

u

a

u

a

unugu*n

a

u u *

au

u c

a

3⬘ss A7 UUAUCGUUUCAG/A

A1

g

a

A2

g

a

B

r

a

r

ag

au

u* r

5⬘ss D1 NL4-3

G/GUGAGU

5⬘ss D1A G/GUAAGA

c

n

g

ugc

g

g *g

u c

gg

g *g

g *gu

5⬘ss D2/G4 G/GUGAAGGGG

u

a g *

a

5⬘ss D3

5⬘ss D4

G/GUAGGA

A/GUAAGU

A1 A2

n

u

u

u

c

g

u

a

g

uc g

c

a

g

g

c*

u

c

g

ac

g

u

g

N

NL4-3

y

u

u

C

O

3⬘ss A4a

UUUUAAAAUUAG/C ACTGTTTTTCAG/A AUCCAUUUUCAG/A UUCAUUGCCAAG/UUUGUUUCAUAACAAAAG/CCTTAG/GCATCTCCTATGGCAG/G

g

B

C

g

D

c g

r

F1

g

a

r

F1

F2

g

a

r

F2

G

g

a

c

G

H

g

u

C D

a c

g

H

J

g

a

J

K

g

a

K

N

c c

g

N

O

c c

n

O

r

g a

c

c

a g

g

g

a

g

FIGURE 2 Sequence alignment of HIV-1 splice sites in different group M clades (A1-K) and groups N and O. Sequences are compared to the pNL4-3 sequence. The locations of the splice sites are indicated by slash marks. The sequences for each clade or group were obtained from


23

more divergent in the group O strains. ESSV, an hnRNP A/B-dependent ESS, contains three (Py/A)UAG motifs and all of these motifs have been shown to contribute to its silencer activity. All three of the (Py/A)UAG motifs are conserved among A1, A2, B, C, G, H, and J clade viruses and two of three motifs are conserved in D, F1, F2, and K clades. Only one (Py/A) UAG motif is conserved in the group N strains and none of the motifs are conserved in the group O strains. The ESS activity of the group O sequence was tested by substituting the pNL4-3 ESSV with the corresponding sequence from the group O virus MVP5180. When assayed either by in vitro splicing experiments ( J. M. Madsen and C. M. Stoltzfus, unpublished data) or by transfection in the context of an infectious HIV-1 plasmid, the group O sequence lacked ESS activity (Madsen and Stoltzfus, 2005). ESS2 has two (Py/A)UAG motifs in clade B viruses but only one of these motifs is conserved in the other group M clades. One (Py/A)UAG motif in ESS2 was sufficient for ESS activity when tested by in vitro splicing assays and by transfection of minigene constructs derived from the clade B SF2 HIV-1 strain. The corresponding sequence to ESS in the group O virus has no (Py/A)UAG motifs and the sequence has no ESS activity when assayed by in vitro splicing assays or by transfection of minigene constructs (Bilodeau et al., 1999). The single (Py/A)UAG motif in ESS3b and the AGAUCC sequence in ESS3a, which are both conserved in the group M viruses, are not conserved in the group O strains. Since none of the known ESS elements are conserved in the group O viruses, it was of interest to compare the splicing patterns of RNA isolated from cells infected with the group O strain MVP5180 with RNA from cells infected with the group M virus NL4-3. In spite of the absence of ESSV repression at 30 ss A2 and ESS2 repression at 30 ss A3, the levels of vpr and tat mRNA species were not significantly increased and the overall balance of spliced to unspliced mRNA was maintained MVP5180-infected cells (Madsen and Stoltzfus, 2005). These data suggest that group O viruses may contain alternative ESS elements that compensate for the lack of ESSV, ESS2, and ESS3.

B. Mutations of HIV-1 regulatory elements inhibit virus replication Identification and characterization of the cis-elements regulating HIV-1 splicing allowed targeting these sequences for mutations in the infectious HIV-1 genome to determine the effects of the cis-elements on the HIV-1 Sequence Database (Los Alamos National Laboratory) and the predominant sequence is shown. An ‘‘r’’ indicates that either A or G is present at the position; a ‘‘y’’ indicates that C or U is present at the position; and an ‘‘n’’ indicates that any of the four nucleotides are found at this position.

NL4-3

ESE-VIF

ESEM1

ESEM2

ESSV

ESS2p

GCAGAGAUCC

UGGAAAG

UGGAAAG

AUAGUUAGCCCUAGG

UGGGU

A1

g

A2

u

B

y

C

c

D

c

g a

F2

c

g

G

c

H

c

J

c

K

a

ESS3a

UAGAAGAAGAA

ac ac

A1

c

g

A2

c

g

B

c

C

c

c c

G

c

aug

n

g

u

a c

a ay

ag

ac

c

c

u

cn

auun

c

rcuga c r

a

aca

uc a

c u

u gaga gcc

g g

acag

g

an

a ***

c gc

ISS UAGUGAAUAGAGUUAGGCAGGGA

a r

rrr

r g

F2

c g

a a

D F1

u

ESS3b

AGAUCCAUUCGAUUAG

g

acu

g

ESE3 NL4-3

g

cc

r

c

c

O

nn

a

u

g

N

GAR ESE GAAGAAGCGGAGACAGCGACGAAGA

ac

uu r

F1

ESS2

ESE2

CCAGUAGAUAUCCUAGACUAGA

g

a

r gr

a

r ca

r a

r

a

r

H

r a

J

c

g

K

c

r

N

c

O

c

g

a

g g g

g

r

g gg

c

a

a cc

a agca gc

r aca

FIGURE 3 Sequence alignment of known HIV-1 splicing elements in different group M clades and groups N and O. Sequences are compared to the pNL4-3 sequence. The sequences for each clade or group were obtained from the HIV-1 Sequence Database (Los Alamos National Laboratory) and the predominant sequence is shown. An ‘‘r’’ indicates that either A or G is present at the position; a ‘‘y’’ indicates that C or U is present at the position; and an ‘‘n’’ indicates that any of the four nucleotides are found at this position. The (G/Py)UAG motifs in the ESS elements are underlined.


25

virus replication. The feasibility of this approach was first shown by Wentz et al. (1997) who studied the replication of the naturally arising point mutant discussed above that activated inclusion of exon 6D. The replication of this mutant in T-cell lines was greatly reduced compared to wild type. This reduction in replication was concomitant with greatly increased levels of tev mRNA and Tev protein as well as a reduction in the levels of Gag and Env. These results suggested that inactivation of the splicing regulatory element may cause a decrease in unspliced and incompletely spliced mRNAs. However, this mutant also expressed fourfold reduced levels of full-length Rev protein. Thus, it was not clear from the results whether the mutant phenotype was primarily due to the to the defect in Rev activity or to excessive splicing of the viral RNA. The importance of an HIV-1 hnRNP A/B-dependent ESS element for virus replication was tested by mutating ESSV, which specifically represses splicing at the vpr 30 ss A2 (Madsen and Stoltzfus, 2005). In 293T cells transfected with infectious pNL4-3 plasmid DNAs, ESSV mutants that did not affect the reading frame of the overlapping vif gene produced an increased level of incompletely spliced vpr mRNA and an almost complete inclusion of noncoding exon 3 which is flanked by 30 ss A2 and 50 ss D3. These mutants also demonstrated a large reduction in unspliced viral RNA, reduced Gag protein levels, and a 10- to 20-fold decrease in production of virus particles. The levels of Env, Rev, and Nef, on the other hand, were comparable or somewhat greater than wild type. Tat mRNA levels were somewhat lower than wild type but cotransfection of the ESSV mutant plasmid with excess amounts of a Tat expression plasmid did not alter the observed virus phenotype (Z. Feng and C. M. Stoltzfus, unpublished data). The results of these experiments indicated that ESSV is required to maintain the appropriate balance of unspliced and spliced mRNA necessary for efficient HIV-1 replication. Consistent with this hypothesis, two types of second site revertants were selected after long-term passage of ESSV mutant virus in Jurkat T cells that restored efficient virus production and balanced splicing (Madsen and Stoltzfus, 2005). The first type of mutation changed the conserved AG at 30 ss A2 such that it was no longer recognized as a splice site. The second type of mutation was a U-to-C change within the conserved GU of the downstream 50 ss D3. The data suggested that, by inhibiting exon 3 definition, this 50 ss D3 mutation acts to inhibit excessive splicing at 30 ss A2 caused by disruption of ESSV. HIV-1 with mutations in ESS2, an hnRNP A/B-dependent ESS which represses splicing at the tat 30 ss A3, exhibits an approximate two- to threefold inhibition of virus production in transfected 293T cells, significantly less inhibition than seen with the ESSV mutants (Z. Feng and C. M. Stoltzfus, in preparation). Interestingly, the ESE2 mutation described by Zahler et al. (2004), which by itself results in an approximately twofold

26

C. Martin Stoltzfus

effect on virus replication, exhibited an approximate 10-fold inhibition of virus production when combined with the ESS2 mutation. These results correlated with increased splicing at 30 ss A3 and a significant reduction in the unspliced mRNA level compared to either the ESE2 or ESS2 single mutants. It appeared from these results that, in the context of the HIV-1 genome expressed in cells, both ESS2 and ESE2 are required to repress splicing at 30 ss A3. The effects on HIV-1 replication of mutations within ESE-Vif and 50 ss D2, both of which regulate splicing at the vif 30 ss A1, have also been tested (Exline et al., 2008). Up mutations of 50 ss D2 and mutations within the GGGG motif silencer caused an excessive splicing phenotype similar to the ESSV mutants. This phenotype was characterized by decreased unspliced RNA, decreased Gag protein levels, and a 10- to 20-fold decrease in virus production. On the other hand, 50 ss D2-down mutations did not significantly affect HIV-1 production in transfected 293T cells or in infected T-cell lines such as CEM-SS cells. CEM-SS cells are referred to as permissive cells for Vif-negative viruses because they do not express ApoBec3G (A3G), which has been shown to inhibit HIV-1 replication at an early step. A3G is a cytidine deaminase which causes C-to-U changes in the HIV-1-negative cDNA strand during reverse transcription, leading to extensive G-to-A mutations in the viral genome. The mechanism by which A3G restricts virus replication is still controversial and may be caused by hypermutation, degradation of newly synthesized HIV-1 DNA, interference with reverse transcription, or a combination of these effects. The restrictive effect of A3G on HIV-1 replication requires packaging of A3G into virions and this is prevented by the expression of Vif, which binds to A3G and targets it for ubiquitination/proteasome-mediated degradation. For a recent review on Vif and APOBEC, the reader is referred to Goila-Gaur and Strebel (2008). Interestingly, in T-cell lines which have been shown to be completely nonpermissive for the replication of vif-minus HIV-1 mutants (e.g., H9 cells), the replication of even the weakest D2-down mutant and the ESE-Vif mutant, both of which produce Vif at levels only approximately 5% that of wild type, were only marginally inhibited. To test the phenotype of the D2-down viruses at elevated A3G levels, a Jurkat T-cell line was created that expressed A3G with a doxycycline-repressible Tet-off promoter. Under no doxycycline conditions, that is, at the highest level of A3G, the A3G-Jurkat T-cell lines produced A3G at levels approximately 15-fold higher than control Tet-off cells. Under these conditions, all of the D2-down and ESE-Vif HIV-1 mutants were shown to be less fit than wildtype virus. The extent of the inhibition of mutant virus replication and their sensitivity to A3G were directly related to their expression levels of Vif. Because 50 ss D2 is highly conserved in all strains of HIV-1 and virus replication does not require 50 ss D2 under permissive conditions,


27

the major function of this splice site may be to modulate the level of vif mRNA splicing and consequently the level of Vif protein (Mandal et al., 2009). If this is the case, it would further suggest that there may be HIV-1 target cells in which the A3G levels are significantly higher than in the normally nonpermissive T-cell lines. Indeed, it has been shown that A3G is upregulated 10-fold in monocyte-derived macrophages treated with IFN-a (Peng et al., 2006; Stopak et al., 2007). A3G was also induced in resting peripheral blood lymphocytes treated with IL-2, IL7, or IL-15 (Stopak et al., 2007). Treatment of the H9 T-cell line with phorbol myristate acetate was shown to induce A3G by approximately 20-fold (Rose et al., 2004). Thus, higher levels of Vif expression may be necessary for efficient HIV-1 replication in those cells in which A3G is upregulated. HIV-1 mutants with the excessive splicing phenotype also exhibited an additional replication defect: a striking increase in the level of Gag precursor relative to products within infected cells (Madsen and Stoltzfus, 2006; Mandal et al., 2008). This was a surprising finding since the locations of the mutations in the viral genome are far from the gag gene. In the case of some of the D2-up mutants, the mutations in D2 also caused changes in the overlapping integrase reading frame. Thus, it was possible that Gag processing could somehow be altered by the mutated integrase. Indeed, it was previously reported that a glutamic acid-to-lysine change at amino acid residue 247 of the integrase reading frame caused a defect in Gag processing and virus particle production that was unique among all C-terminal integrase mutants (Lu et al., 2005). However, this mutation also changed the overlapping 50 ss D2 sequence from G/GUGAAG to G/GUAAG and this change was shown to activate 50 ss D2 (Mandal et al., 2008). To dissect the effect on splicing from the effect on integrase function, second site U-to-A and U-to-G mutations at the U of the conserved GU sequence were constructed to inhibit splicing at 50 ss D2. Because of the redundancy of the genetic code, these mutations did not change the amino acid sequence of the overlapping integrase reading frame. These second site mutations restored the levels of unspliced viral RNA, virus production, and normal Gag processing. Thus, the effect on Gag processing could clearly be shown to be caused by the activation of 50 ss D2 rather than the mutation of integrase in the overlapping reading frame (Mandal et al., 2008). The mechanism by which the excessive splicing mutations cause defects in Gag processing is not yet understood. It is likely that the mutants are not deficient for viral protease since virions with fully processed Gag are produced in the mutant-transfected cells (Mandal et al., 2008). One possible explanation is that particle assembly is affected by reduced amounts of unspliced RNA available for packaging into virions. This also appears to be unlikely since normal levels of virus particles are produced by HIV-1 mutants that do not specifically package viral RNA

28

C. Martin Stoltzfus

(Aldovini and Young, 1990; Lever et al., 1989). It is possible that the low amounts of intracellular Gag produced by the excessive splicing mutants are below the threshold needed to efficiently drive virus assembly. It is also conceivable that unspliced viral RNA produced in the mutantinfected cells is targeted to locations in the cell that are unfavorable for Gag transport and assembly. This scenario, however, would require some quantitative or qualitative difference in the proteins bound to the wild type and mutant unspliced RNA but the nature of such differences and how they would direct the localization of the viral RNA are unclear.

C. Overexpression and siRNA inhibition of cellular splicing factors affect HIV-1 splicing and inhibit virus replication The profound effects on virus replication seen upon mutating HIV-1 splicing elements suggested that similar effects on replication may occur either upon exogenous overexpression of cellular splicing factors binding to these HIV-1 elements or inhibition of cellular splicing factors by treatment with interfering siRNA. This possibility was first addressed in the context of a gag–pol-deleted viral genome in which overexpression of SR proteins SC35, 9G8, and SRp40 were shown to selectively increase splicing at the tat 30 ss A3 at the expense of other viral mRNAs. It was also shown in this study that overexpression of SF2/ASF under the same conditions resulted in increased splicing at 30 ss A2 (Ropers et al., 2004). These data showing the specific effects on splicing at 30 ss A3 were consistent with protein-binding studies indicating that SC35 and SRp40 selectively bind to the region of ESS2 and ESE2 downstream of 30 ss A3 in exon 4 (Hallay et al., 2006). Furthermore, SC35 was shown to selectively activate in vitro splicing of HIV-1 tat gene minigene substrates (Ropers et al., 2004; Zahler et al., 2004). In the context of the infectious virus plasmid pNL4-3, increased splicing at 30 ss A3 in response to SC35 and 9G8 was concomitant with excessive splicing of viral RNA and a greater than 10-fold decrease in virus production. In addition, there was a decrease in intracellular levels of gp160, probably reflecting reduced production of env mRNAs. Similar to the effect of SC35, increased splicing at 30 ss A2 in cells overexpressing SF2/ASF was accompanied by excessive splicing of viral RNA and a drastic reduction of virus production ( Jacquenet et al., 2005). A more complete study has recently been reported describing the effects on HIV-1 RNA metabolism and virus production of overexpressing SR proteins SC35, SF2, and SRp40 and a collection of hnRNP A/B and hnRNP H proteins (Jablonski and Caputi, 2009). As found previously in the context of the subgenomic HIV-1 plasmids, a selective increase in splicing at 30 ss A3 at the expense of other 30 ss was seen when either SC35 or SRp40 were overexpressed. Overexpression of SF2/ASF, on the other


29

hand, resulted in a selective increase in splicing at both 30 ss A1 and A2. Also, as previously shown (Jacquenet et al., 2005), virus production was shown to be greatly reduced by overexpression of either SC35 or SF2/ASF. Overexpression of either hnRNP A1 or to a lesser extent hnRNP A2 resulted in drastic decreases in the level of all spliced viral mRNAs and virion production. The unspliced viral mRNA level, on the other hand, was elevated fourfold by hnRNP A1 and twofold by hnRNP A2 protein overexpression. In addition to effects on splicing, hnRNP A1 and A2 overexpression also inhibited nuclear to cytoplasmic transport of unspliced HIV-1 RNA. Interestingly, overexpression of either hnRNP A1 or A2 proteins had unique effects that was demonstrated by accumulation of different incompletely spliced 4-kb mRNA species. These results suggest that in HIV-1-infected cells alternative splicing of viral RNA is affected differently by hnRNP A1 compared to A2. This contrasts with studies based on in vitro splicing assays using HIV-1 substrates which showed that the splicing inhibition functions of different members of the hnRNP A/B protein family were redundant (Bilodeau et al., 2001; Caputi et al., 1999; Zhu et al., 2001). Use of siRNAs to reduce SR protein levels resulted in a decrease in the level of tat mRNAs in cells treated with SC35 siRNA but not with SRp40 siRNA (Jablonski and Caputi, 2009). Knockdown of hnRNP A1 and A2 proteins had less of an effect on HIV-1 splicing than when these proteins were overexpressed. Jablonski and Caputi suggest this difference may be due to incomplete inhibition achieved by the hnRNP A1 and A2 siRNAs. The splicing factor overexpression and siRNA experiments taken together further support the hypothesis that tight regulation of HIV-1 splicing is required for efficient virus replication and that this regulation can be abrogated by changes in the levels of cellular splicing factors.

D. Changes in expression of cellular splicing factors during HIV-1 infection Because of the dramatic effects on HIV-1 replication seen as a result of mutations of the HIV-1 splicing elements or changes in the expression of cellular splicing factors that bind to these elements, it suggests that changes in levels of splicing factors during virus infection may be a mechanism to regulate HIV-1 gene expression. There is surprisingly little information yet available on levels of SR proteins and hnRNP proteins during infection of T cells with HIV-1. A two- to threefold increase in level of SC35 was observed 2 days after infection of H9 T-cell line but this study was not followed up to determine if this change in SC35 level was accompanied by concomitant changes in the HIV-1 splicing profile (Maldarelli et al., 1998). It has also been found that 9G8 mRNA was downregulated 60 h after HIV-1 infection of the MT4 T-cell line but

30

C. Martin Stoltzfus

again it was not clear if this decrease in 9G8 was accompanied by changes in HIV-1 splicing (Ryo et al., 2000). Macrophages are also a major target for HIV-1 and are an important virus reservoir in infected humans. HIV-1 infections of cultured monocytederived macrophages have been used to study viral gene expression and replication of the virus. Macrophage infections are characterized by several weeks of productive infection followed by a progressive decline over several weeks in virus production. This decline in virus production was correlated with a specific decrease in mRNA species encoding Tat which could be restored by expression of exogenous Tat (Sonza et al., 2002). The decrease in Tat was shown not to be caused by changes in tat mRNA stability but is likely due to changes in the levels of splicing factors. Levels of proteins hnRNP A1, A2, and H were found to decrease relative to uninfected cells for 1–2 weeks after infection followed by a return to uninfected levels during the subsequent 2–3 weeks. There also was a large increase in relative expression of SC35 in the first week of infection followed by a progressive relative decrease in SC35 over the next 4 weeks of infection. The levels of SF2/ASF, on the other hand, remained more constant during infection (Dowling et al., 2008). These results are consistent with previous results discussed in Section III.C, showing a selective effect of SC35 overexpression and knockdown on tat mRNA splicing. Another possible effect of HIV-1 infection are changes in the phosphorylation of SR proteins. These modifications could affect the activity of the proteins as splicing regulators. This possibility was investigated by analysis of SR proteins in pNL4-3-transfected 293T cells (Fukuhara et al., 2006). This study indicated that there was indeed a decrease in phosphorylated SR proteins but further analysis showed this was due to a decrease in the levels of the SR proteins.

IV. STRATEGIES TO TARGET HIV-1 SPLICING WITH ANTIVIRAL DRUGS Because the data highlighted above indicate that regulation of splicing is essential for efficient HIV-1 replication, it suggests that disruption of this exquisitely balanced system is a reasonable approach for development of novel antiviral drugs. Inhibition of virus production could be accomplished either by selectively inhibiting HIV-1 splicing, as seen with D2-down and ESE-Vif mutants, or by selectively enhancing HIV-1 splicing, as seen with the D2-up and ESSV mutants. To date, two general antiviral approaches have been used; both are directed toward specific inhibition of HIV-1 splicing. First, investigators have searched for specific inhibitors of SR proteins and SR protein kinases. Second, antisense nucleic acid approaches have been used to inhibit HIV-1 splicing,


31

either with antisense oligonucleotides or with modified snRNPs to target HIV-1 sequences. Fukuhara et al. (2006) investigated the antiviral activity of a specific isonicotinamide compound termed SRPIN340 which inhibits the SR protein kinases SRPK1 and SRPK2. SRPIN340 was shown to inhibit SRp75 phosphorylation and cause a decrease in the stability of the protein. Virus production in pNL4-3-transfected 293T cells appeared to be limited for phosphorylated SRp75 since overexpression of SRp75 resulted in a 10-fold increase in virus production and this effect was enhanced by coexpression of SRPK2. However, subsequent studies showed that SRPIN340 was not an effective inhibitor of HIV-1 replication when assayed by standard T-cell line infections. It is possible that further testing of compounds related to SRPIN340 may lead to more effective inhibitors of virus replication. Soret et al. (2005) showed that an indole derivative IDC16, which interferes with the enhancer activity of SF2/ASF by binding to the RS domain, specifically suppressed the accumulation of completely spliced HIV-1 mRNA without significant effects on accumulation of unspliced viral RNA. These authors found that HIV-1 virus replication in PBLs and macrophages was inhibited approximately fivefold at IDC16 concentrations that did not affect cell viability. Early events of virus replication (early and late reverse transcription and integration) were not affected in the presence of the drug. Although additional studies must be done to assess the effects of IDC16 on expression of cellular genes, these preliminary studies are interesting and suggest that compounds of this class may have promise as therapeutically important anti-HIV drugs. In early studies, antisense oligonucleotides directed against HIV-1 splice sites were shown to have only limited ability to inhibit virus replication (Goodchild et al., 1988). A more recent study has reported a novel antisense strategy based on derivatives of cellular U7 snRNP targeted to splice sites 30 ss A4, A4c, A4ab, A5, and D4 (Asparuhova et al., 2007). U7 constructs targeted against A5/D4 or GAR ESE/D4 were the most effective of any of the targets in inducing exon skipping and thus, inhibiting the splicing of Tat and Rev mRNAs. The modified U7 constructs were inserted into lentiviral vectors which were then used to create CEM-SS T-cell lines in order to assay the effects on HIV-1 replication. Although most of the constructs tested showed little if any effect on virus replication, the strongest effect on wild-type HIV-1 replication (approximately twofold inhibition) was seen in cells expressing a U7 snRNA targeted to GAR ESE and 50 ss D4. The inhibition was significantly enhanced when a Vif-deficient HIV-1 instead of wild-type virus was targeted in the assay. Based on this result, the authors suggest that the U7 strategy may only be effective in combination with other antivirals targeted to other steps of the virus life cycle.

32

C. Martin Stoltzfus

V. CONCLUSIONS AND PERSPECTIVES We now have a reasonable picture of the cis-elements and trans-acting cellular factors that regulate splicing at each of the HIV-1 alternative splice sites. This picture has been further enhanced by models of RNA secondary structure and the mapping of protein-binding sites in the regions of the HIV-1 30 ss. These models will continue to be further refined by determinations of three-dimensional RNA–protein structures using NMR. The importance for HIV-1 replication of some of the ESS elements and ESE elements have been established by genetic studies. In addition, the highly conserved splice sites downstream of the regulated exons (50 ss D2, D3, and D4) have been shown to be important elements determining the production levels of the incompletely spliced vif, vpr, and env mRNAs, respectively. These studies taken together have established that the exquisite balance of unspliced and spliced mRNA is necessary for efficient virus replication and has provided a strong rationale for developing antivirals specifically targeting HIV-1 splicing. Most of the studies of HIV-1 splicing regulation to date have been performed using HIV-1 strains that belong to the major virus group, group M HIV-1, which represent most of the HIV-1 strains in the world. Sequence comparisons and splicing assays indicated that, although the splice sites of group M and the outlier groups N and O are highly homologous, most of the ESS elements that have been discovered in group M viruses are not conserved at the corresponding positions in the genomes of the group O viruses. These data suggest that the outlier virus strains may contain novel splicing elements at alternative locations in the genome to compensate for the lack of silencers and enhancers that are present in the group M strains. It is now well established that pre-mRNA processing in eukaryotic cells (capping, splicing, and 30 -cleavage/polyadenylation) normally occurs cotranscriptionally (for review, see Kornblihtt et al., 2004; Pandit et al., 2008). This has important implications for retroviruses that depend on the production of large amounts of unspliced and incompletely spliced mRNAs for their survival. It is a particularly relevant issue with a complex retrovirus such as HIV-1 that undergoes extensive splicing with numerous alternative splicing pathways. Little is yet known about the extent to which retroviruses are cotranscriptionally spliced. Using a splice junction in situ hybridization probe, Zhang et al. (1996) could not detect spliced tat mRNA in the nucleus of HeLa cells transfected for 12 h with a subgenomic HIV-1 env expression plasmid. Using another specific probe, on the other hand, these investigators were able to easily detect unspliced RNA in the nucleus in the same cells. Based on this result, it was suggested that efficient splice sites and long transcription units may favor


33

cotranscriptional splicing whereas inefficient splice sites and relatively short transcription units which characterize retroviruses may favor posttranscriptional splicing. These studies need to be confirmed and extended using more sensitive techniques to investigate the possibility of cotranscriptional splicing in the context of the HIV-1 genome. It will be of interest, for instance, to compare the extent of cotranscriptional splicing occurring with wild-type HIV-1 to the excessive splicing mutants described above in which the frequency of splicing is significantly increased. The potential roles of the small exons 2 and 3 in HIV-1 replication also remain an unresolved issue. As discussed above, conclusions based on results from expression of subgenomic constructs have been contradictory. The results in the context of infectious virus indicate that HIV-1 can replicate in T-cell lines under conditions where either exon 2 or 3 is not included into any viral mRNAs. Also in the context of the viral genome, the stability and function of mRNAs in some cell types were not significantly affected by the presence or absence of these small exons. Thus, mRNA species containing the small exons may be byproducts resulting from the necessity for 50 ss D2 and D3 to serve as regulators of vif and vpr mRNA splicing as well as for the maintenance of an adequate supply of unspliced RNA. This is a delicate balancing act where small changes in the U1 snRNP-binding affinities to the downstream 50 ss can dramatically shift the HIV-1 splicing patterns. Because of the dramatic effects of splicing element mutants and effects of cellular splicing factors on virus replication, the search for additional antiviral drugs that specifically inhibit HIV-1 splicing should be pursued. It is possible that additional drugs that target specific SR proteins required for the activity of HIV-1 ESEs will be discovered. Use of antisense nucleic acids to target HIV-1 ESE and ESS as well as specific HIV-1 splice sites should also be tested using new generation antisense oligonucleotides such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. These types of antisense oligonucleotides have been found more stable and effective than standard antisense oligonucleotides (for a recent review, see Karkare and Bhatnagar, 2006).

ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI36073 from the National Institute of Allergy and Infectious Disease. I thank the past and present members of my laboratory and other colleagues for their contributions to the work described in this chapter.

REFERENCES Abbink, T. E., and Berkhout, B. (2008). RNA structure modulates splicing efficiency at the human immunodeficiency virus type 1 major splice donor. J. Virol. 82:3090–3098.

34

C. Martin Stoltzfus

Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M. A. (1986). Production of acquired immunodeficiency syndromeassociated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284–291. Aldovini, A., and Young, R. A. (1990). Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J. Virol. 64:1920–1926. Amendt, B. A., Hesslein, D., Chang, L.-J., and Stoltzfus, C. M. (1994). Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of the human immunodeficiency virus type 1. Mol. Cell. Biol. 14:3960–3970. Amendt, B. A., Si, Z.-H., and Stoltzfus, C. M. (1995). Presence of exon splicing silencers within HIV-1 tat exon 2 and tat/rev exon 3: Evidence for inhibition mediated by cellular factors. Mol. Cell. Biol. 15:4606–4615. Asang, C., Hauber, I., and Schaal, H. (2008). Insights into the selective activation of alternatively used splice acceptors by the human immunodeficiency virus type-1 bidirectional splicing enhancer. Nucl. Acids Res. 36:1450–1463. Ashe, M. P., Griffin, P., James, W., and Proudfoot, N. J. (1995). Poly(A) site selection in the HIV-1 provirus: Inhibition of promoter–proximal polyadenylation by the downstream major splice donor site. Genes Dev. 9:3008–3025. Ashe, M. P., Pearson, L. H., and Proudfoot, N. J. (1997). The HIV-1 50 LTR poly(A) site is inactivated by U1 snRNP interaction with the downstream major splice donor site. EMBO J. 16:5752–5763. Ashe, M. P., Furger, A., and Proudfoot, N. J. (2000). Stem-loop 1 of the U1 snRNP plays a critical role in the suppression of HIV-1 polyadenylation. RNA 6:170–177. Asparuhova, M. B., Marti, G., Liu, S., Serhan, F., Trono, D., and Schumperli, D. (2007). Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping. J. Gene Med. 9:323–334. Benko, D. M., Schwartz, S., Pavlakis, G. N., and Felber, B. K. (1990). A novel human immunodeficiency virus type 1 protein, tev, shares sequences with tat, env, and rev proteins. J. Virol. 64:2505–2518. Berro, R., Kehn, K., de la Fuente, C., Pumfery, A., Adair, R., Wade, J., ColbergPoley, A. M., Hiscott, J., and Kashanchi, F. (2006). Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J. Virol. 80:3189–3204. Berro, R., Pedati, C., Kehn-Hall, K., Wu, W., Klase, Z., Even, Y., Geneviere, A. M., Ammosova, T., Nekhai, S., and Kashanchi, F. (2008). CDK13, a new potential human immunodeficiency virus type 1 inhibitory factor regulating viral mRNA splicing. J. Virol. 82:7155–7166. Bilodeau, P. S., Domsic, J. K., and Stoltzfus, C. M. (1999). Splicing regulatory elements within tat exon 2 of human immunodeficiency virus type 1 (HIV-1) are characteristic of group M but not group O HIV-1 strains. J. Virol. 73:9764–9772. Bilodeau, P. S., Domsic, J. K., Mayeda, A., Krainer, A. R., and Stoltzfus, C. M. (2001). RNA splicing at human immunodeficiency virus type 1 30 splice site A2 is regulated by binding of hnRNP A/B proteins to an exonic splicing silencer element. J. Virol. 75:8487–8497. Black, D. L. (2003). Mechanisms of alternative pre-messenger RNA splicing. Annu. Rev. Biochem. 72:291–336. Bohne, J., and Krausslich, H. G. (2004). Mutation of the major 50 splice site renders a CMV-driven HIV-1 proviral clone Tat-dependent: Connections between transcription and splicing. FEBS Lett. 563:113–118.


35

Bohne, J., Wodrich, H., and Krausslich, H.-G. (2005). Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 50 end. Nucl. Acids Res. 33:825–837. Buratti, E., and Baralle, F. E. (2004). Influence of RNA secondary structure on the pre-mRNA splicing process. Mol. Cell. Biol. 24:10505–10514. Buratti, E., Muro, A. F., Giombi, M., Gherbassi, D., Iaconcig, A., and Baralle, F. E. (2004). RNA folding affects the recruitment of SR proteins by mouse and human polypurinic enhancer elements in the fibronectin EDA exon. Mol. Cell. Biol. 24:1387–1400. Caputi, M., and Zahler, A. M. (2002). SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D. EMBO J. 21:845–855. Caputi, M., Mayeda, A., Krainer, A. R., and Zahler, A. M. (1999). hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J. 18:4060–4067. Caputi, M., Freund, M., Kammler, S., Asang, C., and Schaal, H. (2004). A bidirectional SF2/ASF- and SRp40-dependent splicing enhancer regulates HIV-1 rev, env, vpu, and nef gene expression. J. Virol. 78:6517–6526. Chang, L.-J., and Zhang, C. (1995). Infection and replication of Tat human immunodeficiency viruses: Genetic analyses of LTR and tat mutations in primary and long-term human lymphoid cells. Virology 211:157–169. Chen, C. D., Kobayashi, R., and Helfman, D. M. (1999). Binding of hnRNP H to an exonic splicing silencer is involved in the regulation of alternative splicing of the rat b-tropomyosin gene. Genes Dev. 13:593–606. Cullen, B. R. (2003). Nuclear mRNA export: Insights from virology. Trends Biochem. Sci. 28:419–424. Damgaard, C. K., Tange, T. O., and Kjems, J. (2002). hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure. RNA 8:1401–1415. Damier, L., Domenjoud, L., and Branlant, C. (1997). The D1–A2 and D2–A2 pairs of splice sites from human immunodeficiency virus type 1 are highly efficient in vitro, in spite of an unusual branch site. Biochem. Biophys. Res. Commun. 237:182–187. Dehart, J. L., and Planelles, V. (2008). Human immunodeficiency virus type 1 Vpr links proteasomal degradation and checkpoint activation. J. Virol. 82:1066–1072. DelGatto-Konczak, F., Olive, M., Gesnel, M.-C., and Breathnach, R. (1999). hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol. Cell. Biol. 19:251–260. Domsic, J. K., Wang, Y., Mayeda, A., Krainer, A. R., and Stoltzfus, C. M. (2003). Human immunodeficiency virus type 1 hnRNP A/B-dependent exonic splicing silencer ESSV antagonizes binding of U2AF65 to viral polypyrimidine tracts. Mol. Cell. Biol. 23:8762–8772. Dowling, D., Nasr-Esfahani, S., Tan, C. H., O’Brien, K., Howard, J. L., Jans, D. A., Purcell, D. F., Stoltzfus, C. M., and Sonza, S. (2008). HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages. Retrovirology 5:18. Dyhr-Mikkelsen, H., and Kjems, J. (1995). Inefficient spliceosome assembly and abnormal branch site selection in splicing of an HIV-1 transcript in vitro. J. Biol. Chem. 270:24060–24066. Exline, C. M., Feng, Z., and Stoltzfus, C. M. (2008). Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression. J. Virol. 82:3921–3931.

36

C. Martin Stoltzfus

Fukuhara, T., Hosoya, T., Shimizu, S., Sumi, K., Oshiro, T., Yoshinaka, Y., Suzuki, M., Yamamoto, N., Herzenberg, L. A., Herzenberg, L. A., and Hagiwara, M. (2006). Utilization of host SR protein kinases and RNA-splicing machinery during viral replication. Proc. Natl. Acad. Sci. USA 103: 11329–11333. Goila-Gaur, R., and Strebel, K. (2008). HIV-1 Vif, APOBEC, and intrinsic immunity. Retrovirology 5:51. Goodchild, J., Agrawal, S., Civeira, M. P., Sarin, P. S., Sun, D., and Zamecnik, P. C. (1988). Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 85:5507–5511. Graveley, B. R. (2000). Sorting out the complexity of SR protein functions. RNA 6:1197–1211. Hallay, H., Locker, N., Ayadi, L., Ropers, D., Guittet, E., and Branlant, C. (2006). Biochemical and NMR study on the competition between proteins SC35, SRp40, and heterogeneous nuclear ribonucleoprotein A1 at the HIV-1 Tat exon 2 splicing site. J. Biol. Chem. 281:37159–37174. Han, K., Yeo, G., An, P., Burge, C. B., and Grabowski, P. J. (2005). A combinatorial code for splicing silencing: UAGG and GGGG motifs. PLoS Biol. 3:e158. Hoffman, B. E., and Grabowski, P. J. (1992). U1 snRNP targets an essential splicing factor, U2AF65, to the 30 splice site by a network of interactions spanning the exon. Genes Dev. 6:2554–2568. Jablonski, J. A., and Caputi, M. (2009). Role of cellular RNA processing factors in human immunodeficiency virus type 1 mRNA metabolism, replication, and infectivity. J. Virol. 83:981–992. Jacquenet, S., Mereau, A., Bilodeau, P. S., Damier, L., Stoltzfus, C. M., and Branlant, C. (2001). A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H. J. Biol. Chem. 276:40464–40475. Jacquenet, S., Decimo, D., Muriaux, D., and Darlix, J.-L. (2005). Dual effect of the SR proteins ASF/SF2, SC35, and 9G8 on HIV-1 RNA splicing and virion production. Retrovirology 2:33. Kammler, S., Leurs, C., Freund, M., Krummheuer, J., Seidel, K., Tange, T. O., Lund, M. K., Kjems, J., Scheid, A., and Schaal, H. (2001). The sequence complementarity between HIV-1 50 splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA. RNA 7:421–434. Kammler, S., Otte, M., Hauber, I., Kjems, J., Hauber, J., and Schaal, H. (2006). The strength of the HIV-1 30 splice sites affects Rev function. Retrovirology 3:89. Karkare, S., and Bhatnagar, D. (2006). Promising nucleic acid analogs and mimics: Characteristic features and applications of PNA, LNA, and morpholino. Appl. Microbiol. Biotechnol. 71:575–586. Kim, S., Byrn, R., Groopman, J., and Baltimore, D. (1989). Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression. J. Virol. 63:3708–3713. Klotman, M. E., Kim, S., Buchbinder, A., DeRossi, A., Baltimore, D., and WongStaal, F. (1991). Kinetics of expression of multiply spliced RNA in early human immunodeficiency virus type 1 infection of lymphocytes and monocytes. Proc. Natl. Acad. Sci. USA 88:5011–5015. Kornblihtt, A. R., Mata, M. D. L., Fededa, J. P., Munoz, M. J., and Nogues, G. (2004). Multiple links between transcription and splicing. RNA 10:1489–1498. Krummheuer, J., Lenz, C., Kammler, S., Scheid, A., and Schaal, H. (2001). Influence of the small leader exons 2 and 3 on human immunodeficiency virus type 1 gene expression. Virology 286:276–289.


37

Kuramitsu, M., Hashizume, C., Yamamoto, N., Asuma, A., Kamata, M., Yamamoto, N., Tanaka, Y., and Aida, Y. (2005). A novel role for Vpr of human immunodeficiency virus type 1 as a regulator of splicing of cellular pre-mRNA. Microbes Infect. 7:1150–1160. Lever, A., Gottlinger, H., Haseltine, W., and Sodroski, J. (1989). Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J. Virol. 63:4085–4087. Lu, X., Heimer, J., Rekosh, D., and Hammarskjold, M.-L. (1990). U1 small nuclear RNA plays a direct role in the formation of a rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proc. Natl. Acad. Sci. USA 87:7598–7602. Lu, R., Ghory, H. Z., and Engelman, A. (2005). Genetic analyses of conserved residues in the carboxyl-terminal domain of human immunodeficiency virus type 1 integrase. J. Virol. 79:10356–10368. Luo, Y., Yu, H., and Peterlin, B. M. (1994). Cellular protein modulates effects of human immunodeficiency virus type 1 Rev. J. Virol. 68:3850–3856. Lutzelberger, M., Reinert, L. S., Das, A. T., Berkhout, B., and Kjems, J. (2006). A novel splice donor site in the gag–pol gene is required for HIV-1 RNA stability. J. Biol. Chem. 281:18644–18651. Madsen, J. M., and Stoltzfus, C. M. (2005). An exonic splicing silencer downstream of 30 splice site A2 is required for efficient human immunodeficiency virus type 1 replication. J. Virol. 79:10478–10486. Madsen, J. M., and Stoltzfus, C. M. (2006). A suboptimal 50 splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication. Retrovirology 3:10. Maldarelli, F., Xiang, C., Chamoun, G., and Zeichner, S. L. (1998). The expression of the essential nuclear splicing factor SC35 is altered by human immunodeficiency virus infection. Virus Res. 53:39–51. Mandal, D., Feng, Z., and Stoltzfus, C. M. (2008). Gag-processing defect of human immunodeficiency virus type 1 integrase E246 and G247 mutants is caused by activation of an overlapping 50 splice site. J. Virol. 82:1600–1604. Mandal, D., Exline, C. M., Feng, Z., and Stoltzfus, C. M. (2009). Regulation of vif mRNA splicing by human immunodeficiency virus type 1 requires 50 splice site D2 and an exonic splicing enhancer to counteract cellular restriction factor APOBEC3G. J. Virol. 83:6067–6078. Marchand, V., Mereau, A., Jacquenet, S., Thomas, D., Mougin, A., Gattoni, R., Stevenin, J., and Branlant, C. (2002). A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. J. Mol. Biol. 323:629–652. Matlin, A. J., Clark, F., and Smith, C. W. (2005). Understanding alternative splicing: Towards a cellular code. Nat. Rev. Mol. Cell Biol. 6:386–398. Mayeda, A., Screaton, G. R., Chandler, S. D., Fu, X.-D., and Krainer, A. R. (1999). Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements. Mol. Cell. Biol. 19:1853–1863. Michael, N. L., Morrow, P., Mosca, J., Vahey, M., Burke, D. S., and Redfield, R. R. (1991). Induction of human immunodeficiency virus type 1 expression in chronically infected cells is associated primarily with a shift in RNA splicing patterns. J. Virol. 65:1291–1303. Muesing, M. A., Smith, D. H., and Capon, D. J. (1987). Regulation of mRNA accumulation by a human immunodeficiency virus transactivator protein. Cell 48:691–701.

38

C. Martin Stoltzfus

O’Reilly, M. M., McNally, M. T., and Beemon, K. L. (1995). Two strong 50 splice sites and competing suboptimal 30 splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. Virology 213:373–385. Pandit, S., Wang, D., and Fu, X. D. (2008). Functional integration of transcriptional and RNA processing machineries. Curr. Opin. Cell Biol. 20:260–265. Peng, G., Lei, K. J., Jin, W., Greenwell-Wild, T., and Wahl, S. M. (2006). Induction of APOBEC3 family proteins, a defensive maneuver underlying interferoninduced anti-HIV activity. J. Exp. Med. 203:41–46. Petersen-Mahrt, S. K., Estmer, C., Ohrmalm, C., Matthews, D. A., Russell, W. C., and Akusjarvi, G. (1999). The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation. EMBO J. 18:1014–1024. Pollard, V. W., and Malim, M. H. (1998). The HIV-1 Rev protein. Annu. Rev. Microbiol. 52:491–532. Pongoski, J., Asai, K., and Cochrane, A. (2002). Positive and negative modulation of human immunodeficiency virus type 1 Rev function by cis and trans regulators of viral RNA splicing. J. Virol. 76:5108–5120. Price, D. H. (2000). P-TEFb, a cyclin-dependent kinase controlling degradation by RNA polymerase II. Mol. Cell. Biol. 20:2629–2634. Purcell, D. F. J., and Martin, M. A. (1993). Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication and infectivity. J. Virol. 67:6365–6378. Riggs, N. L., Little, S. J., Richman, D. D., and Guatelli, J. C. (1994). Biological importance and cooperativity of HIV-1 regulatory gene splice acceptors. Virology 202:264–271. Robberson, B. L., Cote, G. J., and Berget, S. M. (1990). Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol. Cell. Biol. 10:84–94. Ropers, D., Ayadi, L., Gattoni, R., Jacquenet, S., Damier, L., Branlant, C., and Stevenin, J. (2004). Differential effects of the SR proteins 9G8, SC35, ASF/SF2, and SRp40 on the utilization of the A1 to A5 splicing sites of HIV-1 RNA. J. Biol. Chem. 279:29963–29973. Rose, K. M., Marin, M., Kozak, S. L., and Kabat, D. (2004). Transcriptional regulation of APOBEC3G, a cytidine deaminase that hypermutates human immunodeficiency virus. J. Biol. Chem. 279:41744–41749. Ryo, A., Suzuki, Y., Arai, M., Kondoh, N., Wakatsuki, T., Hada, A., Shuda, M., Tanaka, K., Sato, C., Yamamoto, M., and Yamamoto, N. (2000). Identification and characterization of differentially expressed mRNAs in HIV type 1-infected human T cells. AIDS Res. Hum. Retroviruses 16:995–1005. Salfeld, J., Gottlinger, H., Sia, R., Park, R., Sodroski, J., and Haseltine, W. (1990). A tripartite HIV-1 tat–env–rev fusion protein. EMBO J. 9:965–970. Saliou, J. M., Bourgeois, C. F., Ayadi-Ben Mena, L., Ropers, D., Jacquenet, S., Marchand, V., Stevenin, J., and Branlant, C. (2009). Role of RNA structure and protein factors in the control of HIV-1 splicing. Front Biosci. 14:2714–2729. Schwartz, S., Felber, B. K., Benko, D. M., Fenyo, E.-M., and Pavlakis, G. N. (1990). Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1. J. Virol. 64:2519–2529. Sharp, P. M., Bailes, E., Chaudhuri, R. R., Rodenburg, C. M., Santiago, M. O., and Hahn, B. H. (2001). The origins of acquired immune deficiency syndrome viruses: Where and when? Philos. Trans. R. Soc. Lond. B Biol. Sci. 356:867–876. Si, Z.-H., Amendt, B. A., and Stoltzfus, C. M. (1997). Splicing efficiency of human immunodeficiency virus type 1 Tat RNA is determined by both a suboptimal


39

30 splice site and a 10 nucleotide exon splicing silencer element located within tat exon 2. Nucl. Acids Res. 25:861–867. Si, Z.-H., Rauch, D., and Stoltzfus, C. M. (1998). The exon splicing silencer in human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in spliceosome assembly. Mol. Cell. Biol. 18:5404–5413. Sonza, S., Mutimer, H. P., O’Brien, K., Ellery, P., Howard, J. L., Axelrod, J. H., Deacon, N. J., Crowe, S. M., and Purcell, D. F. (2002). Selectively reduced tat mRNA heralds the decline in productive human immunodeficiency virus type 1 infection in monocyte-derived macrophages. J. Virol. 76:12611–12621. Soret, J., Bakkour, N., Maire, S., Durand, S., Zekri, L., Gabut, M., Fic, W., Divita, G., Rivalle, C., Dauzonne, D., Nguyen, C. H., Jeanteur, P., and Tazi, J. (2005). Selective modification of alternative splicing by indole derivatives that target serine–arginine-rich protein splicing factors. Proc. Natl. Acad. Sci. USA 102:8764–8769. Staffa, A., and Cochrane, A. (1995). Identification of positive and negative splicing regulatory elements within the terminal tat/rev exon of HIV-1. Mol. Cell. Biol. 15:4597–4605. Stoltzfus, C. M., and Madsen, J. M. (2006). Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing. Curr. HIV Res. 4:43–55. Stopak, K. S., Chiu, Y. L., Kropp, J., Grant, R. M., and Greene, W. C. (2007). Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells. J. Biol. Chem. 282:3539–3546. Swanson, A. K., and Stoltzfus, C. M. (1998). Overlapping cis sites used for splicing of HIV-1 env/nef and rev mRNAs. J. Biol. Chem. 273:34551–34557. Tange, T. O., and Kjems, J. (2001). SF2/ASF binds to a splicing enhancer in the third HIV-1 tat exon and stimulates U2AF binding independently of the RS domain. J. Mol. Biol. 312:649–662. Tange, T. O., Jensen, T. H., and Kjems, J. (1996). In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2associated protein, p32. J. Biol. Chem. 271:10066–10072. Terada, Y., and Yasuda, Y. (2006). Human immunodeficiency virus type 1 Vpr induces G2 checkpoint activation by interacting with the splicing factor SAP145. Mol. Cell. Biol. 26:8149–8158. Varani, L., Hasegawa, M., Spillantini, M. G., Smith, M. J., Murrell, J. R., Ghetti, B., Klug, A., Goedert, M., and Varani, G. (1999). Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17. Proc. Natl. Acad. Sci. USA 96:8229–8234. Wang, Z., and Burge, C. B. (2008). Splicing regulation: From a parts list of regulatory elements to an integrated splicing code. RNA 14:802–813. Wentz, M. P., Moore, B. E., Cloyd, M. W., Berget, S. M., and Donehower, L. A. (1997). A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production. J. Virol. 71:8542–8551. Zahler, A. M., Damgaard, C. K., Kjems, J., and Caputi, M. (2004). SC35 and heterogeneous nuclear ribonucleoprotein A/B proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate HIV-1 tat exon 2 splicing. J. Biol. Chem. 279:10077–10084. Zhang, G., Zapp, M. L., Yan, G., and Green, M. R. (1996). Localization of HIV-1 RNA in mammalian nuclei. J. Cell Biol. 135:9–18.

40

C. Martin Stoltzfus

Zheng, Z.-M. (2004). Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression. J. Biomed. Sci. 11:278–294. Zheng, Y. H., Yu, H. F., and Peterlin, B. M. (2003). Human p32 protein relieves a post-transcriptional block to HIV replication in murine cells. Nat. Cell Biol. 5:611–618. Zhu, J., Mayeda, A., and Krainer, A. R. (2001). Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins. Mol. Cell 8:1351–1361.

CHAPTER

2 New Insights into Flavivirus Nonstructural Protein 5 Andrew D. Davidson

Contents

I. Introduction II. The Methyltransferase Domain A. 50 -RNA cap formation B. MTase enzymatic activities C. MTase structure D. MTase structure–function studies E. A model for flavivirus cap methylation III. The RNA-Dependent RNA Polymerase Domain A. Flavivirus RNA synthesis B. RdRp activity of NS5 C. RdRp structure D. Structure–function analysis IV. NS5 Interactions A. NS5 intramolecular interactions B. The interaction of NS5 with viral RNA C. The interaction of NS3 and NS5 D. The interaction of NS5 with host proteins V. NS5 Phosphorylation VI. NS5 Localization A. Cellular localization of NS5 B. NS5 nuclear localization VII. Emerging Roles for NS5 in Viral Pathogenesis VIII. Conclusions and Future Perspectives Acknowledgments References

42 44 44 45 52 57 60 61 61 62 67 74 75 75 76 77 78 79 81 81 82 85 89 92 92

Department of Cellular and Molecular Medicine, School of Medical and Veterinary Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74002-3

#


41

42

Abstract

Andrew D. Davidson

Disease caused by flavivirus infections is an increasing world health problem. Flavivirus nonstructural protein 5 (NS5) possesses enzymatic activities required for capping and synthesis of the viral RNA genome and is essential for virus replication. NS5 is comprised of two domains. The N-terminal domain binds GTP and can perform two biochemically distinct methylation reactions required for RNA cap formation. The C-terminal domain contains RNA-dependent RNA polymerase activity. As such, NS5 is an interesting target against which antiviral drugs could be developed and research toward this goal has accelerated our understanding of NS5 structure and function in recent years. The production and purification of recombinant versions of either the full-length NS5 or the two individual NS5 domains has led to detailed enzymatic studies on NS5 and the determination of structures of the two NS5 domains. In turn, studies using a combination of structural, biochemical, and reverse genetic approaches are revealing how NS5 performs its multifunctional roles in genome replication. Aside from its localization in the membranebound replication complex, NS5 can be found free in the cytoplasm and for some flaviviruses in the nucleus of virus-infected cells. NS5 is phosphorylated which may potentially regulate NS5 function and trafficking. Recently, NS5 of a number of flaviviruses has been shown to interact with cellular pathways involved in the host immune response, suggesting that NS5 may play a role in viral pathogenesis. This chapter reviews recent advances in our understanding of the multifunctional roles played by NS5 in the virus lifecycle.

I. INTRODUCTION Flaviviruses are small enveloped RNA viruses that comprise one of the three genera in the Flaviviridae family together with the Hepaciviruses and the Pestiviruses. The Flavivirus genus contains at least 53 recognized viral species, which are predominantly transmitted by arthropod vectors. There are 40 flaviviruses capable of causing disease in humans (Gubler et al., 2007). A number of these are medically important pathogens causing significant mortality and morbidity including; the four serotypes of dengue virus (DENV types 1–4), Japanese encephalitis virus ( JEV), tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and yellow fever virus (YFV). Control measures against flaviviruses are limited to vaccines against JEV, TBEV, and YFV and vector control. Currently, there are no antiviral compounds in clinical use against flaviviruses. Difficulties in controlling flaviviral vectors, particularly mosquitoes, societal changes and lapses in vaccine coverage have made DENV, JEV, and WNV among the most important examples of emerging and reemerging viruses (Mackenzie et al., 2004).

Flavivirus NS5

43

The flavivirus particle consists of two outer membrane proteins, envelope (E) and membrane (M, processed from the precursor prM), surrounding a nucleocapsid containing the capsid (C) protein and a positive sense single-stranded RNA genome. The genome is approximately 11 kb in size with a type I cap structure at the 50 -end but lacks a 30 -polyadenylate tail. The viral RNA contains a single long open reading frame flanked by 50 - and 30 -untranslated regions (UTRs). The UTRs contain a number of cis-acting signals required for viral replication including conserved stem-loop structures. Complementary sequences in the 50 - and 30 -terminal regions (TRs) have been identified that are able to interact and cyclize the genome, a prerequisite for genome replication (reviewed in Markoff, 2003; Villordo and Gamarnik, 2009). The open reading frame is translated as a single polyprotein which is cleaved by a combination of cellular signal peptidase and the virally encoded two component serine protease NS2B/NS3. Proteolysis yields the three structural proteins (C, prM, and E) and seven nonstructural (NS) proteins; NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The nonstructural proteins are all involved in viral RNA replication with NS3 and NS5 possessing the enzymatic activities required for RNA capping and genome replication. In addition, specific nonstructural proteins have been shown to play roles in viral assembly and perturbation of host defense mechanisms (Lindenbach and Rice, 2003; Lindenbach et al., 2007). Flavivirus infection results in extensive reorganization and proliferation of cytoplasmic endoplasmic reticular (ER) membranes, leading to the formation of characteristic structures, late in infection, described as vesicle packets (VPs), convoluted membranes (CM), and paracrystalline arrays (PC). Ultrastructural, immunological, and biochemical studies have shown that newly synthesized viral RNA is found in VPs in association with NS1, NS2A, NS3, NS4A, NS4B, and NS5 while NS2B, NS3, and NS4A are found in CM structures (Mackenzie et al., 1998; Miller et al., 2006; Westaway et al., 1997). It has been proposed that the VPs are double-membrane structures that house the replication complex and are the sites of RNA synthesis, the newly synthesized RNA is then exported to the CM/PC for translation and proteolytic processing (Mackenzie, 2005; Westaway et al., 2002, 2003). NS5 is the largest (900 amino acids) and most conserved of the flaviviral proteins. NS5 plays a key role in viral replication, containing enzymatic activities required for capping and synthesis of the RNA genome. Sequence analysis first suggested that NS5 is comprised of an methyltransferase N-terminal S-adenosyl-L-methionine-dependent domain (Koonin, 1993) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain (Koonin, 1991; Poch et al., 1989; Rice et al., 1985; Sumiyoshi et al., 1987). The N-terminal domain has since been shown to have the ability to bind GTP (Egloff et al., 2002) and perform two biochemically distinct methylation reactions required for RNA cap formation

44

Andrew D. Davidson

(Egloff et al., 2002; Ray et al., 2006). Recombinant versions of NS5, including the C-terminal domain alone, have been demonstrated to have RdRp activity in in vitro assays (Ackermann and Padmanabhan, 2001; Guyatt et al., 2001; Selisko et al., 2006; Steffens et al., 1999; Tan et al., 1996). The X-ray crystal structures of the two individual domains have been determined for representative flaviviruses (Assenberg et al., 2007; Egloff et al., 2002; Malet et al., 2007; Mastrangelo et al., 2007; Yap et al., 2007a; Zhou et al., 2007). Recent advances in our understanding of the enzymatic activities of NS5, coupled with structure–function and interaction studies are beginning to reveal how NS5 performs its multifunctional roles in viral replication, making NS5 an interesting target for antiviral drug development (reviewed in (Dong et al., 2008c; Malet et al., 2008; Rawlinson et al., 2006)). In addition to its role in the viral replication complex, recent studies have shown that NS5 may also play a role in pathogenesis. NS5 is not only found associated with the membrane-bound replication complex but free in the cytoplasm and for some flaviviruses in the nucleus where it potentially interacts with host factors. The NS5 of specific flaviviruses can perturb interferon signaling and cytokine production (Best et al., 2005; Lin et al., 2006; Medin et al., 2005; Werme et al., 2008). NS5 is known to be phosphorylated, providing a mechanism by which NS5 enzymatic activity, molecular interactions, and trafficking could be regulated (Lindenbach et al., 2007). This chapter will focus on reviewing recent advances in our understanding of NS5 replicative function and trafficking and the possible role of NS5 in pathogenesis. As the functional and structural properties of the N- and C-terminal domains have largely been investigated in isolation, studies on each domain will be reviewed separately.

II. THE METHYLTRANSFERASE DOMAIN A. 50 -RNA cap formation Cellular and many viral mRNAs contain a modified 50 -terminal guanosine ‘‘cap’’ structure covalently linked to the 50 -end of the mRNA. The formation of the 50 -RNA cap structure requires three sequential enzymatic reactions: (1) the 50 -terminal triphosphate of the nascent RNA is hydrolyzed to a diphosphate by the enzyme RNA triphosphatase, (2) the RNA is capped with GMP in a 50 –50 -triphosphate linkage by mRNA guanyltransferase, and (3) the guanosine is methylated at the N7 position by a (guanine-N7)-methyltransferase (N7 MTase) using S-adenosyl7 L-methionine (AdoMet) as a methyl donor to form a type 0 (m GpppN) cap structure. Nucleotides adjacent to the cap structure may be further methylated by nucleoside-20 -O-methyltransferases (20 -O-MTase), to give type I (m7GpppNm) or type II cap structures (m7GpppNmNm) (Furuichi

Flavivirus NS5

45

and Shatkin, 2000; Shuman, 2001). Vector-borne flaviviruses have identical type I cap structures (m7GpppAmG) (Cleaves and Dubin, 1979; Wengler et al., 1978) as the first two nucleotides (AG) of the genome are strictly conserved (Markoff, 2003). Formation of the flavivirus cap structure is believed to involve NS3 which has RNA triphosphatase activity (Bartelma and Padmanabhan, 2002; Benarroch et al., 2004b; Wengler, 1993) and NS5 that has both N7 and 20 -O-MTase activities (Egloff et al., 2002; Ray et al., 2006). As yet, the source of the mRNA guanyltransferase activity has not been identified. Although the mechanism by which the cap structure is formed is functionally conserved in eukaryotes and many viruses, the architecture of the capping enzymes and sequence of capping reactions varies (Furuichi and Shatkin, 2000). In the simplest case, such as for yeast, each of the enzymatic activities required for RNA capping resides in an individual protein. In contrast, for vaccinia virus, the best characterized viral system, capping is performed by a heterodimeric enzyme. The larger D1 subunit has RNA triphosphatase and mRNA guanyltransferase activities, while full N7 MTase activity requires the formation of a complex with the smaller D12 subunit (Shuman, 1995). A third protein, VP39, possesses 20 -O-MTase activity. For dsRNA viruses of the family Reoviridae and negative strand RNA viruses of the order Mononegavirales, all of the enzymatic activities have been detected in single large multidomain proteins (Furuichi and Shatkin, 2000; Hercyk et al., 1988; Ogino et al., 2005; Ramadevi et al., 1998). Structural studies on Reoviridae proteins have assigned distinct enzymatic activities to individual protein domains (Reinisch et al., 2000; Sutton et al., 2007). Interestingly, the L protein of vesicular stomatitis virus (VSV), similar to the flavivirus NS5, exhibits both N7 and 20 -O-MTase activities but contains a single AdoMet-binding site (Li et al., 2006).

B. MTase enzymatic activities The identification of a sequence motif that is conserved in AdoMetdependent MTases first suggested that the N-terminal region of NS5 may have MTase activity (Koonin, 1993). Further bioinformatic analysis delineated a potential MTase domain in the first 296 amino acids of the DENV-2 NS5 (DENV-2 MTase) (Fig. 1). Examination of the MTase activity of the bacterially expressed DENV-2 MTase, using short capped (GpppAC5 and m7GpppAC5) and noncapped (pppAC5) RNA substrates, showed that the MTase had cap-dependent 20 -O-MTase activity (Egloff et al., 2002). Under the experimental conditions used, N7 MTase activity was not detectable. More recently, the MTase activities of a bacterially expressed, full-length WNV NS5 and a truncated NS5, containing the MTase domain (amino acids 1–300), were examined using a capped RNA transcript corresponding to the first 190 nucleotides of the WNV

MA1 DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA

MA2

MB1

MA3

MaX

X

Mb2

DV-1 DV-3 DV-2 DV-4 JEV MVEV WNV KUNV YFV TBEV LGTV MEAV MoDV CFA

MaA

Mb1

+ 20 + 40 + 60# + 80 + ---GTGAQGETLGEKWKRQLNQLSKSEFNTYKRSGIIEVDRSEAKEGLKRGE-PTKHA--VSRGTAKLRWFVERNLVKPEGKVIDLGCGRGGWSYYCAGLKKV ---GTGSQGETLGEKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGE-TTHHA--VSRGSAKLQWFVERNMVVPEGRVIDLGCGRGGWSYYCAGLKKV ---GTGNIGETLGEKWKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGE-TDHHA--VSRGSAKLRWFVERNMVTPEGKVVDLGCGRGGWSYYCGGLKNV ---GTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGS-KIKHA--VSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNV ----GRPGGRTLGEQWKEKLNAMSREEFFKYRREAIIEVDRTEARRARRENNIVGGHP--VSRGSAKLRWLVEKGFVSPIGKVIDLGCGRGGWSYYAATLKKV ----GRAGGRTLGEQWKEKLNAMGKEEFFSYRKEAILEVDRTEARRARREGNKVGGHP--VSRGTAKLRWLVERRFVQPIGKVVDLGCGRGGWSYYAATMKNV ----GGAKGRTLGEVWKERLNQMTKEEFTRYRKEAIIEVDRSAAKHARKEGNVTGGHP--VSRGTAKLRWLVERRFLEPVGKVIDLGCGRGGWCYYMATQKRV ----GGAKGRTLGEVWKERLNQMTKEEFIRYRKEAITEVDRSAAKHARKERNITGGHP--VSRGTAKLRWLVERRFLEPVGKVIDLGCGRGGWCYYMATQKRV ----GSANGKTLGEVWKRELNLLDKRQFELYKRTDIVEVDRDTARRHLAEGKVDTGVA--VSRGTAKLRWFHERGYVKLEGRVIDLGCGRGGWCYYAAAQKEV ----GGSEGDTLGDLWKRRLNNCTREEFFVYRRTGILETERDKARELLRRGETNVGLA--VSRGTAKLAWLEERGYATLKGEVVDLGCGRGGWSYYAASRPAV ----GGSEGDTLGDMWKARLNSCTKEEFFAYRRAGVMETDREKARELLKRGETNMGLA--VSRGTSKLAWMEERGYVTLKGEVVDLGCGRGGWSYYAASRPAV ---GPGSTGASLGMMWKDKLNAMTKEEFTRYKRAGVMETDRKEARDYLKRGDGKTGLS--VSRGTAKLAWMEERGYVELTGRVVDLGCGRGGWSYYAASRPHV ---GICSSAPTLGEIWKRKLNQLDAKEFMAYRRRFVVEVDRNEAREALAKGKTNTGHA--VSRGTAKLAWIDERGGVELKGSVVDLGCGRGGWSYYAASQPNV MHIAARALGAVAPFNQFRALEKSTTIGLGMKWKMTLNALDGDAFTRYKSRGVNETERGDYVSRGGLKLNEIISKYEWRPSGRVVDLGCGRGGWSQRAVMEETV * **** * * * ********* *

Mb3

I

MaD

Mb4

Mb5 #

MaE

100 + 120 + 140 # + 160 + 180 + TEVKGYTKGGPGHEEPIPMATYGWNLVKLYSGKDVFFTPPEKCDTLLCDIGESSPNPTIEEGRTLRVLKMVEPWLRGN---QFCIKILNPYMPSVVETLEQMQ TEVRGYTKGGPGHEEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTVEESRTIRVLKMVEPWLKNN---QFCIKVLNPYMPTVIEHLERLQ REVKGLTKGGPGHEEPIPMSTYGWNLVRLQSGVDVFFTPPEKCDTLLCDIGESSPNPTVEAGRTLRVLNLVENWLNNNT--QFCIKVLNPYMPSVIEKMEALQ TEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKP--EFCIKVLNPYMPTVIEELEKLQ QEVRGYTKGGAGHEEPMLMQSYGRNLVSLKSGVDVFYKPSEPSDTLFCDIGESSPSPEVEEQRTLRVLEMTSDWLHRGP-REFCIKVLCPYMPKVIEKMEVLQ QEVRGYTKGGPGHEEPMLMQSYGWNIVTMKSGVDVFYKPSEISDTLLCDIGESSPSAEIEEQRTLRILEMVSDWLSRGP-KEFCIKILCPYMPKVIEKLESLQ QEVRGYTKGGPGHEEPQLVQSYGWNIVTMKSGVDVFYRPSECCDTLLCDIGESSSSAEVEEHRTIRVLEMVEDWLHRGP-REFCVKVLCPYMPKVIEKMELLQ QEVRGYTKGGPGHEEPQLVQSYGWNIVTMKSGVDVFYRPSECCDTLLCDIGESSSSAEVEEHRTLRVLEMVEDWLHRGP-KEFCVKVLCPYMPKVIEKMELLQ SGVKGFTLGRDGHEKPMNVQSLGWNIITFKDKTDIHRLEPVKCDTLLCDIGESSSSSVTEGERTVRVLDTVEKWLACGV-DNFCVKVLAPYMPDVLEKLELLQ MSVRAYTIGGKGHEAPKMVTSLGWNLIKFRSGMDVFSMQPHRADTVMCDIGESSPDAAVEGERTRKVILLMEQWKNRNPTAACVFKVLAPYRPEVIEALHRFQ MSVRAYTIGGKGHESPRMVTSLGWNLIKFRAGMDVFSMEPHRADAILCDIGESNPDAVVEGERSRRVILLMEQWKNRNPTATCVFKVLAPYRPEVIEALHRFQ MDVRAYTLGVGGHEVPRITESYGWNIVKFKSRVDIHTLPVERTDVIMCDVGESSPKWSVESERTIKILELLEKWKVKNPSADFVVKVLCPYSVEVMERLSVMQ REVKAYTLGTSGHEKPRLVETFGWNLITFKSKVDVRKMEPFQADTVLCDIGESNPTAAVEASRTLTVLNVISRWLEYNQGCGFCVKVLNPYSCDVLEALMKMQ SSALGFTIGGAEKENPQRFVTKGYNLATLKTGVDVHRLTPFRCDTIMCDIGESDPSPIKEKTRTLKVLQLLENWLLVNPGAHFVCKILSPYSLEVLRKIESLQ * * * * * * * * ** *** * * * * * ** * *

II

III

IV

FIGURE 1

V

(Continued)

97 97 97 97 97 97 97 97 97 97 97 98 98 103

VI

VII

197 197 198 198 199 199 199 199 199 200 200 201 201 206


MaE Mb6 MB2 Pa1 MA4 Mb6 200 + # 220 + 240 + 260 + 280 + RKHGGMLVRNPLSRNSTHEMYWVSCGTGNIVSAVNMTSRMLLNRFTMAHRK-PTYERDVDLGAGTRHVAVEPEV-ANLDIIGQRIENIKNGHKSTWHYDEDNP RKHGGMLVRNPLSRNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRR-PTIEKDVDLGAGTRHVNAEPET-PNMDVIGERIKRIKEEHNSTWHYDDENP RKHGGALVRNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRHKK-ATYEPDVDLGSGTRNIGIESEI-PNLDIIGKRIEKIKQEHETSWHYDQDHP RKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRK-PTYEKDVDLGAGTRSVSTETEK-PDMTIIGRRLQRLQEEHKETWHYDQENP RRFGGGLVRLPLSRNSNHEMYWVSGAAGNVVHAVNMTSQVLLGRMDRTVWRGPKYEEDVNLGSGTRAVGKGEVH-SNQEKIKKRIQKLKEEFATTWHKDPEHP RRFGGGLVRVPLSRNSNHEMYWVSGASGNIVHAVNMTSQVLIGRMDKKIWKGPKYEEDVNLGSGTRAVGKGVQH-TDYKRIKSRIEKLKEEYAATWHTDDNHP RRYGGGLVRNPLSRNSTHEMYWVSRASGNVVHSVNMTSQVLLGRMEKRTWKGPQYEEDVNLGSGTRAVGKPLLN-SDTSKIKNRIERLRREYSSTWHHDENHP RRYGGGLVRNPLSRNSTHEMYWVSRASGNVVHSVNMTSQVLLGRMEKKTWKGPQYEEDVNLGSGTRAVGKPLLN-SDTSKIKNRIERLRREYSSTWHHDENHP RRFGGTVIRNPLSRNSTHEMYYVSGARSNVTFTVNQTSRLLMRRMRRPTGK-VTLEADVILPIGTRSVETDKGP-LDKEAIEERVERIKSEYMTSWFYDNDNP LQWGGGLVRTPFSRNSTHEMYYSTAVTGNIVNSVNVQSRKLLARFGD--QRGPTKVPELDLGVGTRCVVLAEDK-VKEQDVQERIRALREQYSETWHMDEEHP LQWGGGLVRTPFSRNSTHEMYFSTAITGNIVNSVNIQSRKLLARFGD--QRGPTRVPEIDLGVGTRCVVLAEDK-VKEKDVMERIQALKDQYCDTWHEDHEHP RKWGGGLVRNPYSRNSTHEMYFTSRAGGNIIGAVTACTERLLGRMAR--RDGPVVVPELNLGTGTRCVTLAEDK-VSRDLIDERLAKIKSQYAASWLEDENHP RRFGGGLIRVPLSRNSTHEMYFVSGIKNNIMGNVTAVSRQLLKRMEE--QGGERVVPDYKFSTGTRSNLTQKIE-VPEEEVQMRVDKIKAEKSGTWCFDSNHP HLYNGRLVRLSHSRNSSVEMYYISGARSNVVRTTYMTLAALMARFSR--HLDSVVLPSPVLPKGTRADPAASVASMNTSDMMDRVERLMNENRGTWFEDQQHP * * **** *** * * * *** * * * *

VIII


298 298 299 299 301 301 301 301 300 300 300 301 301 307

Interdomain Linker

Pa3 Pa5 Pa4 Pa6 Pa7 Pa2 L2 L1 Pb1 301 + 320 + 340 + 360 + 380 + YKTWAYHGSYEVKPSGSASSMVNGVVRLLTKPWDVIPMVTQIAMTDTTPFGQQRVFKEKVDTRTPKAKRGTAQIMEVTARWLWGFLSRNK-KPRICTREEFTR YKTWAYHGSYEVKATGSASSMINGVVKLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTPRPLPGTRKVMGITAEWLWRTLGRNK-RPRLCTREEFTK YKTWAYHGSYETKQTGSASSMVNGVVRLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTQEPKEGTKKLMKITAEWLWKELGKKK-TPRMCTREEFTR YRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKK-NPRLCTREEFIS YRTWTYHGSYEVKATGSASSLVNGVVKLMSKPWDAIANVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPAGAKEVLNETTNWLWAHLSREK-RPRLCTKEEFIK YRTWTYHGSYEVKPSGSASTLVNGVVRLLSKPWDAITGVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPQGVKTVMDETTNWLWAYLARNK-KARLCTREEFVK YRTWNYHGSYDVKPTGSASSLVNGVVRLLSKPWDTITNVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPEGVKYVLNETTNWLWAFLAREK-RPRMCSREEFIR YRTWNYHGSYEVKPTGSASSLVNGVVRLLSKPWDTITNVTTMAMTDTTPFGQQRVFKEKVDTKAPEPPEGVKYVLNETTNWLWAFLAREK-RPRMCSREEFIR YRTWHYCGSYVTKTSGSAASMVNGVIKILTYPWDRIEEVTRMAMTDTTPFGQQRVFKEKVDTRAKDPPAGTRKIMKVVNRWLFRHLAREK-NPRLCTKEEFIA YRTWQYWGSYRTAPTGSAASLINGVVKLLSWPWNAREDVVRMAMTDTTAFGQQRVFKDKVDTKAQEPQPGTRVIMRAVNDWILERLAQKS-KPRMCSREEFIA YRTWQYWGSYKTAATGSSASLLNGVVKLLSWPWNAREDVVRMAMTDTTAFGQQRVFKDKVDTKAQEPQPGTKIIMRAVNDWLLERLVKKS-RPRMCSREEFIA YRTWQYWGSYRCADSGSAASLINGIVKMMSWPWNNREDVCLMAMTDTTAFGQQRVFKDKVDTKAQEPRVGTRVVMRTVNNWLLERLSRKS-KPRLCTREEFIQ YRTWNYHGSYRVRDVGTRASAVNHVVKLLSWPWGKMEKVLAMSMTDTTAFGQQRVFKQKVDTKAPEPNIQVKKVMRKVFKWLIERIKTKGGKVRTCTKEEFIQ YKSFKYFGSFVTDDVKVGGQAVNPLVRKIMWPWETLTSVVGFSMTDVSTYSQQKVLREKVDTVIPPHPQHIRRVNRTITKHFIRLFKNRNLRPRILSKEEFVA * * ** * ** * *** ** * **** * ***

“bNLS”

FIGURE 1

“a/bNLS”

(Continued)

400 400 401 401 403 403 403 403 402 402 402 403 404 410


Pa9 Pa7 Pa8 Pa10 Pa11 L3 403 + 420 + 440 + 460 + 480 + 500 KVRSNAAIGAVFVDENQWNSAKEAVEDERFWDLVHRERELHKQGKCATCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFMNEDHWFSRENSL KVRTNAAMGAVFTEENQWDSAKAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGVRYLEFEALGFLNEDHWFSRENSY KVRSNAALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKCETCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSL KVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSW KVNSNAALGAVFAEQNQWSTAREAVDDPRFWEMVDEERENHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARYLEFEALGFLNEDHWLSRENSG KVNSHAALGAMFEEQNQWKNAREAVEDPKFWEMVDEERECHLRGECRTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWMSRENSG KVNSNAALGAMFEEQNQWRSAREAVEDPKFWEMVDEEREAHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWLGRKNSG KVNSNAALGAMFEEQNQWRSAREAVEDPKFWEMVDEEREAHLRGECHTCIYNMMGKREKKPGEFGKAKGSRAIWFMWLGARFLEFEALGFLNEDHWLGRKNSG KVRSHAAIGAYLEEQEQWKTANEAVQDPKFWELVDEERKLHQQGRCRTCVYNMMGKREKKLSEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWASRENSG KVKSNAALGAWSDEQNRWASAREAVEDPAFWRLVDEERERHLMGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRESSG KVRSNAALGAWSDEQNKWKSAREAVEDPEFWSLVEAERERHLQGRCAHCVYNMMGKREKKLGEFGVAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASRASSG KVRSNAAIGAWLDEQNQWKNAREAVEDPRFWRMVDEERELHLQGRCATCVYNMMGKREKKAGEFGKAKGSRAIWYMWLGSRFLEFEALGFLNEDHWASREKSG KVRSHAAIGAWSSDMEGWSSAVEAVDDPRFWNMVQKERDLHLQGKCEMCVYNLMGKSEKKPGDFGVAKGSRTIWYMWLGSRFLEFESFGFLNEEHWASRELSG NVRNDAAVGSWSRDVP-WRDVQEAIQDQCFWDLVGKERALHLQGKCEMCIYNTMGKKEKKPSLAGEAKGSRTIWYMWLGSRFLEFEALGFLNADHWVSREHFP * ** * * * * ** * ** * * * * ** *** *** * ***** ** **** * **** ** * ** *

503 503 504 504 506 506 506 506 505 505 505 506 507 512

F


Pa12 a13 Pa15 Pa14 Pb3 Pb2 506 + 520 + 540 + 560 + 580 + 600 SGVEGEGLHKLGYILRDISKIPGGNMYADDTAGWDTRITEDDLQNEAKITDIMEPE--HALLATSIFKLTYQNKVVRVQRPA--KNG-TVMDVISRRDQRGSG SGVEGEGLHKLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKIIQQMDPE--HRQLANAIFKLTYQNKVVKVQRPT--PTG-TVMDIISRKDQRGSG SGVEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNHMEGE--HKKLAEAIFKLTYQNKVVRVQRPT--PRG-TVMDIISRRDQRGSG SGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPH--HKILAKAIFKLTYQNKVVKVLRPT--PRG-AVMDIISRKDQRGSG GGVEGSGVQKLGYILRDIAGKQGGKMYADDTAGWDTRITRTDLENEAKVLELLDGE--HRMLARAIIELTYRHKVVKVMRPA--AEGKTVMDVISREDQRGSG GGVEGAGIQKLGYILRDVAQKPGGKIYADDTAGWDTRITQADLENEAKVLELMEGE--QRTLARAIIELTYRHKVVKVMRPA--AGGKTVMDVISREDQRGSG GGVEGLGLQKLGYILREVGTRPGGKIYADDTAGWDTRITRADLENEAKVLELLDGE--HRRLARAIIELTYRHKVVKVMRPA--ADGRTVMDVISREDQRGSG GGVEGLGLQKLGYILREVGTRPGGRIYADDTAGWDTRITRADLENEAKVLELLDGE--HRRLARAIIELTYRHKVVKVMRPA--ADGRTVMDVISREDQRGSG GGVEGIGLQYLGYVIRDLAAMDGGGFYADDTAGWDTRITEADLDDEQEILNYMSPH--HKKLAQAVMEMTYKNKVVKVLRPA--PGGKAYMDVISRRDQRGSG AGVEGISLNYLGWHLKKLSTLNGGLFYADDTAGWDTKVTNADLEDEEQILRYMEGE--HKQLATTIMQKAYHAKVVKVARPS--RDGGCIMDVITRRDQRGSG AGVEGISLNYLGWHLKKLASLSGGLFYADDTAGWDTKITNADLDDEEQILRYMDGD--HKKLAATVLRKAYHAKVVRVARPS--REGGCVMDIITRRDQRGSG GGVEGMGLHYLGWLVKDLAELEGGKLYADDTAGWDTRVTNSDLEDEEEILNHLEGE--HKKLAEAIMKLAYHAKVVKVARPA--SDGGTVMDIISRRDQRGSG GGVEGIPLNYLGYHLREMAQKPG-VLYADDTAGWDTRITMADLEDEGMLLDMMSGE--HKKLASALFSKAYKVKVALCPRPG--PKGGTLMDVISRTDQRGSG GGVGGVGVNYFGYYLKDIA-SRGKYLIADDIAGWDTKISEEDLEDEEALLTALTEDPYHRALMAATMRLAYQNIVAMFPRTHSKYGSGTVMDVVGRRDQRGSG ** * * * *** ***** ** * * * * * ** * ******

A

B

FIGURE 1

(Continued)

601 601 602 602 605 605 605 605 604 604 604 605 605 614


a17 Pa18 Pa16 a19 Pa20 Pb4 Pb5 604 + 620 + 640 + 660 + 680 + 700 QVGTYGLNTFTNMEAQLIRQMESEGIFSPSELETP-NLAER-VLDWLKKHGTERLKRMAISGDDCVVKPIDDRFATALTALNDMGKVRKDIPQWEPSKGWNDW QVGTYGLNTFTNMEAQLVRQMEGEGVLTKADLENP-HLLEKKITQWLETKGVERLKRMAISGDDCVVKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDW QVGTYGLNTFTNMEAQLIRQMEGEGVFKSIQHLT--VTEEIAVQNWLARVGRERLSRMAISGDDCVVKPLDDRFASALTALNDMGKVRKDIQQWEPSRGWNDW QVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNP-KGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNW QVVTYALNTFTNIAVQLVRLMEAEGVIGPQHLEQLPRKTKIAVRTWLFENGEERVTRMAISGDDCVVKPLDDRFATALHFLNAMSKVRKDIQEWKPSHGWHDW QVVTYALNTFTNIAVQLVRLMEAEAVIGPDDIESIERKKKFAVRTWLFENAEERVQRMAVSGDDCVVKPLDDRFSTALHFLNAMSKVRKDIQEWKPSQGWYDW QVVTYALNTFTNLAVQLVRMMEGEGVIGPDDVEKLTKGKGPKVRTWLFENGEERLSRMAVSGDDCVVKPLDDRFATSLHFLNAMSKVRKDIQEWKPSTGWYDW QVVTYALNTFTNLAVQLVRMMEGEGVIGPDDVEKLTKGKGPKVRTWLSENGEERLSRMAVSGDDCVVKPLDDRFATSLHFLNAMSKVRKDIQEWKPSTGWYDW QVVTYALNTITNLKVQLIRMAEAEMVIHHQHVQDCDESVLTRLEAWLTEHGCDRLKRMAVSGDDCVVRPIDDRFGLALSHLNAMSKVRKDISEWQPSKGWNDW QVVTYALNTLTNIKVQLIRMMEGEGVIEAADAHNP---RLLRVERWLKEHGEERLGRMLVSGDDCVVRPLDDRFGKALYFLNDMAKTRKDIGEWEHSAGFSSW QVVTYALNTITNIKVQLVRMMEGEGVIEVADSHNP---RLLRVEKWLEEHGEERLSRMLVSGDDCVVRPVDDRFSKALYFLNDMAKTRKDTGEWEPSTGFASW QVVTYALNTITNIKVQLIRMMEGEGVIGPADMTEP---RIIRVERWLERHGEERLGRLLVSGDDCVVKPIDDRFAEAVHFLNDMSKTRKDIGEWSPSVGYTNW QVVTYALNTLTNIKVQLIRMAEAEGVLGATFEDFG-------IDRWLQEHGEDRVERMLVSGDDCVVNAIDERFGSSLNWLNAMEKVRKDIDLWKPSPSFRNW QVVTYALNTITNGKVQVARVLESEGLLQAD---------ESVLDAWLEKHLEEALGNMVIAGDDVVVSTDNRDFSSALEYLELTGKTRKNVPQGAPSRMESNW ** ** *** ** * * * * ** *** ** * * * ** * *

B


C

D

Pa21 Pa22 Pa23 Pb6 Pb 7 705 + 720 + 740 + 760 + 780 + 800 QQVPFCSHHFHQLIMKDGREIVVPCRNQDELVGRARVSQGAGWSLRETACLGKSYAQMWQLMYFHRRDLRLAANAICSAVPVDWVPTSRTTWSIHAHHQWMTT QQVPFCSHHFHELIMKDGRKLVVPCRPQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMTT TQVPFCSHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWSLRETACLGKSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHAKHEWMTT QEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTT QQVPFCSNHFQEIVMKDGRSIVVPCRGQDELIGRARISPGAGWNVKDTACLAKAYAQMWLLLYFHRRDLRLMANAICSAVPVDWVPTGRTSWSIHSKGEWMTT QQVPFCSNHFQEVIMKDGRTLVVPCRGQDELIGRARISPGSGWNVRDTACLAKAYAQMWLVLYFHRRDLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTT QQVPFCSNHFTELIMKDGRTLVVPCRGQDELVGRARISPGAGWNVRDTACLAKSYAQMWLLLYFHRRDLRLMANAICSAVPVNWVPTGRTTWSIHAGGEWMTT QQVPFCSNHFTELIMKDGRTLVTPCRGQDELVGRARISPGAGWNVRDTACLAKSYAQMWLLLYFHRRDLRLMANAICSAVPVNWVPTGRTTWSIHAGGEWMTT ENVPFCSHHFHELQLKDGRRIVVPCREQDELIGRGRVSPGNGWMIKETACLSKAYANMWSLMYFHKRDMRLLSLAVSSAVPTSWVPQGRTTWSIHGKGEWMTT EEVPFCSHHFHELVMKDGRTLVVPCRDQDELVGRARISPGCGWSVRETACLSKAYGQMWLLSYFHRRDLRTLGLAINSAVPADWVPTGRTTWSIHASGAWMTT EEVPFCSHHFHELVMKDGRALVVPCRDQDELVGRARVSPGCGWSVRETACLSKAYGQMWLLSYFHRRDLRTLGFAICSAVPVDWVPTGRTTWSIHASGAWMTT EEVPFCSHHFHRLVMKDGRELIVPCRDQDELIGRARVSPGCGWTVRETAGLSKAYAQMWLLSYFHRRDLRLMGFGICSAVPVDWVPTGRTTWSIHGKGEWMTT ERVEFCSNHFHEMTMKDGRVIVAPCRGQTELIARGTVNQGGCVGVESTGCLAKAYAQMWLLLYFHRRDLRTLALAVMSAVPSNWIPTGRTTWSLMVKGEWMTD EKVEFCSHHYHEMSLKDGRIIIAPCRHENEVLGRSRLQKGGVVSISESACMAKAYAQMWALYYFHRRDLRLGFIAISSAVPTNWFPLGRTSWSVHQYHEWMTT * *** * **** *** * * * * * ** *** ** * * ** * * ** ** ***

E

Priming loop

FIGURE 1 (Contined)

702 703 703 704 708 708 708 708 707 704 704 705 701 708

805 806 806 807 811 811 811 811 810 807 807 808 804 811

50

899 900 900 900 905 905 905 905 905 903 903 906 898 906

Priming loop

FIGURE 1 Comparative alignment of the flavivirus NS5 amino acid sequences. Secondary structural elements are indicated by boxes (a-helices), arrows (b-sheets), or dots (important loop regions) above the sequences. Elements in the MTase and POL domains follow those described for the DENV-2 MTase (Egloff et al., 2002) and DENV-3 POL (Yap et al., 2007a,b) structures, respectively. The elements are labeled as M for the MTase or P for the POL domains. Conserved MTase sequence motifs I–X (Malone et al., 1995) based on the DENV-2 MTase structure (Egloff et al., 2002) are shown below the sequence alignment as are the positions of conserved RdRp motifs A–F defined by Poch et al. (1989). The positions of four catalytic amino acids conserved among 20 -O-MTases are indicated by # above the sequence. Invariant amino acids are marked below the sequences as ‘‘*’’. The virus sequences and their GenBank accession numbers are as follows: DENV-1 strain Western Pacific 74 (DV-1) (DVU88535), DENV-3 strain Singapore (DV-3) (AY662691), DENV-2 strain NGC (DV-2) (AF038403), DENV-4 strain 814669 (DV-4) (M14931), Japanese encephalitis virus (JEV) strain JaOArS982 (M18370), Murray Valley encephalitis virus (MVEV) strain 1–51 (AF161266), WNV strain NY 2000-crow3356 (AF404756), WNV strain Kunjin (KUNV) (AY274504), yellow fever virus (YFV), strain 17D (X03700), tick-borne encephalitis virus (TBEV) strain Neudorfl (U27495), Langat virus (LGTV) strain E5 (AF253420), Meaban virus (MEAV) (DQ235144), Modoc virus (MoDV) (AJ242984), and Cell fusing agent (CFA) (M91671). The alignments were performed using ClustalW (Thompson et al., 1994).

Andrew D. Davidson


Pa 24 a 25 Pa 26 Pa 27 808 + 820 + 840 + 860 + 880 + EDMLSVWNRVWIEENPWM--EDKTHVSSWEDVPYLGKREDRWCGSLIGLTARATWATNIQVAINQVRRLIGNEN-----YLDFMTSMKRFKNESDPEGALW-EDMLTVWNRVWIEENPWM--EDKTPVTTWENVPYLGKREDQWCGSLIGLTSRATWAQNIPTAIQQVRSLIGNEE-----FLDYMPSMKRFRKEEESEGAIW-EDMLTVWNRVWIQENPWM--EDKTPVESWEEIPYLGKREDQWCGSLIGLTSRATWAKNIQTAINQVRSLIGNEE-----YTDYMPSMKRFRREEEEAGVLW-EDMLKVWNRVWIEDNPNM--TDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKNIHTAITQVRNLIGKEE-----YVDYMPVMKRYSAPSESEGVL--EDMLQVWNRVWIEENEWM--MDKTPITSWTDVPYVGKREDIWCGSLIGTRSRATWAENIYAAINQVRAVIGKEN-----YVDYMTSLRRYEDVLIQEDRVI-EDMLSVWNRVWILENEWM--EDKTTVSDWTEVPYVGKREDIWCGSLIGTRTRATWAENIYAAINQVRSVIGKEK-----YVDYVQSLRRYEETHVSEDRVL-EDMLEVWNRVWIEENEWM--EDKTPVEKWSDVPYSGKREDIWCGSLIGTRARATWAENIQVAINQVRAIIGDEK-----YVDYMSSLKRYEDTTLVEDTVL-EDMLEVWNRVWIEENEWM--EDKTPVEKWSDVPYSGKREDIWCGSLIGTRARATWAENIQVAINQVRSIIGDEK-----YVDYMSSLKRYEDTTLVEDTVL-EDMLEVWNRVWITNNPHM--QDKTMVKKWRDVPYLTKRQDKLCGSLIGMTNRATWASHIHLVIHRIRTLIGQEK-----YTDYLTVMDRYSVDADLQLGELIEDMLDVWNRVWILDNPFM--QNKERVMEWRDVPYLPKAQDMLCSSLVGRRERAEWAKNIWGAVEKVRKMIGPEK-----FKDYLSCMDRHDLHWELRLESSII EDMLEVWNRVWIYDNPFM--EDKTRVDEWRDTPYLPKSQDILCSSLVGRGERAEWAKNIWGAVEKVRRMIGPEH-----YRDYLSSMDRHDLHWELKLESSIF EDMLEVWNRVWIEDNPFMPCEKKRWITDWRDVPYLPKAQDQICGSLIGTSSRASWAENIWSTVEKVRGMVGAEN-----YRDYLSVMDRYGGGTPVPMTSDIL EDMLAVWNRVWIEDNPFM--EDKREVERWSEVPYLPRNQDKSCGSLIGTTARAEWAKLLPGAVEKVRNIFGKQR-----FRNYLRNMGRYESQEEAPFSMY-DDMLRVWNDVWVHNNPWM--LNKESIESWDDIPYLHKKQDITCGSLIGVKERATWAREIENSVISVRRIIDAETGVLNTYKDELSVMSRYRRGNDVI-----*** *** ** * * * * ** * * ** * ** ** * *

Flavivirus NS5

51

genome as a substrate. Both proteins exhibited 20 -O-MTase activity, and in addition, N7 MTase activity (Ray et al., 2006). MTase activity could not be detected using a capped nonspecific RNA substrate, demonstrating that both MTase activities relied on the presence of specific flaviviral sequences. Examination of the MTase activities of the corresponding MTase domains of DENV-1, YFV, and Powassan virus (PWV) using the WNV RNA substrate, revealed that all of the proteins possessed both N7 and 20 -O-MTase activities (Dong et al., 2007; Zhou et al., 2007). Furthermore, kinetic analysis of the methylation of the substrate RNA (GpppA-RNA) by the WNV, DENV-1, and YFV MTases resulted in the initial detection of the guanine N7-methylated product (m7GpppA-RNA). The 20 -O-methylated product (m7GpppAm-RNA) was detected only after sufficient N7-methylated substrate had accumulated, suggesting that flavivirus cap methylation occurs in the order GpppA ! m7GpppA ! m7GpppAm (Ray et al., 2006; Zhou et al., 2007). Analysis of the substrate preferences of the WNV MTase showed that the N7 MTase was equally active on nonmethylated (GpppA-RNA) or 20 -O-methylated (GpppAm-RNA) substrates, whereas the 20 -O-MTase was more active on a N7-methylated substrate (m7GpppARNA) than a nonmethylated substrate (GpppA-RNA). The preference of the 20 -O-MTase for a N7-methylated substrate may therefore determine the sequential order of cap methylation (Dong et al., 2008a). The DENV-2 MTase has recently been shown to exhibit N7 MTase activity using a capped RNA transcript corresponding to the first 211 nucleotides of the DENV-2 genome as a substrate (Kroschewski et al., 2008). As the assay conditions were similar to those used for the WNV N7 MTase activity assay, the results confirm that flavivirus N7 MTase activity is reliant on the use of a viral-specific RNA substrate. Detailed analysis of the RNA substrate requirements for WNV MTase activity, by mutation of the 190-nucleotide RNA substrate, has revealed that distinct viral sequences are required for N7 and 20 -O-MTase activities (Dong et al., 2007). The first 190 nucleotides of the flavivirus genome contains three stem-loop structures termed ‘‘stem-loop A’’ (SLA), ‘‘stem-loop B’’ (SLB), and ‘‘capsid hairpin’’ (cHP) that are highly conserved (Markoff, 2003; Villordo and Gamarnik, 2009). N7 MTase activity was reliant on a substrate containing at least the first 74 nucleotides of the WNV genome, which includes three 50 -terminal nucleotides, the second (G), and third (U) of which had to be of wild-type sequence and SLA. Systematic mutagenesis of the stem-loop structure revealed that N7 activity primarily relied on the presence of two specific helical stem regions at the base of the stem-loop structure but not sequences within the stemloop structure. In contrast, 20 -O-MTase activity required a substrate of at least 20 nucleotides in length, containing the wild-type viral sequence at the first (A) and second (G) nucleotides (Dong et al., 2007). The importance of the 50 -terminal nucleotides for both N7 and 20 -O-MTase activities

52

Andrew D. Davidson

was supported by the finding that the binding of small RNAs to the DENV-2 MTase was increased when the RNA substrates contained the first two authentic viral nucleotides (Egloff et al., 2007). It should, however, be noted that studies using different experimental systems to assay flavivirus MTase activities have shown that whereas N7 MTase activity appears to be strictly dependent on a viral RNA substrate containing at least the 50 -conserved structure SLA and specific buffer conditions, the template and buffer conditions for 20 -O-methylation show more variability. WNV 20 -O-MTase activity was optimal at pH 10 using a viral substrate consisting of the 50 -terminal 20 nucleotides and 5–10 mM MgCl2. By contrast, for other flaviviral MTases, 20 -O-MTase activity has been more often demonstrated using short (500 uM), and NS5 (Nomaguchi et al., 2003). A similar high concentration of GTP, compared to the other nucleotides, has also been found to be required for de novo initiation of RNA synthesis by the RdRp proteins of BVDV (Ranjith-Kumar et al., 2002), HCV (Luo et al., 2000), and other viruses (Kao et al., 2001; van Dijk et al., 2004). Structural studies have now shown that all of the Flaviviridae RdRps have a binding site for GTP (Bressanelli et al., 2002; Choi et al., 2004; Yap et al., 2007b) that is postulated to stabilize the priming nucleotide. The initiation of DENV-2 RNA synthesis by self-priming at higher temperatures suggested that an exogenous primer may also function to prime RNA synthesis at higher temperatures. Accordingly, addition of the primer AGAA to the RdRp assays performed at 35 C caused a significant shift toward the synthesis of the 1 product rather than the hairpin product. A four nucleotide primer was found to be optimal for RNA synthesis, suggesting that this

Flavivirus NS5

65

may be the size of the RNA product formed before the transition to RNA elongation (Nomaguchi et al., 2003). Based on these studies, it was proposed that the RdRp could exist in two states (Ackermann and Padmanabhan, 2001; Nomaguchi et al., 2003). At lower temperature, the RdRp was in a closed form which could not accommodate a template with a folded back 30 -end and synthesized RNA de novo on a single-stranded RNA template. Initial de novo synthesis or an increase in temperature caused a change in the conformation of the RdRp to a more mobile open form that either elongated the de novo initiation product or could accommodate a folded back 30 -structure to initiate synthesis of the hairpin-like RNA. However, as previous analysis of flaviviral RNA species had not detected any hairpin structure (Chu and Westaway, 1985) it was concluded that this form of initiation was an artifact of in vitro RdRp assays (Kao et al., 2001). Therefore, the change in RdRp conformation from a closed to an open form, observed with temperature change in vitro, most likely reflects the conformational changes that occur in the transition from de novo initiation of RNA synthesis to elongation of RNA. To identify putative rate-limiting steps that may occur during NS5 RNA synthesis, RNA products synthesized by the DENV-2 and WNVKUN POLs and the HCV and BVDV RdRps were compared over time (Selisko et al., 2006). RNA synthesis by the HCV RdRp is known to undergo several distinct rate-limiting steps during the transition from de novo initiation to elongation. Analysis of the accumulation of reaction products of different sizes produced by the different POLs using an oligo(C) template showed that the HCV RdRp and WNVKUN POL produced a higher percentage of short abortive products compared to the BVDV RdRp and DENV-2 POL. This suggested that there were fewer ratelimiting steps in the transition from de novo RNA synthesis to elongation by the DENV-2 POL compared to that of WNVKUN and the HCV RdRp. This could be explained by the DENV-2 POL possessing a higher conformational flexibility than the WNV POLKUN and HCV RdRp.

4. Template requirements for RNA synthesis The demonstration that extracts from DENV-2-infected cells had RdRp activity using an exogenous subgenomic RNA template containing the viral 50 - and 30 -TRs (You and Padmanabhan, 1999) has led to studies investigating the template requirements of recombinant flavivirus NS5. Analysis of the template requirements for () strand RNA synthesis using DENV-2 RdRp assays based on either extracts from DENV-2-infected cells or a recombinant DENV-2 NS5 (Ackermann and Padmanabhan, 2001) revealed that the 30 -TR could only act as a template when the 50 -TR was present, either in cis or in trans. By contrast, the 50 -TR alone could be used as a template to synthesize both 1 de novo initiated and

66

Andrew D. Davidson

2 hairpin products. A functional interaction between the 50 - and 30 -TRs, mediated by complementary regions in the two regions that are known as cyclization (CYC) sequences was shown to be required for the initiation of () strand synthesis from the 30 -TR. The CYC sequences are critical for virus viability in vivo (Corver et al., 2003; Khromykh et al., 2001; Kofler et al., 2006). In addition to the CYC sequences, highly conserved flavivirus stem-loop structures in the 50 - and 30 -TRs were found to be important for RNA synthesis using the 30 -TR template. (You and Padmanabhan, 1999; You et al., 2001). Analysis of the template requirements of a bacterially expressed WNV NS5 showed that a subgenomic WNV RNA transcript containing the WNV 50 - and 30 -TRs was active as a template for () strand RNA synthesis, confirming the results of the DENV-2 NS5 study. The WNV NS5 also had RNA synthesizing activity using a template of () strand polarity complementary to the subgenomic template (Nomaguchi et al., 2004). In contrast to the products obtained using the (þ) strand template, nearly all of the product synthesized from the () strand RNA template was the result of de novo initiation. In addition, the use of capped (þ) strand transcripts was found to inhibit the synthesis of the 2 hairpin product but had little effect on RNA synthesis initiated de novo or on RNA synthesis using a capped () strand template. The use of (þ) and () strand templates with mutated CYC sequences showed that whereas the presence of the 50 -CYC and to a lesser extent the 30 -CYC was required for RNA synthesis using the (þ) transcript, mutation of the CYC sequences had no effect on RNA synthesis using the () strand template. Furthermore, analysis of the efficiency of RNA synthesis from the individual (þ) 30 -TR, () 30 -TR, and (þ) 50 -TRs revealed that as for DENV-2, the (þ) 30 -TR alone was inactive in RNA synthesis. RNA synthesis from the () strand 30 -TR template resulted primarily in de novo initiated 1 products whereas the use of the (þ) strand 50 -TR resulted in the production of a mixture of 2 and 1 products. These results suggested that cyclization of the genome was an important prerequisite for () but not for (þ) strand RNA synthesis by flavivirus NS5. Investigation of the specific viral RNA sequences recognized by the DENV-2 POL (NS5 amino acids 270–900) revealed that NS5 bound specifically to the conserved 50 -stem-loop structure SLA (Filomatori et al., 2006). As in previous studies, the DENV-2 POL was able to use a template consisting of either the 50 -terminal 160 nucleotides or the 50 -terminal 160 nucleotides and the (þ) 30 -TR, but not the (þ) 30 -TR alone, to synthesize RNA. Mutation and deletion analysis of the 50 -terminal 160 nucleotides, in combination with electrophoretic mobility shift and filter-binding assays, using the DENV-2 POL, localized the element responsible for the 50 template activity to the highly conserved SLA structure. Mutagenesis of SLA in the context of the DENV-2 genome and replicons revealed that

Flavivirus NS5

67

mutations that disrupted the base of the stem or a top loop abolished or severely decreased viral replication but not initial translation (Filomatori et al., 2006). A physical interaction between the DENV-2 POL and the 50 -TR, mediated by SLA, was confirmed by atomic force microscopy and RNA-binding assays. The DENV-2 POL did not interact with the 30 -TR confirming that it is not an active template for RNA synthesis. However, the 50 -TR with an intact SLA could promote RNA synthesis in trans on a (þ) 30 -TR template. Based on these results, it was proposed that SLA acts a promoter to recruit NS5 for RNA synthesis. Following genome cyclization via long-range interactions between sequences in the 50 - and 30 -TRs, the NS5-SLA complex is positioned adjacent to the 30 -end of the genome facilitating de novo synthesis of the () strand genomic RNA. In contrast to DENV-2 and WNV NS5, JEV NS5 was shown to initiate RNA synthesis de novo using templates corresponding to both the (þ) and () 30 -regions. The yield of product from the 30 1-kb (þ) template was detectable but much less than that using the () template (Kim et al., 2007; Yu et al., 2007). NS5 was shown to bind to and use the terminal 83 nucleotides of the genome to produce a template size product. Sizing of the product showed it was 81 nucleotides in length suggesting that internal initiation had occurred (Kim et al., 2007). It may be the case for JEV NS5 that the 30 -terminal 83 nucleotides are sufficient for the initiation of () RNA synthesis but that RNA synthesis is enhanced by genome cyclization. The difference in template specificity could also be explained by the reaction conditions used in the various experimental systems but requires further investigation.

C. RdRp structure 1. Overall NS5 RdRp structure The crystal structures of the WNV and DENV-3 RdRp domains (described as POL domains below) have recently been determined. Truncated WNV NS5 fusion proteins consisting of a N-terminal hexahistidine tag fused to either amino acids 273–905 (POL1) or 316–905 (POL2) of NS5, expressed in E. coli were soluble and could be crystallized. The ˚ resolution and used as X-ray structure of POL2 was determined to 2.35 A ˚ a basis to determine the POL1 structure to 3 A resolution by molecular replacement (Malet et al., 2007). Plasmid constructs encoding N-terminal truncations of NS5 for all four DENV serotypes were expressed in E. coli and screened for proteins suitable for crystallization. A truncated DENV-3 protein containing amino acids 273–900 of NS5 yielded crystals that ˚ resolution (Yap et al., 2007a). Using the structure of diffracted at 1.85 A the WNV POL as a guide, the DENV-3 POL structure was determined by molecular replacement (Yap et al., 2007b). Not surprisingly, the overall fold of the WNV and DENV-3 POL structures is well conserved and most

68

Andrew D. Davidson

closely related to RdRp structures determined for the Flaviviridae members, HCV (Ago et al., 1999; Bressanelli et al., 1999, 2002; Lesburg et al., 1999) and BVDV (Choi et al., 2004, 2006). However, superimposed on the overall core structure are a number of important differences which distinguish the flavivirus RdRp from those of other viruses. The structures of the WNV and DENV-3 POLs revealed that they have a roughly spherical shape and adopt an architecture typical of previously characterized viral RdRp structures that resemble a ‘‘cupped right hand’’ with subdomains that have been termed the ‘‘fingers,’’ ‘‘palm,’’ and ‘‘thumb’’ ( Joyce and Steitz, 1995; Ng et al., 2008; Fig. 3A). Similar to other viral RdRps, the fingers and thumb subdomains of the flavivirus POL interconnect via the N-terminal region of the RdRp and through loops protruding from the fingers domain to encircle the active site on the palm domain, forming a closed structure (Ferrer-Orta et al., 2006). Two tunnels that run perpendicular to each other can be observed on the structure. One tunnel is located at the interface between the fingers and thumb domain and is predicted to allow access of the single-stranded RNA template to the active site on the palm domain. A second tunnel runs perpendicular to the first, intersecting at the active site and opens to the back of the structure, allowing diffusion of dNTPs to the active site (Fig. 3B).

2. The palm subdomain The palm subdomain is the most highly conserved feature of RdRp structures and contains the active site. Four of the six conserved sequence motifs (A to F (Fig. 1)) that define RdRps are located in the palm domain and residues from these motifs are involved in the binding of metal ions, nucleotides and RNA, and phosphoryl transfer. The palm contains three strictly conserved aspartic acid residues (located in motifs A and C containing the sequences Asp–X4–Asp and Gly–Asp–Asp, respectively) that coordinate two Mg2þ ions and catalyze phosphoryl transfer ( Joyce and Steitz, 1995; Ng et al., 2008). The palm domain of the flavivirus POL structures closely resembles those of other structurally characterized RdRps; however, some important differences were observed (Malet et al., 2007; Yap et al., 2007b). Typically, the palm domain is comprised of a central b-sheet formed from three antiparallel b-strands surrounded by a-helices. By contrast, the central b-sheet of the flavivirus POL consists of only two b-strands (Fig. 1; b4 and b5; unless stated the nomenclature used and amino acid numbering follows that described for the DENV-3 structure (Yap et al., 2007b)) surrounded by eight a-helices. The two b-strands were much shorter than those found in other RdRp structures ˚ compared with 20 A ˚ in the HCV and BVDV RdRps). The active (i.e., 10 A site Gly–Asp–Asp residues (Motif C) are located in a turn between the b4 and b5 strands. Conversely, there was an unusually long insertion between motifs B and C which is absent in the HCV and BVDV RdRp

Flavivirus NS5

69

RNA template tunnel

A

Priming loop Thumb

Fingers

Palm

B a/bNLS

bNLS

Fingers

Fingers Thumb

180° Palm

“Front”

dNTP access tunnel

Palm

“Rear”

RNA entry C

Core MTase subdomain

Thumb Fingers

N-term subdomain

RNA exit

Palm

FIGURE 3 Structure of the NS5 POL domain. (A) A schematic representation of the X-ray structure of the DENV-3 POL (PDB code: 2J7U; Yap et al., 2007a,b) is shown with the fingers, palm, and thumb subdomains colored in blue, green, and salmon, respectively. The a/bNLS and bNLS are colored in yellow and purple, respectively. The priming loop is colored in black and arrowed. (B) Front and rear surface views of the DENV-3 POL structure. The coloring scheme is the same as in (A). (C) A hypothetical model of the overall WNV NS5 structure generated as described in Malet et al. (2007).

70

Andrew D. Davidson

structures. Structural comparison of the flavivirus POLs with those of other viruses identified the three conserved Asp residues as Asp-533 (motif A), Asp-663, and Asp-664 (motif C). However, neither of the flaviviral POL structures contained metal ions coordinated to the Asp residues in a catalytic conformation. Soaking of the DENV-3 and WNV POL crystals in MgCl2 resulted in the binding of a Mg2þ ion to Asp-533 and Asp-664 in a noncatalytic conformation. The function of the noncatalytic metal ion is unknown. By analogy to other RdRp structures in which noncatalytic metal ions have been observed, it was suggested that it may play a role in the initiation of RNA synthesis (Malet et al., 2007).

3. The fingers subdomain The fingers subdomain serves to shape a tunnel that guides the template RNA to the active site cleft. Similar to the HCV and BVDV RdRps, the fingers subdomain of flavivirus POLs contained a core domain and extended loops termed the ‘‘fingertips’’ that interconnect with the thumb subdomain to enclose the active site (Bressanelli et al., 1999; Choi et al., 2004). However, major differences in the fingers subdomain of flaviviruses and those of HCV and BVDV were found in (a) the N-terminal region of the fingers subdomain, (b) a loop region termed the ‘‘G loop,’’ and (c) the orientation of secondary structures encompassing conserved motif F (Malet et al., 2007, 2008; Yap et al., 2007b). The fingers subdomain also contains a region that has been shown for DENV-2 to contain functional nuclear localization sequences, the so-called ‘‘bNLS’’ and ‘‘a/bNLS’’ (Brooks et al., 2002; Forwood et al., 1999; see Section VI.B). Elements in this region play an important role in the RdRp structure. In the flavivirus, POL structures there is an extra N-terminal stretch of 35-amino acids containing an a-helix (a1) and a b-strand (b1) (Fig. 1), which is absent in the HCV and BVDV structures. Interestingly, the WNV POL2 protein, which had a 44-amino acid N-terminal truncation compared to POL1, lacked RdRp activity, despite the proteins having very similar structures. The b1 strand in the additional N-terminal region contributes to the formation of a three-stranded b-sheet that is absent in the HCV and BVDV RdRps. It was predicted that the b-sheet may stabilize the fingers domain and contribute to the formation of the RNA template tunnel therefore playing an important role in the structure of the POL domain (Malet et al., 2007). The coordinates for the model were kindly provided by Dr. Bruno Canard and colleagues. The WNV POL subdomains are colored as for the DENV-3 POL in (A). The WNV MTase is shown with the N-terminal, core MTase, and C-terminal subdomains colored in red, cyan, and yellow, respectively. The directions for the entry of the template and exit of the nascent RNA are shown. All schematics were produced using PYMOL (DeLano, 2002).

Flavivirus NS5

71

A flexible loop (termed L1) extended from the N-terminal structure in the fingers subdomain to connect with three a-helices (a2–a4) that encompass a helix-turn-helix motif, and lay on top of the thumb domain. Another loop (L2) extended back to helix a5 in the fingers subdomain, providing an interdomain link (Figs. 1 and 3A and B). Interestingly, a-helices 2–5 are all located in a region shown for DENV-2 to bind both the intracellular tranport protein importin-b and NS3 (termed the ‘‘bNLS’’; Johansson et al., 2001; see Section VI.B). Loop L2 and helix a5, in particular, are found in a 20-amino acid stretch that is highly conserved between flaviviruses (Fig. 1) and predicted to be mobile in the structure. It was suggested that the two flexible loops are likely to be important in transmitting conformational changes between the fingers and thumb domains and maintaining the POL structure (Yap et al., 2007b). Furthermore, the loops may serve to modulate a conformational change in the POL, from a closed to an open structure. For DENV-2, a region adjacent to the bNLS, termed the ‘‘a/bNLS’’ has been shown to contain a functional nuclear localization signal and bind to importin-a/b (Brooks et al., 2002; Forwood et al., 1999). The DENV-3 POL structure revealed that the a/ bNLS is comprised of helix a6, located between the fingers and palm subdomains and helix a7 which is buried in the finger subdomain. Extending from helix a7 is a highly mobile loop (L3) termed the ‘‘G loop’’ as it corresponds to a loop found in primer-dependent RdRps containing a conserved motif (G motif) (Malet et al., 2008). However in the flavivirus POL, loop L3 is found in a unique conformation compared to other characterized RdRps. The loop protrudes toward the active site and is well placed to regulate access of the ssRNA substrate at the entrance to the template channel and could contribute to closure of the active site. The position of the loop coincides with the positioning of C-terminal regions of the HCV and BVDV RdRps. For HCV, the C-terminal region is known to be able to regulate RdRp activity (Leveque et al., 2003; Vo et al., 2004) suggesting loop L3 may play a similar role. Motif F is a conserved feature unique to RdRps that contains positively charged residues that mediate interactions with incoming NTPs (Bruenn, 2003; Ferrer-Orta et al., 2006; Lesburg et al., 1999). In the flavivirus POL, motif F is found in a second fingertip loop. This region was partially disordered and amino acids 454–466 preceding motif F were not visible in the POL structures. However, it was observed that the ordered part of the fingertip contained an a-helix rather than a b-strand present in other RdRps. In addition, this structure was orientated perpendicular to the corresponding structures of other RdRps, shifting the localization of motif F such that it cannot bind incoming NTPs. It was suggested that a conformational shift in the POL would have to occur for motif F to play a role in catalysis (Malet et al., 2007).

72

Andrew D. Davidson

The largest variation between the WNV and DENV-3 structures occurred in the fingers subdomain at residues 296–301, 419–423, 543–545, and 556–559. In addition to local changes, the overall orientation of the fingers subdomain of the WNV POL was rotated closer to the thumb subdomain in comparison to the DENV-3 POL, giving rise to a more closed conformation for the WNV POL. Overall, the fingers subdomain of the flavivirus POL is highly mobile and by comparison to other RdRps, has some important structural differences that necessitate conformational changes occur, before RNA synthesis can be initiated (Malet et al., 2008).

4. The thumb subdomain The C-terminal 187 amino acids of the flavivirus RdRp constitute the thumb subdomain, the most diverse feature among viral RdRp structures. The size and complexity of the thumb subdomain distinguishes RdRps that initiate RNA synthesis de novo from those that require a primer, which have a much smaller thumb subdomain (Ferrer-Orta et al., 2006). The thumb subdomain of RdRps that initiate RNA synthesis de novo possess two features not found in primer-dependent RdRps: (1) a characteristic loop (often termed the initiation or priming loop) that is predicted to form a platform stabilizing the RNA initiation complex and (2) unique C-terminal regions that can fold back into the active site cleft to regulate RNA synthesis (Ng et al., 2008; van Dijk et al., 2004). While the flavivirus thumb subdomain resembles those of other RdRps that initiate synthesis de novo, its overall topology is distinct from that of the HCV thumb subdomain, and more closely resembles the BVDV thumb subdomain. Aside from two antiparallel b-strands that form the interface between the thumb and palm subdomains and constitute conserved motif E, the thumb subdomain consists of a-helices connected by large loops. Two of these loops are of particular importance for flaviviral RNA synthesis. The loop connecting a21 and a22 projects toward the fingers domain and in association with the fingertips contributes to the shape of the RNA template tunnel. A second loop, connecting a23 and a24, was identified as the flavivirus priming loop (amino acids 792–804; Fig. 1; Malet et al., 2007; Yap et al., 2007b). Although clearly recognizable on the structure, it was previously not possible to identify the priming loop by sequence and secondary structure analysis (Kao et al., 2001). The flavivirus priming loop originates from the same part of the thumb subdomain as in the HCV and BVDV RdRps, but is larger in size. Unlike the priming loop of HCV, which takes the form of a b-hairpin, the flavivirus priming loop contains no structural elements. The priming loop is stabilized by internal electrostatic (Thr-794, Ser-796, Glu-807, Arg-815) and base-stacking (Arg-749, Trp-787) interactions with amino acids that are well conserved in the flavivirus POL sequence. The priming loop is likely

Flavivirus NS5

73

to shape the upper part of the template tunnel and, in association with loop L3 from the fingers subdomain, regulate the entry and exit of the template from the active site (Yap et al., 2007b). The C-terminal regions of HCV, BVDV, Norwalk virus, and bacteriophage F6 can all fold back into the active site cleft. In the case of HCV, this has been shown to regulate RNA synthesis (Leveque et al., 2003; Vo et al., 2004). The C-terminal regions of the flavivirus POLs were not visible in the structures. However, the distance between the last visible residues (Met-883 and Leu-899 for DENV-3 and WNV, respectively) and the active site appeared to be too great for the C-terminus to fold back into it. In addition, a C-terminally truncated WNV POL, lacking the last 23 amino acids, had normal RdRp activity, indicating that the C-terminus of flavivirus NS5 does not regulate RNA synthesis, unlike the HCV and BVDV RdRps. It was suggested that the L3 loop, extending from the fingers subdomain may instead perform this function (Malet et al., 2007).

5. Initiation of RNA synthesis The determination of the structures of a number of viral RdRps in complex with metal ions, ssRNA and/or nucleoside triphosphates has identified specific regions of RdRps important for the initiation of RNA synthesis (van Dijk et al., 2004). Although it has not yet been possible to determine the structure of a flavivirus POL in complex with ssRNA, the structure of the DENV-3 POL in complex with the nucleoside analog 30 dGTP has been determined (Yap et al., 2007b). Biochemical studies have previously shown that flavivirus de novo RNA synthesis requires high concentrations of rGTP. By analogy with other RdRps, it was suggested that rGTP is required for formation of the preinitiation complex (Nomaguchi et al., 2003). The 30 dGTP molecule was found to bind in the ˚ from the active site. Three amino acid vicinity of the priming loop, 7 A residues, strictly conserved in flaviviruses (Ser-710, Arg-729, and Arg-737) were found to bind to the triphosphate component of 30 dGTP. Superimposition of the DENV-3 complex with other RdRps bound to rGTP suggested that Trp-795 stabilized 30 dGTP by base stacking. Based on the structures of the HCV and F6 RdRps bound to ssRNA and rNTPs, the flavivirus RdRp initiation complex has been modeled (Malet et al., 2007; Yap et al., 2007b). A ssRNA template of 5–7 nucleotides could be modeled into a template tunnel formed between the fingers and thumb subdomains such that the 30 -end of the RNA was placed in the catalytic site in a position suitable for interaction with rNTPs involved in the initiation of RNA synthesis. The tunnel is shaped by loops L1, L2, and L3 projecting from the fingers subdomain and the priming loop and the loop connecting a21 and a22, projecting from the thumb subdomain. Binding of the ssRNA was stabilized by electrostatic interactions made with residues in the fingers subdomain. Residue Trp-795, in the priming

74

Andrew D. Davidson

loop, was well placed to stabilize an interaction between the priming nucleotide and the RNA template, therefore providing a platform for the initiation of RNA synthesis. Although a strand of ssRNA could fit in the template tunnel, the tunnel was not wide enough to accommodate a RNA duplex. This supports biochemical studies, suggesting that conformational changes in the RdRp, from a closed to an open form are required during RNA synthesis.

6. Structure of the full-length NS5 Despite intensive effort, it has not yet been possible to determine the structure of the full-length NS5 for any flavivirus. However, the elucidation of multiple flavivirus structures for the MTase and POL domains, in combination with genetic data has facilitated the production of a model for the full-length WNV NS5 using an in silico docking approach (Malet et al., 2007; Fig. 3C). Reverse genetic analysis of the DENV-2 MTase domain led to the identification of a genetic interaction between the MTase and POL domains (Kroschewski et al., 2008; Malet et al., 2007). Mutation of the DENV-2 MTase residues Lys-46, Arg-47, and Glu-49 to Ala, in the context of the viral genome abolished virus replication. Repeated attempts to rescue virus containing this mutation, resulted in the identification of a compensatory mutation in the POL domain (Leu-512 to Val) that restored virus replication. As the MTase and POL mutations lay outside of active site regions, it was proposed that they defined regions in the two domains that interact. Using this data, a homology model of the WNV MTase (based on the DENV-2 MTase structure) and the WNV POL were docked in silico. Spatial constraints imposed by the C-terminus of the MTase and N-terminus of the POL domains (amino acids 264 and 278, respectively) were applied during the modeling process. The model of the full-length WNV NS5 places the RNA-binding region of the MTase in close proximity to the RNA exit tunnel of the POL, suggesting that capping of the newly synthesized positive strand RNA could occur as it leaves the POL domain.

D. Structure–function analysis By contrast to the MTase, there have been fewer studies specifically investigating the structure–function relationship of the flavivirus POL domain, although studies on other viral RdRps can be more easily extrapolated to the flavivirus POL. The essential requirement of conserved RdRp motifs A–D for NS5 function has been confirmed by mutagenesis, either by assaying the RdRp activity of wild-type and mutant recombinant NS5 (Yu et al., 2007) or by the introduction of mutations into the viral genome (Khromykh et al., 1998; Westaway et al., 2002). Eighty clustered charged to Ala mutations have been introduced into the NS5 gene of

Flavivirus NS5

75

DENV-4 and their effects on viral replication examined to identify attenuated viruses (Hanley et al., 2002). A number of mutations conferring lethal or temperature sensitive viral phenotypes map to regions of NS5 predicted to be important for function by structural analysis. The recent determination of the WNV and DENV-3 POL structures has raised many questions regarding the importance of specific POL elements for RNA synthesis that can now be tested using biochemical and genetic approaches.

IV. NS5 INTERACTIONS The key processes in viral replication performed by NS5 undoubtedly require intramolecular changes to the conformation of NS5 itself, in addition to interactions with viral and presumably host proteins and viral RNA in the replication complex. As described earlier, both the MTase and RdRp activities of NS5 require specific interactions with the viral genome which have been mapped. Interactions between the MTase and POL have also been identified. The multifunctional NS3 protein contains RNA helicase, nucleoside triphosphatase (NTPase), and RNA triphosphatase activities, which are assumed to act in combination with the MTase and RdRp activities of NS5 to replicate and cap the viral genome. A number of studies have detected an association between NS3 and NS5 and regions of the proteins which interact have been identified. In addition, interactions with an increasing number of cellular proteins not currently known to be directly required for replication, are being defined, suggesting that NS5 plays a number of roles in the infected host cell.

A. NS5 intramolecular interactions The enzymatic activities of the MTase and POL domains are active in isolation; however, recent evidence suggests that the conformation or enzymatic activity of one domain may influence the function of the other. As described earlier (see Section III.C.6) reverse genetic analysis of the DENV-2 MTase domain identified a genetic interaction between residues Lys-46, Arg-47, and Glu-49 in the MTase domain and Leu-512 in the POL domain (Malet et al., 2007). In silico docking of the two domains fitted a MTase loop containing residues Lys-46, Arg-47, and Glu-49 into a groove formed by the fingers and thumb subdomains. A loop protruding from the palm subdomain containing residue Leu-512 was located at the base of the groove suggesting a direct interaction between the residues could occur, although structural analysis could not provide an explanation for the effects mutations at these residues had on NS5 function. Reverse genetic analysis has also identified a genetic interaction between

76

Andrew D. Davidson

the WNV MTase and POL domains (Zhang et al., 2008). Mutation of the catalytic site residue Asp-146 to Ala in the WNV MTase abolished both N7 and 20 -O-MTase activities and viral replication. Examination of the effects of additional mutations at Asp-146 revealed that the introduction of a Ser substitution restored minimal levels of N7 MTase activity and allowed low-level replication. Continued passaging of the mutant virus resulted in the isolation of large plaque variants which were found to have second site mutations in the MTase (Lys-61 to Gln/Thr) and POL domains (Trp-751 to Arg) and the 50 -stem-loop structure (G35U or a U insertion at nucleotide 38). Analysis of the mutations revealed that the substitutions at Lys-61 increased N7 MTase activity while the change Trp751 to Arg enhanced RdRp activity. Trp-751 was shown to be surface exposed, at the opening of the template tunnel, but unlikely to directly interact with residue Asp-146 in the MTase domain, suggesting that the increase in RdRp activity may compensate for decreased cap methylation.

B. The interaction of NS5 with viral RNA A number of studies have investigated the interaction of NS5 with the 50 - and 30 -TRs. As described earlier (see Section III.B.4), a conserved stemloop structure (SLA) at the 50 -end of the flavivirus genome has been shown to be essential for both MTase and RdRp activities and to bind recombinant full-length WNV NS5 (Dong et al., 2007) and DENV-2 POL (Filomatori et al., 2006) in vitro. Mapping of the site of interaction of WNV NS5 with the 50 -terminal 190 nucleotides of the genome by RNA foot print analysis showed that NS5 protected the lower half of the SLA stem-loop structure and a number of nucleotides in SLB. Analysis of NS5 binding when the 50 -TR was complexed with the 30 -TR revealed an identical pattern, except that nucleotides in SLB were no longer protected. The results suggested that NS5 binds to the 50 -TR primarily through SLA. The importance of the nucleotides implicated in NS5 binding to viral replication was confirmed by mutagenesis studies using a WNV replicon system (Dong et al., 2008d). The full-length WNV NS5 and the DENV-2 POL were not found to bind specifically to the 30 -TR, by contrast, an interaction between JEV NS5 and the 30 -TR has been detected. A UV crosslinking assay was used to identify proteins in JEV-infected BHK-21 cell lysates that bound to the 30 -terminal 585 nucleotides of the JEV RNA genome (Chen et al., 1997). Two proteins identified as the NS3 and NS5 proteins by immunoprecipitation were found to bind the RNA. The NS5-binding site was mapped to the terminal 83 nucleotides of the RNA, containing a conserved stem-loop. This result was supported by the finding that the 30 -terminal 83 nucleotides could be used as a template for RNA synthesis by a recombinant JEV NS5 (Kim et al., 2007). Interestingly, it was reported that although WNV NS5 bound

Flavivirus NS5

77

to the 50 -TR, the WNV POL showed minimal binding (Dong et al., 2008d). Overall, these results suggest that there may be flavivirus-specific differences in the interaction of NS5 with the 50 - and 30 -TRs.

C. The interaction of NS3 and NS5 Interaction of NS3 and NS5 during viral infection was first demonstrated for DENV-2 by radioimmunoprecipitation using antibodies against the NS3 and NS5 proteins (Kapoor et al., 1995). NS3 and NS5 also interacted in HeLa cells coinfected with recombinant vaccinia viruses expressing the two proteins, showing that additional viral proteins were not required for the interaction. A recombinant C-terminally hexahistidine-tagged NS5, immobilized to Ni-agarose beads, bound recombinant NS3 present in cell lysates, demonstrating that NS3 and NS5 interacted in vitro. In the study of Kapoor et al. (1995) two forms of NS5 were detected, a hyperphosphorylated form that was predominantly localized to the nucleus of infected cells and a hypophosphorylated form present in the cytoplasm. NS3 only interacted with the cytoplasmic hypophosphorylated form, suggesting that differential phosphorylation may regulate the interaction between NS3 and NS5 (see Section V). The association between NS3 and NS5 in vivo has since been confirmed by coimmunoprecipitation experiments using lysates from JEV- (Chen et al., 1997) and DENV-1 (Cui et al., 1998)-infected cells and NS3- and NS5-specific antisera. The physical association of NS3 and NS5 potentially leads to an alteration in the enzymatic activities of one or both proteins. The NTPase activity of a recombinant full-length DENV-1 NS3 was stimulated by the addition of purified recombinant DENV-1 NS5 to the assays, an effect that was specific to NS5 among the viral proteins (Cui et al., 1998). The stimulatory effect of NS5 on NS3 NTPase activity was confirmed using recombinant full-length DENV-2 NS5 and NS3 (Yon et al., 2005). The DENV-2 NS5 stimulated NS3 NTPase activity in a dose-dependent manner until a 1:1 stoichiometry was reached, after which there was no effect, suggesting that a NS3/NS5 complex was the active unit for NTPase activity. The presence of the DENV-2 NS5 was also found to stimulate the 50 -RNA triphosphatase activity of NS3 by fivefold, providing evidence that flavivirus cap formation involves a complex between NS3 and NS5 (Yon et al., 2005). Using the yeast two-hybrid system, it was shown that amino acids 303–618 of the DENV-2 NS3, located in the C-terminal helicase domain, interacted with amino acids 320–368 of DENV-2 NS5 located in the RdRp domain (Johansson et al., 2001). Interestingly, the nuclear import factor importin-b was also found to interact with the same region of NS5 using the yeast two-hybrid system. Competitive binding analysis using the fulllength NS3 and importin-b showed that the NS5-binding sites for NS3 and importin-b either overlap or are closely related. NS5 residues 320–368

78

Andrew D. Davidson

encompass a 20-amino acid stretch that is highly conserved among all flaviviruses. In contrast to the interacting NS3 and NS5 regions defined by biochemical studies, a genetic interaction between the DENV-2 NS3 protease domain and the NS5 MTase domain has also been demonstrated. Mutation of NS5 amino acids Glu-192, Lys-193, and Glu-195 to Ala in the context of a DENV-2 infectious clone led to undetectable levels of virus replication but had little effect on N7 and 20 -O-MTase activities (Kroschewski et al., 2008). The mutated residues are surface exposed in helix aE of the MTase, well removed from the AdoMet- and GTP-binding sites (Fig. 2A). Repeated attempts at virus recovery resulted in the isolation of a virus containing the clustered Glu-192, Lys-193, and Glu-195 to Ala mutation and additionally an Ala to Gly substitution at amino acid residue 70 in the protease domain of NS3, suggesting that helix aE of the MTase, encompassing residues 187–202, interacts with the NS3 protease domain. This finding is supported by a previous study showing that NS5 amino acids 1–316 and NS3 amino acids 1–178 could not be complemented in trans using a WNVKUN replicon system (Khromykh et al., 1999, 2000); therefore, the genetic analysis may have identified regions of NS3 and NS5 that interact in cis during the formation of the replication complex in vivo. Substitution of Glu-192 and Glu-193 with Ala in a DENV-4 infectious cDNA clone abolished viral replication confirming the importance of helix aE for virus replication (Hanley et al., 2002).

D. The interaction of NS5 with host proteins There are a number of points during the virus lifecycle where NS5 is believed to interact with host proteins. It has been proposed that NS5 interacts with viral RNA sequences and proteins during the formation and functioning of the replication complex (Villordo and Gamarnik, 2009; Westaway et al., 2003). Host proteins also presumably play a role in the viral replication complex. Experiments analyzing the interaction of the DENV-4 50 - and 30 -TRs with cellular and viral proteins led to the identification of an interaction between NS5 and the La protein (Garcia-Montalvo et al., 2004). UV crosslinking experiments using RNA transcripts representing the DENV-4 50 - and 30 -TRs and lysates from infected U937 monocytic cells identified seven proteins that interacted with both the 50 - and 30 -TRs, one of the proteins was the La protein, a protein previously shown to interact with the 30 -TR of the DENV-4 genome in human monocytes (YocupicioMonroy et al., 2003). The La protein could be immunoprecipitated from lysates from infected cells both in complex with the DENV-4 50 - and 30 -TRs and NS5 (Garcia-Montalvo et al., 2004). A recombinant La protein was found to inhibit DENV RdRp activity in a dose-dependent manner, both in assays using recombinant DENV-2 NS5 or extracts from DENV-4-infected C6/36 cells, suggesting that the La protein may play a role in regulating positive and negative strand synthesis (Yocupicio-Monroy et al., 2007).

Flavivirus NS5

79

NS5 is found not only in the replication complex but also free in the cytoplasm and for some flaviviruses in the nucleus of infected cells (see Section VI). In addition, NS5 is known to be phosphorylated (see Section V). This suggests that NS5 interacts at least transiently with host proteins involved in intracellular trafficking and phosphorylation/ dephosphorylation. Evidence for such interactions is described in Sections V and VI. Recent investigations have also shown that NS5 can influence cellular host immune responses leading to the identification of flavivirus-specific interactions with host proteins. These interactions are described in Section VII.

V. NS5 PHOSPHORYLATION For a number of flaviviruses, NS5 has been shown to be phosphorylated. As the phosphorylation of a protein can change its enzymatic activity, subcellular localization or ability to interact with other macromolecules (Cohen, 2000), phosphorylation provides a means to regulate the multifunctional roles of NS5 in the virus lifecycle. It is well established that the function of replicative proteins of negative strand viruses can be regulated by phosphorylation and there is accumulating evidence that this may also be the case for positive strand RNA viruses ( Jakubiec and Jupin, 2007). Immunoprecipitation of NS5 from flavivirus-infected cells metabolically labeled with 32P-orthophosphate established that NS5 of DENV-2 (Kapoor et al., 1995), YFV (Reed et al., 1998), and WNVKUN (Mackenzie et al., 2007) can be phosphorylated during viral infection. In addition, the TBEV NS5 present in lysates from infected cells or immunoprecipitates could be phosphorylated using an in vitro kinase assay (Morozova et al., 1997). Ectopic expression of the DENV-2 and YFV NS5 genes in mammalian cells resulted in the production of phosphorylated NS5, showing that NS5 phosphorylation can occur in the absence of other virus proteins. Phosphoamino acid analyses of 32P-labeled NS5 from DENV-2-, TBEV-, and YFV (Kapoor et al., 1995; Morozova et al., 1997; Reed et al., 1998)infected cells and Western blot analysis of the WNVKUN NS5, using phospho-specific antibodies, revealed that phosphorylation is primarily restricted to Ser, although a low level of Thr phosphorylation was also observed for YFV. Phosphorylation of DENV-2 NS5 was proposed to occur on at least four distinct Ser residues. More recently, mass spectrometry has been used in combination with site-specific mutagenesis to identify a specific amino acid residue (Ser-56) that is phosphorylated in YFV NS5 (Bhattacharya et al., 2008). Mass spectrometry analysis of a recombinant hexahistidine-tagged YFV NS5, expressed in and purified from HEK-293 cells, resulted in the identification of six phosphopeptides containing Ser and/or Thr residues. Detailed analysis of the phosphorylation

80

Andrew D. Davidson

of one of the peptides (amino acids 49–61), by mutagenesis and biochemical analysis, identified Ser-56 as a phosphoamino acid. Interestingly, Ser56 has been shown to be essential for MTase activity (Dong et al., 2008b; Kroschewski et al., 2008; see Section II.D.2). Substitution of Ser-56 with Ala (ablating phosphorylation) or Asp (mimicking phosphorylation) both in a recombinant bacterially expressed YFV MTase protein and in the context of a YFV replicon, abolished 20 -O-MTase activity and replication of the YFV replicon, respectively, confirming the importance of Ser-56 in the viral lifecycle (Bhattacharya et al., 2008). It is tempting to speculate that phosphorylation of Ser-56, a residue strictly conserved among flaviviruses and essential for MTase activity, may regulate the function of NS5. However, it remains to be demonstrated whether Ser-56 is phosphorylated during the virus lifecycle and manifests a change in NS5 function in vivo. Little is known concerning the kinases or phosphatases that may regulate the phosphorylation of NS5. A study comparing the phosphorylation of the NS5A proteins of HCV and BVDV with YFV NS5, using an in vitro kinase assay, suggested that the same or closely related kinases phosphorylated all three proteins and that the kinase responsible may be a member of the CMGC family, which includes casein kinase II (CKII) and proline-directed kinases such as the mitogen-activated protein kinases (MAPKs), glycogen synthase kinase 3 (GSK3), and cyclin-dependent kinases (CDKs). In addition, in vitro phosphorylation of YFV NS5 was found to be much more sensitive to the broad spectrum kinase inhibitor staurosporine, indicating a number of kinases may be involved in its phosphorylation (Reed et al., 1998). However, subsequent studies on the HCV 5A protein have shown that casein kinase I-a (CKI-a) rather than a CMGC kinase is most likely to phosphorylate the 5A protein in vivo (Huang et al., 2007; Quintavalle et al., 2007). Differentially phosphorylated forms of DENV-2 NS5 could be detected in both infected cells and cells expressing NS5 alone. Cell fractionation experiments revealed that hypophosphorylated NS5 was confined to the cytoplasm whereas a hyperphosphorylated form was found predominantly in the nucleus. As mentioned earlier, NS3 associated with the hypo but not the hyperphosphorylated form of NS5 (Kapoor et al., 1995). These results led to a model predicting that the association of NS3 and NS5 in the viral replication complex is regulated by phosphorylation. Hyperphosphorylation of NS5 results in dissociation of NS5 from NS3 and in the case of DENV-2, nuclear import of NS5 (Kapoor et al., 1995). However, phosphorylation of NS5 may also inhibit NS5 nuclear import. Examination of the DENV-2 NS5 sequence led to the identification of a consensus CKII site (Thr-395/Arg/Glu/Glu) within a 37-amino acid stretch containing a functional nuclear localization signal. A fusion protein consisting of the 37-amino acid stretch fused to b-galactosidase could

Flavivirus NS5

81

be phosphorylated in vitro by CKII; however, phosphorylation inhibited rather than enhanced nuclear import (Forwood et al., 1999).

VI. NS5 LOCALIZATION A. Cellular localization of NS5 Fractionation of WNV-infected cells to enrich for RdRp activity led to progressive depletion of NS5 from membrane fractions but had little effect on the RdRp activity exhibited by these fractions (Grun and Brinton, 1986, 1987, 1988). Further studies demonstrated that WNVKUN RdRp activity was predominantly associated with cytoplasmic ‘‘heavy’’ membrane fractions which could be sedimented at >16,000g (termed the ‘‘16K fraction’’) (Chu and Westaway, 1987, 1992; Chu et al., 1992). The heavy membrane fraction was found to be enriched for NS3, NS2A, NS2B, and NS4A. By contrast, NS5 was either depleted or could not be detected in the heavy membrane fractions. The majority of NS5 was found in soluble fractions which retained little RdRp activity. More recently, 16K membrane fractions produced from 35S-labeled JEV-, DENV-2-, and WNV-infected mammalian cells were found to possess RdRp activity which was associated with detectable amounts of labeled NS3 and NS5 (Uchil and Satchidanandam, 2003a,b). Extensive treatment of the 16K membrane fraction with trypsin decreased NS3 and NS5 to undetectable amounts whereas there was no reduction in RdRp activity. When the 16K membrane fractions were first solubilized with the ionic detergent sodium deoxycholate before trypsin treatment, RdRp activity was destroyed. Collectively these results suggested that only very small amounts of catalytically active NS3 and NS5 are found in the membrane-bound replication complex and are required for RdRp activity. Whereas NS3 is primarily membrane associated, NS5 may be localized in soluble cytoplasmic or nuclear fractions. Immunolocalization studies have shown that, during infection, the NS5 of DENV-2 (Kapoor et al., 1995; Mackenzie et al., 2007; Malet et al., 2007; Miller et al., 2006; Pryor et al., 2007), YFV (Buckley et al., 1992), and JEV (Uchil et al., 2006) can be detected in the nucleus of a range of mammalian cell types. Nuclear localization of DENV-2 NS5, in infected Vero and BHK-21 cells, could be detected as early as 14–16 h postinfection and increased as the infections progressed (Miller et al., 2006; Pryor et al., 2007). By contrast, NS5 of WNV strains Kunjin and Sarafend could not be detected in the nucleus of infected cells either by immunofluorescence assay or by immunogold labeling (Mackenzie et al., 2007; Malet et al., 2007). Detailed studies of DENV-2 NS5 have shown that the nuclear localization of NS5 is an active process dependent on the cellular nuclear

82

Andrew D. Davidson

import and export pathways (Alvisi et al., 2008; Brooks et al., 2002; Forwood et al., 1999; Pryor et al., 2006; see Section VI.B). Recently, it has been suggested that nuclear-localized NS5 may be involved in the synthesis of viral RNA. In early studies investigating flaviviral RdRp activity, a proportion of the RdRp activity was often found in crude nuclear pellets. However, it was not determined whether the RdRp activity was contained within the nucleus or was associated with contaminating outer nuclear membranes that are a rich source of RdRp activity. The possible involvement of the nucleus in flavivirus RdRp activity was re-examined by Uchil et al. (2006). Examination of the RdRp activity of cytoplasmic, heavy membrane, and nuclear fractions from mammalian cells infected with JEV, WNV, and DENV revealed that while the heavy membrane fractions contained the majority of the RdRp activity, as previously reported, 30–40% of the RdRp activity was associated with the nuclear pellet. Treatment of virus-infected cells with the microtubule depolymerizing drug nocodazole, to separate cytoplasmic membranes from the nucleus, did not affect the proportion of the RdRp activity found in the nuclear fraction. Biochemical analysis using techniques that separate nuclei from nuclear membranes revealed that the JEV NS3 and NS5 proteins and newly synthesized RNA were present in the nuclear preparations (Uchil et al., 2006). NS5 was found diffusely throughout the nucleus whereas NS3 and viral RNA were tightly localized to the inner nuclear membrane. By contrast, colocalization studies using WNVKUN-infected cells have detected NS3, NS5, and newly synthesized RNA only in the cytoplasm in association with perinuclear membranes (Mackenzie et al., 2007). Further investigations are required to determine whether nuclear-localized NS5 is involved in flavivirus RNA synthesis.

B. NS5 nuclear localization The nuclear localization of proteins greater than 45 kDa is an active process requiring recognition of a nuclear localization signal (NLS) or nuclear export signal (NES) on the cargo protein by members of the importin superfamily. In the ‘‘classical’’ NLS import pathway, the positively charged NLS is first recognized by importin-a which serves as an adaptor to indirectly link the cargo protein to importin-b. The complex is then translocated through the nuclear pore complex. Binding of Ran-GTP to importin-b on the nucleoplasmic side of the pore results in dissociation of the complex and release of the cargo protein in the nucleus. Typically, NLSs recognized by importin-a consist of either a single stretch of basic amino acids (monopartite NLS) such as that for the SV40 large T antigen (PKKKRKV) or two clusters of basic amino acids separated by 10–12 amino acids (bipartite NLS). However, most importin family members,

Flavivirus NS5

83

including importin-b, can interact directly with a NLS on the cargo protein and translocate it through the nuclear pore complex. Conversely, proteins can also be actively exported from the nucleus by specific importin superfamily members termed exportins. Typically, a leucine-rich NES on the protein is recognized by an exportin complexed with Ran-GTP. Disassociation of Ran-GTP occurs upon hydrolysis of GTP to GDP, leading to nuclear export. The best characterized exportin is CRM-1, responsible for export of the HIV-1 Rev protein (Alvisi et al., 2008; Conti and Izaurralde, 2001; Lange et al., 2007). Following the finding that DENV-2 NS5 could be detected in the nucleus of infected cells (Kapoor et al., 1995), analysis of the DENV-2 NS5 sequence identified three clusters of basic amino acids (Lys-371/ 372, Lys-388/389/390, and Arg-401/Lys-402) encompassed within amino acids 369–405 that resembled one or more bipartite NLSs (Fig. 1). Fusion of the 37-amino acid region N-terminally to b-galactosidase (NS5NLS-b-gal) resulted in the nuclear localization of b-galactosidase, both in vivo in microinjected cells and in vitro in mechanically perforated cells, confirming that DENV-2 NS5 contained a functional NLS (Forwood et al., 1999). The NS5-NLS-b-gal protein bound a mouse importina/b-heterodimer with high affinity in an ELISA-based binding assay, suggesting that nuclear transport of NS5 depended on the conventional importin-a/b import pathway. Site-directed mutagenesis and deletion analysis of the 37-amino acid region containing the NLS identified a minimal NLS (amino acids 369–389, termed the a/bNLS), which retained the b-gal nuclear-targeting ability and importin-a/b-binding activity of amino acids 369–405. Within this region, Lys-371/372 and to a lesser extent Lys-388/389 were found to be most important for the function of the NLS (Brooks et al., 2002). A second functional NLS was identified adjacent to the a/bNLS. Yeast two-hybrid analysis identified an interaction between amino acids 320–368 of the DENV-2 NS5 (termed the bNLS) and importin-b1 (Johansson et al., 2001). A fusion protein consisting of the bNLS fused N-terminally to b-galactosidase (NS5-bNLS-b-gal) accumulated in the nucleus and bound importin-b1 with high affinity in an ELISA assay (Brooks et al., 2002; Johansson et al., 2001). As mentioned previously, the C-terminal region of NS3 was also found to interact with NS5 amino acids 320–368 and compete with importin-b for binding of the bNLS using pulldown assays. Interestingly, the bNLS contains a stretch of 20 amino acids (amino acids 342–361; Fig. 1) that are highly conserved among flaviviruses. Although the bNLS and a/bNLS were found individually to be functional, a b-gal fusion protein containing both sequences showed markedly reduced nuclear accumulation and binding to importin-b1 and importina/b. This suggested that in the context of the full-length NS5, the function of the NLSs may be regulated by their conformation or interaction with

84

Andrew D. Davidson

other molecules (e.g. NS3). To investigate whether both NLSs were required for nuclear localization of the full-length NS5 protein and assess the role of the NLSs in the viral lifecycle, the NLSs were mutated, in the context of both a plasmid encoding the green fluorescent protein N-terminally fused to the full-length NS5 (GFP-NS5) and a DENV-2 infectious cDNA clone (Pryor et al., 2007). Two clusters of charged residues in the bNLS (Arg-353/Lys-358 and Glu-357/Lys-358/Asp-360), predicted to play a role in the binding of importin-b (Brooks et al., 2002) were mutated to Ala. The mutations had little effect on the nuclear localization of the transiently expressed GFP-NS5; however, when introduced into the viral genome, the mutations abolished viral replication. By contrast, mutation of each of the two basic clusters of amino acids, previously shown to be important for the function of the a/bNLS (Lys-371/372 and Lys-387/388/ 389) confirmed that both clusters were required for nuclear localization of GFP-NS5 with the second cluster being most important. Introduction of the individual clustered mutations into the viral genome resulted in the production of viable viruses whereas the introduction of both clusters abolished viral replication. Analysis of the localization of NS5 in cells infected with virus containing the Lys-387/388/389 to Ala mutations revealed that there was a delay in NS5 localization compared to the wild-type virus that was most pronounced early in the infection. The delay in NS5 localization was found to correlate with a delay in viral growth and a 100-fold decrease in peak viral titer, suggesting that NS5 nuclear localization plays a role in DENV-2 replication. Although originally proposed to lie in an interdomain linker region of NS5 (Forwood et al., 1999), structural studies have now shown that the bNLS and a/bNLS are actually important structural components of the RdRp domain (Fig. 3B; Malet et al., 2007; Yap et al., 2007b; see Section III. C.3). Therefore, it is not surprising that when mutations in the bNLS were introduced into the viral genome they abolished virus replication without affecting nuclear localization. In addition, it is possible that mutations in the a/bNLS could also affect viral replication through mechanisms distinct to its role in nuclear localization. The accumulation of DENV-2 NS5 in the nucleus is not only dependent on nuclear import but also on nuclear export. The drug leptomycin B inhibits CRM-1-dependent nuclear export. Treatment of DENV-2-infected Vero cells with leptomycin B resulted in an increase in the accumulation of nuclear-localized NS5, particularly early in the infection which correlated with an increase in virus production (Pryor et al., 2006). This effect was also shown when a GFP-NS5 was expressed alone in Vero cells. An NES has been identified in the bNLS and it has been reported that site-specific mutagenesis of the NES results in increased NS5 accumulation and nonresponsiveness to leptomycin B (Alvisi et al., 2008; Pryor et al., 2006; Rawlinson et al., 2009).

Flavivirus NS5

85

The role of NS5 in the nucleus has not yet been elucidated. As NS5 nuclear localization is not a property shared by all flaviviruses, it cannot be strictly required for replication. It has been shown that DENV-2 NS5 can induce the production of the cytokine IL-8 (see Section VII). A mutation in the a/bNLS which decreased nuclear localization of NS5 and virus production also altered IL-8 secretion, providing evidence that the nuclear trafficking of NS5 may influence host cell processes (Pryor et al., 2007). Further studies on NS5 localization using a range of flaviviruses and cell types relevant to disease are required to define the role of NS5 localization in the virus lifecycle.

VII. EMERGING ROLES FOR NS5 IN VIRAL PATHOGENESIS A number of recent studies suggest that NS5 has the ability to interfere with key processes involved in the host immune response, but that interestingly, the pathways involved may be flavivirus specific. The interferon (IFN) response is a key host defense to viral infection including flaviviruses. Like many viruses, flaviviruses have evolved strategies to evade the IFN response. A common theme is the ability of flaviviruses to block cellular signaling by the Janus-activated kinase– signal transducer and activator of transcription (JAK–STAT) pathway in response to IFN stimulation. A number of different flavivirus proteins have been shown to be capable of inhibiting IFN signaling including, recently, the NS5 of JEV, LGTV, and TBEV. IFN-a and -b (Type I IFNs) and IFN-g (Type II IFN) bind to heterodimeric receptors on the cell surface known as the type I IFN receptor (comprised of the IFNAR1 and IFNAR2 subunits) and type II IFN receptor (comprised of the IFNGR1 and IFNGR2 subunits), respectively. The subunits of the receptors are constitutively associated with distinct cellular tyrosine kinases belonging to the JAK family; IFNAR1 and IFNAR2 are associated with tyrosine kinase 2 (Tyk2) and JAK1, respectively, while IFNGR1 and IFNGR2 are associated with JAK1 and JAK2, respectively. Binding of the IFNs to their specific receptors causes rearrangement and oligomerization of receptor subunits leading to autophosphorylation and activation of the associated tyrosine kinase, which in turn regulates the phosphorylation and activation of STAT proteins. The phosphorylated STAT proteins form homodimers or heterodimers with other STAT proteins (STAT1–STAT2 for IFN-a/b and STAT1–STAT1 for IFN-g) and then translocate to the nucleus to activate the transcription of IFN-stimulated genes (ISGs). The production of the ISG gene products leads to an antiviral state in the cell (Platanias, 2005; Randall and Goodbourn, 2008).

86

Andrew D. Davidson

IFN-a/b signaling was found to be inhibited in JEV-infected cells due to a block in Tyk2 phosphorylation (Lin et al., 2004). By contrast, in LGTVinfected cells, the phosphorylation of Tyk2 and JAK1 was blocked, leading to inhibition of both IFN-a/b and IFN-g signaling (Best et al., 2005). Analysis of STAT1 phosphorylation and nuclear translocation in response to IFN-a/b treatment of cells expressing the individual JEV and LGTV gene products revealed that NS5 alone inhibited JAK–STAT signaling (Best et al., 2005; Lin et al., 2006). Further analysis of the effects of NS5 on JAK–STAT signaling showed that JEV NS5 inhibited Tyk2 phosphorylation and the downstream induction of several IFN-a-inducible gene products while LGTV NS5 could inhibit transcription from IFN-a/b and IFN-g-responsive reporter gene constructs. To determine whether the effects of NS5 on JAK–STAT signaling were the result of a direct physical interaction with components of the IFN signaling pathways, protein interaction studies were done. Immunoprecipitation of the IFN receptor subunits from Vero cells transiently expressing the LGTV NS5 demonstrated that NS5 bound to the IFN-a/b receptor subunit IFNAR2 and possibly the IFN-g receptor subunit IFNGR2 (Best et al., 2005). The interaction of LGTV NS5 with IFNAR2 and IFNGR2 was confirmed in a more relevant context using lysates from LGTV-infected human and murine monocyte-derived dendritic cells. Once again, the LGTV NS5 coprecipitated with IFNAR2 but not JAK1, Tyk2 or STAT1. The interaction of LGTV NS5 with IFNAR2 occurred both with and without IFN treatment. The JEV NS5 was found not to interact with Tyk2 or JAK1 using a mammalian two-hybrid system (Lin et al., 2006). However, pretreatment of JEV NS5-expressing cells with sodium orthovanadate, a broad spectrum inhibitor of protein tyrosine phosphatases that are known to negatively regulate JAK–STAT signaling, resulted in suppression of the effects of NS5 on IFN signaling. Based on these results, it was suggested that JEV NS5 might activate inhibitors of JAK–STAT signaling rather than directly perturbing the components themselves. The minimal regions of the JEV and LGTV NS5 proteins required for inhibition of JAK–STAT signaling were defined by examining STAT1 phosphorylation and/or nuclear translocation in response to IFN treatment of cells expressing truncated versions of NS5. A truncated JEV NS5 protein containing the N-terminal 1–762 amino acids was capable of inhibiting STAT1 nuclear translocation, similar to the full-length protein. However, deletion of the C-terminal region to residue 667 reduced the block to STAT1 nuclear translocation while further deletion of either the C-terminus to residue 584 or the N-terminal 83 or 166 amino acids abolished the ability of the truncated NS5 proteins to inhibit STAT1 nuclear translocation (Lin et al., 2006). These results suggested that JEV NS5 did not require functional MTase or RdRp activities to inhibit IFN signaling. The examination of 11 N- and C-terminally truncated LGTV NS5 proteins

Flavivirus NS5

87

defined a minimal region of NS5 (amino acids 355–735) that was required for wild-type inhibition of JAK–STAT signaling (Park et al., 2007). A truncated LGTV NS5 comprising amino acids 342–735 was then subjected to extensive random and site-directed mutagenesis including potential phosphorylation and active site residues and the resultant proteins screened for their ability to inhibit JAK–STAT signaling. Two regions were found to contribute to the inhibition of JAK–STAT signaling when evaluated in the context of the full-length NS5; residues 374–380 with Arg-376 and Asp-380 being most critical and residues 624–647 with residues Glu-326, Glu-328, and Trp-647 being the most critical. When residues 374–380 and 624–647 were modeled on the WNV POL crystal structure, it was found that the two regions lay in close proximity and were surface exposed at the junction of the finger and palm subdomains, suggesting that this region of the protein may be directly involved in binding the IFNAR2 receptor complex (Park et al., 2007). Interestingly, residues 374–389 in LGTV NS5 overlaps the a/bNLS identified in DENV-2 NS5 (see Section VI.B), suggesting that this region of NS5 may be specifically suited to interact with host proteins. Neither NS5 phosphorylation nor RdRp activity appeared to be required for NS5-mediated inhibition of JAK–STAT signaling. It will be of interest to determine whether mutation of these regions abrogate IFNAR2 binding and effect the sensitivity of LGTV to IFNs when introduced into the viral genome. Using a different approach, it has recently been shown that the TBEV NS5 is also able to interfere with JAK–STAT signal transduction. The TBEV NS5 was found to interact with the human scribble (hScrib) protein in a yeast two-hybrid screen (Werme et al., 2008). hScrib is highly concentrated at epithelial cell junctions where it is involved in establishing and maintaining cell polarity (Dow et al., 2003) and belongs to the LAP family of adaptor proteins which are characterized by a combination of 16 leucine repeats at the N-terminal region of the protein and either 1 or 4 PDZ domains at the C-terminal region (Santoni et al., 2002). Each PDZbinding domain mediates binding of the protein to C-terminally located sequences in proteins conforming to the PDZ-binding motif (S/T–X–L/ V/I). The TBEV NS5 protein contains a PDZ-binding motif in its C-terminal region; however, deletion analysis of TBEV NS5 followed by site-specific mutagenesis showed that Tyr-222 and Ser-223 and not the residues in the PDZ-binding motif mediated hScrib binding. The association between NS5 and hScrib was confirmed by pull-down assays using bacterially expressed proteins and lysates from HeLa cells expressing NS5. Investigation of the colocalization of endogenous hScrib and transiently expressed wild-type and mutant (Tyr-222/Ser-223 to Ala) NS5 in MDCK cells showed that there was substantial enrichment of wild-type but not mutant NS5 at the cell–cell contacts where hScrib was found. Knockdown of hScrib led to relocalization of NS5 to the site of the mutant

88

Andrew D. Davidson

NS5, demonstrating that hScrib was able to target NS5 to the cell periphery. To determine if the membrane-localized NS5 may play a role in perturbing IFN signaling, HeLa cells expressing the wild-type and mutant TBEV NS5 were examined for their IFN responsiveness by the analysis of STAT-1 phosphorylation and reporter gene assays. The results showed that both IFN-a/b and IFN-g signaling pathways were inhibited by NS5 whereas the mutant NS5 had a reduced ability to block JAK–STAT signaling. Overall, the results of this work suggested that the association between hScrib and NS5 was important for inhibition of JAK–STAT signaling (Werme et al., 2008). In comparison to the TBEV, JEV, and LGTV NS5, the NS5 proteins of DENV-2 and 4, which were included as controls in the studies described previously, did not inhibit JAK–STAT signaling (see Footnote for recent studies showing that DENV-2 NS5 can perturb type I IFN signalling)1. However, DENV-2 NS5 has been shown to be capable of inducing the production of the cytokine interleukin-8 (IL-8). Elevated levels of IL-8 have been detected in the serum of DENV-infected patients (Juffrie et al., 2000; Medin et al., 2005; Raghupathy et al., 1998) and in the culture supernatants of a variety of DENV-infected cultured cells including dendritic cells and monocytes (Bosch et al., 2002; Moreno-Altamirano et al., 2004), which are of relevance to DENV infection in vivo. Expression of individual DENV genes in HEK-293 cells identified NS5 alone as being capable of increasing IL-8 gene expression and secretion (Medin et al., 2005). A number of transcription factors are required to activate IL-8 transcription including, activating protein 1 (AP-1), NF-kB and CAAT/ enhancer-binding protein (c/EBP). Using promoter reporter constructs activated by the three transcription factors, it was shown that NS5induced IL-8 expression was predominantly reliant on c/EBP and to a lesser extent NF-kB while DENV-2 infection activated all three factors suggesting NS5 alone was not responsible for full IL-8 induction (Medin et al., 2005). To determine whether the localization of NS5 played a role in IL-8 induction, IL-8 secretion from HEK-293 cells infected with DENV2 or a recombinant virus containing a mutation in the NS5 a/bNLS, which delayed NS5 localization was compared. It was found that delayed nuclear accumulation of NS5 led to an increase in IL-8 secretion and decrease in virus production. The effects on IL-8 secretion were confirmed by transient expression of the wild-type and mutant NS5 (Pryor et al., 2007). The results suggested that an increase in cytoplasmic NS5 may contribute to IL-8 induction. However, it remains to be determined how the change in NS5 nuclear localization alters the IL-8 response. 1

During the publication of this article, two manuscripts (listed below) describing the interaction of DENV-2 NS5 with STAT-2 have been published. Both articles provide evidence that like the NS5 of JEV, LGTV and TBEV, the DENV-2 NS5 can also perturb type I IFN signalling. (Ashour et al., 2009; Mazzon et al., 2009)

Flavivirus NS5

89

Nitric oxide radicals released during inflammatory processes have been shown to inhibit DENV replication in a strain-specific fashion (Charnsilpa et al., 2005; Ubol et al., 2008). Using in vitro RdRp assays based on both crude extracts from DENV-2-infected cells and bacterially expressed DENV-2 NS5, it was shown that de novo synthesis of RNA was inhibited by nitric oxide radicals, suggesting that NS5 may play a role in nitric oxide sensitivity (Takhampunya et al., 2006). Based on this premise the NS5 genes from DENV strains showing differences in susceptibility or resistance to nitric oxide were sequenced. Although sequence changes were detected, a firm correlation with nitric oxide sensitivity was not established (Ubol et al., 2008). Collectively, these studies demonstrate that NS5 has a role outside of the replication complex in viral-infected cells. The effects of NS5 on the host cell described to date involve perturbation of the host immune response and therefore NS5 has the potential to play a role in viral pathogenesis. Future studies using relevant cell types and disease models will be required to establish the contribution of NS5 to viral pathogenesis.

VIII. CONCLUSIONS AND FUTURE PERSPECTIVES Flavivirus NS5 is essential for virus replication, possessing a number of viral-specific enzymatic properties. NS5 is therefore a very interesting target against which antiviral drugs can be developed and research in this field has accelerated our understanding of NS5 structure and function in recent years. The production and purification of recombinant versions of either the full-length NS5 or the two individual NS5 domains has led to detailed enzymatic studies on NS5 and the determination of the structures of the two individual NS5 domains. The original prediction, over 20 years ago, that NS5 was the viral RdRp has been well substantiated. In addition, NS5 is now known to play a major role in RNA cap formation as it can bind the cap structure and perform two sequential methylation reactions required for the formation of a type I cap structure. The structural characterization of the MTase and POL domains of NS5 has had a major impact on our understanding of NS5. The structures provide a basis for functional studies and understanding the enzymatic properties of NS5 at the atomic level. Enzymatic assays have now been developed using recombinant MTase and POLs that can be adapted for high throughput screening of antiviral compounds. Coupled with in silico structure-based compound screening and structure-guided drug design, compounds have already been identified which hold promise as antiflaviviral agents. It is anticipated that in the future, our increased understanding of NS5 will translate into antiviral therapies against flaviviruses that are urgently needed.

90

Andrew D. Davidson

As for other viral proteins involved in RNA capping, the study of NS5 has illustrated the many variations on a theme that exist for formation of the RNA cap structure. Typically, different proteins are required for N7 and 20 -O-methylation of the cap structure. N7 and 20 -O-MTases are structurally distinct and methylate RNA using different reaction mechanisms. However, for flaviviruses, both activities reside in NS5 although there is only one binding site for AdoMet, the methyl donor. Structure–function studies have been instrumental in identifying regions of NS5 involved in each of the MTase activities and suggesting mechanisms to explain how NS5 may perform both methylation reactions. It has recently been proposed that separate molecules of NS5 may perform the individual methylation reactions. In addition, cap formation also requires RNA triphosphatase, and guanyltransferase activities. NS3 has been shown to possess RNA triphosphatase activity. The guanyltransferase activity required for flavivirus cap formation has not yet been identified, although it has been suggested that NS5 may also carry out this function. By analogy with other capping systems, it may be that the full complement of enzyme activities required for flavivirus cap formation will only be identified through examination of a multiprotein complex rather than the individual protein constituents. The next challenge in understanding the role of NS5 in capping will therefore be to determine how NS3 and NS5 interact to facilitate cap formation and determine the stoichiometry of NS5 in the capping complex. Intriguingly, studies have shown that N7 but not 20 -O-MTase activity is strictly required for virus viability. Little is known concerning the role of 20 -O-methylation in the RNA cap structure either for flaviviruses or in general. Animal studies suggest that the 20 -O-MTase activity of NS5 may play a role in viral pathogenesis which is an interesting future area of research. Despite intensive research, it has not yet been possible to produce a structure for the entire NS5. The determination of the structures for the two individual NS5 domains has had a major impact on our understanding of NS5 as the enzymatic functions of the two domains are independently active. However, evidence suggests that NS5 undergoes a number of conformational changes when carrying out its enzymatic activities and that the two domains operate in concert during flaviviral replication. In the absence of a full-length NS5 structure, a speculative structural model has been produced using an in silico docking approach. The model suggests that once the newly synthesized RNA exits the POL domain, it can engage with the MTase domain. Viral sequences and structures in the 50 -TR are required for N7 and to a lesser extent 20 -O-MTase activities, providing a mechanism to discriminate between (þ) strand RNA viral transcripts and () strand viral RNA transcripts that

Flavivirus NS5

91

are not believed to be capped. However, the relationship between RNA capping and RNA synthesis is currently unknown and it is not clear how many viral nucleotides are synthesized before the nascent transcript is capped. Current and future research will be directed at understanding how the full-length NS5 functions to coordinately synthesize and cap the RNA genome in combination with other viral and presumably host proteins. Many studies have demonstrated that flaviviral RNA synthesis occurs in association with host perinuclear ER membranes. However, it appears that only catalytic amounts of NS5 are required for genome replication. Viral RNA sequences and regions of NS3 that are believed to interact with NS5 during viral replication have been defined using in vitro studies, but the exact composition of the replication complex and the interactions that take place within it, in virally infected cells remains unknown, as does the contribution of host proteins. Much of the NS5 in flavivirus-infected cells is found free in the cytoplasm or for a number of flaviviruses in the nucleus. It is not known whether only a specific subset of NS5 molecules are recruited to the replication complex or alternatively if modification of NS5 in the replication complex leads to its redistribution in the host cell. Recent studies have shown that aside from its role in replication, NS5 can interact with host macromolecules involved in the host immune response, raising the possibility that NS5 may be involved in viral pathogenesis. Little is still known about either the trafficking of NS5 in infected cells and its interaction with host proteins or the how these processes are regulated. Phosphorylation is one means by which NS5 function could be regulated and a potential NS5 phosphorylation site has recently been identified that has the potential to regulate MTase function. The role of NS5 in the nucleus is presently unclear, although recent studies on DENV-2 suggest NS5 localization is important for virus replication. Nuclear-localized NS5 has the potential to alter host processes or play a role in viral RNA synthesis. The trafficking of NS5 to the nucleus has been studied in detail for DENV-2, but it remains to be determined whether the trafficking mechanisms established for DENV-2 are common to other flaviviruses. Compared to the enzymatic and structural properties of NS5, there are likely to be many more flaviviral-specific differences in the way in which NS5 interacts with the host cell. The elucidation of the role NS5 in pathogenesis will ultimately require studies in relevant systems in the context of virus infection. Due to the pleiotropic functions of NS5 in viral replication this will not be trivial, as disrupting one function of NS5 may have unforeseen consequences on other functions. Nevertheless, the investigation of NS5–host cell interactions is an exciting area of research which promises to expand our understanding of the role played by NS5 in the flavivirus lifecycle.

92

Andrew D. Davidson

ACKNOWLEDGMENTS The author wishes to acknowledge research support from the Medical Research Council, UK; the Novartis Institute for Tropical Diseases, Singapore; and the National Health and Medical Research Council of Australia. The author is grateful to Stuart Siddell for critical reading of the manuscript; former laboratory members Rebecca Butcher and Helga Kroschewski for their input into flavivirus NS5 research; and Siew Pheng Lim, Subhash Vasudevan, Peter Wright, David Jans, Melinda Pryor, Marie-Pierre Egloff, Barbara Selisko, Bruno Canard and Mike Jacobs for engaging in stimulating collaborative investigations.

REFERENCES Ackermann, M., and Padmanabhan, R. (2001). De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. J. Biol. Chem. 276:39926–39937. Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K., and Miyano, M. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7:1417–1426. Alvisi, G., Rawlinson, S. M., Ghildyal, R., Ripalti, A., and Jans, D. A. (2008). Regulated nucleocytoplasmic trafficking of viral gene products: A therapeutic target? Biochim. Biophys. Acta 1784:213–227. Ashour, J., Laurent-Rolle, M., Shi, P. Y., and Garcia-Sastre, A. (2009). NS5 of dengue virus mediates STAT2 binding and degradation. J Virol. 83:5408–5418. Assenberg, R., Ren, J., Verma, A., Walter, T. S., Alderton, D., Hurrelbrink, R. J., Fuller, S. D., Bressanelli, S., Owens, R. J., Stuart, D. I., and Grimes, J. M. (2007). Crystal structure of the Murray Valley encephalitis virus NS5 methyltransferase domain in complex with cap analogues. J. Gen. Virol. 88:2228–2236. Bartelma, G., and Padmanabhan, R. (2002). Expression, purification, and characterization of the RNA 50 -triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 299:122–132. Bartholomeusz, A. I., and Wright, P. J. (1993). Synthesis of dengue virus RNA in vitro: Initiation and the involvement of proteins NS3 and NS5. Arch. Virol. 128:111–121. Benarroch, D., Egloff, M. P., Mulard, L., Guerreiro, C., Romette, J. L., and Canard, B. (2004). A structural basis for the inhibition of the NS5 dengue virus mRNA 20 -O-methyltransferase domain by ribavirin 50 -triphosphate. J. Biol. Chem. 279:35638–35643. Benarroch, D., Selisko, B., Locatelli, G. A., Maga, G., Romette, J. L., and Canard, B. (2004). The RNA helicase, nucleotide 50 -triphosphatase, and RNA 50 -triphosphatase activities of Dengue virus protein NS3 are Mg2þdependent and require a functional Walker B motif in the helicase catalytic core. Virology 328:208–218. Best, S. M., Morris, K. L., Shannon, J. G., Robertson, S. J., Mitzel, D. N., Park, G. S., Boer, E., Wolfinbarger, J. B., and Bloom, M. E. (2005). Inhibition of interferonstimulated JAK–STAT signaling by a tick-borne flavivirus and identification of NS5 as an interferon antagonist. J. Virol. 79:12828–12839. Bhattacharya, D., Hoover, S., Falk, S. P., Weisblum, B., Vestling, M., and Striker, R. (2008). Phosphorylation of yellow fever virus NS5 alters methyltransferase activity. Virology 380:276–284. Bosch, I., Xhaja, K., Estevez, L., Raines, G., Melichar, H., Warke, R. V., Fournier, M. V., Ennis, F. A., and Rothman, A. L. (2002). Increased production of interleukin-8 in primary human monocytes and in human epithelial and endothelial cell lines after dengue virus challenge. J. Virol. 76:5588–5597.

Flavivirus NS5

93

Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., De Francesco, R., and Rey, F. A. (1999). Crystal structure of the RNAdependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. USA 96:13034–13039. Bressanelli, S., Tomei, L., Rey, F. A., and De Francesco, R. (2002). Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J. Virol. 76:3482–3492. Brooks, A. J., Johansson, M., John, A. V., Xu, Y., Jans, D. A., and Vasudevan, S. G. (2002). The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals. J. Biol. Chem. 277:36399–36407. Bruenn, J. A. (2003). A structural and primary sequence comparison of the viral RNA-dependent RNA polymerases. Nucleic Acids Res. 31:1821–1829. Buckley, A., Gaidamovich, S., Turchinskaya, A., and Gould, E. A. (1992). Monoclonal antibodies identify the NS5 yellow fever virus non-structural protein in the nuclei of infected cells. J. Gen. Virol. 73:1125–1130. Charnsilpa, W., Takhampunya, R., Endy, T. P., Mammen, M. P., Jr., Libraty, D. H., and Ubol, S. (2005). Nitric oxide radical suppresses replication of wild-type dengue 2 viruses in vitro. J. Med. Virol. 77:89–95. Chen, C. J., Kuo, M. D., Chien, L. J., Hsu, S. L., Wang, Y. M., and Lin, J. H. (1997). RNA-protein interactions: Involvement of NS3, NS5, and 30 noncoding regions of Japanese encephalitis virus genomic RNA. J. Virol. 71:3466–3473. Choi, K. H., Groarke, J. M., Young, D. C., Kuhn, R. J., Smith, J. L., Pevear, D. C., and Rossmann, M. G. (2004). The structure of the RNA-dependent RNA polymerase from bovine viral diarrhea virus establishes the role of GTP in de novo initiation. Proc. Natl. Acad. Sci. USA 101:4425–4430. Choi, K. H., Gallei, A., Becher, P., and Rossmann, M. G. (2006). The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain. Structure 14:1107–1113. Chu, P. W., and Westaway, E. G. (1985). Replication strategy of Kunjin virus: Evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. Virology 140:68–79. Chu, P. W., and Westaway, E. G. (1987). Characterization of Kunjin virus RNA-dependent RNA polymerase: Reinitiation of synthesis in vitro. Virology 157:330–337. Chu, P. W., and Westaway, E. G. (1992). Molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of Kunjin virus RNA. Arch. Virol. 125:177–191. Chu, P. W., Westaway, E. G., and Coia, G. (1992). Comparison of centrifugation methods for molecular and morphological analysis of membranes associated with RNA replication of the flavivirus Kunjin. J. Virol. Methods 37:219–234. Cleaves, G. R., and Dubin, D. T. (1979). Methylation status of intracellular dengue type 2 40 S RNA. Virology 96:159–165. Cleaves, G. R., Ryan, T. E., and Schlesinger, R. W. (1981). Identification and characterization of type 2 dengue virus replicative intermediate and replicative form RNAs. Virology 111:73–83. Cohen, P. (2000). The regulation of protein function by multisite phosphorylation— A 25 year update. Trends Biochem. Sci. 25:596–601. Conti, E., and Izaurralde, E. (2001). Nucleocytoplasmic transport enters the atomic age. Curr. Opin. Cell Biol. 13:310–319.

94

Andrew D. Davidson

Corver, J., Lenches, E., Smith, K., Robison, R. A., Sando, T., Strauss, E. G., and Strauss, J. H. (2003). Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization. J. Virol. 77:2265–2270. Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K., Chan, Y. C., and Fu, J. (1998). Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein. Virology 246:409–417. DeLano, W. L. (2002). The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA, USA De la Pena, M., Kyrieleis, O. J., and Cusack, S. (2007). Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase. EMBO J. 26:4913–4925. Dong, H., Ray, D., Ren, S., Zhang, B., Puig-Basagoiti, F., Takagi, Y., Ho, C. K., Li, H., and Shi, P. Y. (2007). Distinct RNA elements confer specificity to flavivirus RNA cap methylation events. J. Virol. 81:4412–4421. Dong, H., Ren, S., Li, H., and Shi, P. Y. (2008). Separate molecules of West Nile virus methyltransferase can independently catalyze the N7 and 20 -O methylations of viral RNA cap. Virology 377:1–6. Dong, H., Ren, S., Zhang, B., Zhou, Y., Puig-Basagoiti, F., Li, H., and Shi, P. Y. (2008). West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism. J. Virol. 82:4295–4307. Dong, H., Zhang, B., and Shi, P. Y. (2008). Flavivirus methyltransferase: A novel antiviral target. Antiviral Res. 80:1–10. Dong, H., Zhang, B., and Shi, P. Y. (2008). Terminal structures of West Nile virus genomic RNA and their interactions with viral NS5 protein. Virology 381:123–135. Dow, L. E., Brumby, A. M., Muratore, R., Coombe, M. L., Sedelies, K. A., Trapani, J. A., Russell, S. M., Richardson, H. E., and Humbert, P. O. (2003). hScrib is a functional homologue of the Drosophila tumour suppressor Scribble. Oncogene. 22:9225–9230. Edward, Z., and Takegami, T. (1993). Localization and functions of Japanese encephalitis virus nonstructural proteins NS3 and NS5 for viral RNA synthesis in the infected cells. Microbiol. Immunol. 37:239–243. Egloff, M. P., Benarroch, D., Selisko, B., Romette, J. L., and Canard, B. (2002). An RNA cap (nucleoside-20 -O-)-methyltransferase in the flavivirus RNA polymerase NS5: Crystal structure and functional characterization. EMBO J. 21:2757–2768. Egloff, M. P., Decroly, E., Malet, H., Selisko, B., Benarroch, D., Ferron, F., and Canard, B. (2007). Structural and functional analysis of methylation and 50 RNA sequence requirements of short capped RNAs by the methyltransferase domain of Dengue virus NS5. J. Mol. Biol. 372:723–736. Fabrega, C., Hausmann, S., Shen, V., Shuman, S., and Lima, C. D. (2004). Structure and mechanism of mRNA cap (guanine-N7) methyltransferase. Mol. Cell 13:77–89. Fauman, E. B., Blumenthal, R. M., and Cheng, X. (1999). Structure and evolution of AdoMet-dependent methyltransferases. In ‘‘S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions’’ (X. Cheng and R. M. Blumenthal, eds.), pp. 1–38. World Scientific Publishing, Singapore. Ferrer-Orta, C., Arias, A., Escarmis, C., and Verdaguer, N. (2006). A comparison of viral RNA-dependent RNA polymerases. Curr. Opin. Struct. Biol. 16:27–34.

Flavivirus NS5

95

Filomatori, C. V., Lodeiro, M. F., Alvarez, D. E., Samsa, M. M., Pietrasanta, L., and Gamarnik, A. V. (2006). A 50 RNA element promotes dengue virus RNA synthesis on a circular genome. Genes Dev. 20:2238–2249. Forwood, J. K., Brooks, A., Briggs, L. J., Xiao, C. Y., Jans, D. A., and Vasudevan, S. G. (1999). The 37-amino-acid interdomain of dengue virus NS5 protein contains a functional NLS and inhibitory CK2 site. Biochem. Biophys. Res. Commun. 257:731–737. Furuichi, Y., and Shatkin, A. J. (2000). Viral and cellular mRNA capping: Past and prospects. Adv. Virus Res. 55:135–184. Garcia-Montalvo, B. M., Medina, F., and del Angel, R. M. (2004). La protein binds to NS5 and NS3 and to the 50 and 30 ends of Dengue 4 virus RNA. Virus Res. 102:141–150. Grun, J. B., and Brinton, M. A. (1986). Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. J. Virol. 60:1113–1124. Grun, J. B., and Brinton, M. A. (1987). Dissociation of NS5 from cell fractions containing West Nile virus-specific polymerase activity. J. Virol. 61:3641–3644. Grun, J. B., and Brinton, M. A. (1988). Separation of functional West Nile virus replication complexes from intracellular membrane fragments. J. Gen. Virol. 69:3121–3127. Gubler, D., Kuno, G., and Markoff, L. (2007). Flaviviruses. In ‘‘Fields Virology’’ (D. M. Knipe and P. M. Howley, eds.), 5th Edn., vol. 1, pp. 1153–1252. Lippincott-Raven Publishers, Philadelphia. Guyatt, K. J., Westaway, E. G., and Khromykh, A. A. (2001). Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. J. Virol. Methods 92:37–44. Hager, J., Staker, B. L., Bugl, H., and Jakob, U. (2002). Active site in RrmJ, a heat shock-induced methyltransferase. J. Biol. Chem. 277:41978–41986. Hanley, K. A., Lee, J. J., Blaney, J. E., Jr., Murphy, B. R., and Whitehead, S. S. (2002). Paired charge-to-alanine mutagenesis of dengue virus type 4 NS5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes. J. Virol. 76:525–531. Hausmann, S., Zheng, S., Fabrega, C., Schneller, S. W., Lima, C. D., and Shuman, S. (2005). Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase: Methyl acceptor specificity, inhibition by S-adenosylmethionine analogs, and structure-guided mutational analysis. J. Biol. Chem. 280:20404–20412. Hercyk, N., Horikami, S. M., and Moyer, S. A. (1988). The vesicular stomatitis virus L protein possesses the mRNA methyltransferase activities. Virology 163:222–225. ˚ Hodel, A. E., Gershon, P. D., Shi, X., and Quiocho, F. A. (1996). The 1.85 A structure of vaccinia protein VP39: A bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85:247–256. Hodel, A. E., Gershon, P. D., and Quiocho, F. A. (1998). Structural basis for sequence-nonspecific recognition of 50 -capped mRNA by a cap-modifying enzyme. Mol. Cell 1:443–447. Huang, Y., Staschke, K., De Francesco, R., and Tan, S. L. (2007). Phosphorylation of hepatitis C virus NS5A nonstructural protein: A new paradigm for phosphorylation-dependent viral RNA replication? Virology 364:1–9. Jakubiec, A., and Jupin, I. (2007). Regulation of positive-strand RNA virus replication: The emerging role of phosphorylation. Virus Res. 129:73–79.

96

Andrew D. Davidson

Johansson, M., Brooks, A. J., Jans, D. A., and Vasudevan, S. G. (2001). A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3. J. Gen. Virol. 82:735–745. Joyce, C. M., and Steitz, T. A. (1995). Polymerase structures and function: Variations on a theme? J. Bacteriol. 177:6321–6329. Juffrie, M., van Der Meer, G. M., Hack, C. E., Haasnoot, K., Sutaryo, K., Veerman, A. J., and Thijs, L. G. (2000). Inflammatory mediators in dengue virus infection in children: Interleukin-8 and its relationship to neutrophil degranulation. Infect. Immun. 68:702–707. Kao, C. C., Singh, P., and Ecker, D. J. (2001). De novo initiation of viral RNA-dependent RNA synthesis. Virology 287:251–260. Kapoor, M., Zhang, L., Ramachandra, M., Kusukawa, J., Ebner, K. E., and Padmanabhan, R. (1995). Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5. J. Biol. Chem. 270:19100–19106. Khromykh, A. A., Kenney, M. T., and Westaway, E. G. (1998). Trans-complementation of flavivirus RNA polymerase gene ns5 by using kunjin virus replicon-expressing BHK cells. J. Virol. 72:7270–7279. Khromykh, A. A., Sedlak, P. L., and Westaway, E. G. (1999). trans-Complementation analysis of the flavivirus Kunjin ns5 gene reveals an essential role for translation of its N-terminal half in RNA replication. J. Virol. 73:9247–9255. Khromykh, A. A., Sedlak, P. L., and Westaway, E. G. (2000). cis- and trans-Acting elements in flavivirus RNA replication. J. Virol. 74:3253–3263. Khromykh, A. A., Meka, H., Guyatt, K. J., and Westaway, E. G. (2001). Essential role of cyclization sequences in Flavivirus RNA replication. J. Virol. 75:6719–6728. Kim, Y. G., Yoo, J. S., Kim, J. H., Kim, C. M., and Oh, J. W. (2007). Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase. BMC Mol. Biol. 8:59. Kofler, R. M., Hoenninger, V. M., Thurner, C., and Mandl, C. W. (2006). Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses. J. Virol. 80:4099–4113. Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J. Gen. Virol. 72:2197–2206. Koonin, E. V. (1993). Computer-assisted identification of a putative methyltransferase domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. J. Gen. Virol. 74:733–740. Kroschewski, H., Lim, S. P., Butcher, R. E., Yap, T. L., Lescar, J., Wright, P. J., Vasudevan, S. G., and Davidson, A. D. (2008). Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain. J. Biol. Chem. 283:19410–19421. Lange, A., Mills, R. E., Lange, C. J., Stewart, M., Devine, S. E., and Corbett, A. H. (2007). Classical nuclear localization signals: Definition, function, and interaction with importin alpha. J. Biol. Chem. 282:5101–5105. Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F., and Weber, P. C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 6:937–943. Leveque, V. J., Johnson, R. B., Parsons, S., Ren, J., Xie, C., Zhang, F., and Wang, Q. M. (2003). Identification of a C-terminal regulatory motif in hepatitis

Flavivirus NS5

97

C virus RNA-dependent RNA polymerase: Structural and biochemical analysis. J. Virol. 77:9020–9028. Li, J., Wang, J. T., and Whelan, S. P. (2006). A unique strategy for mRNA cap methylation used by vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA 103:8493–8498. Lim, S. P., Wen, D., Yap, T. L., Yan, C. K., Lescar, J., and Vasudevan, S. G. (2008). A scintillation proximity assay for dengue virus NS5 20 -O-methyltransferasekinetic and inhibition analyses. Antiviral Res. 80:360–369. Lin, R. J., Liao, C. L., Lin, E., and Lin, Y. L. (2004). Blocking of the alpha interferon-induced Jak-Stat signaling pathway by Japanese encephalitis virus infection. J. Virol. 78:9285–9294. Lin, R. J., Chang, B. L., Yu, H. P., Liao, C. L., and Lin, Y. L. (2006). Blocking of interferon-induced Jak-Stat signaling by Japanese encephalitis virus NS5 through a protein tyrosine phosphatase-mediated mechanism. J. Virol. 80:5908–5918. Lindenbach, B. D., and Rice, C. M. (2003). Molecular biology of flaviviruses. Adv. Virus Res. 59:23–61. Lindenbach, B. D., Thiel, H. J., and Rice, C. M. (2007). Flaviviridae: The viruses and their replication. In ‘‘Fields Virology’’ (D. M. Knipe and P. M. Howley, eds.), 5th Edn., vol. 1, pp. 1101–1152. Lippincott-Raven Publishers, Philadelphia. Luo, G., Hamatake, R. K., Mathis, D. M., Racela, J., Rigat, K. L., Lemm, J., and Colonno, R. J. (2000). De novo initiation of RNA synthesis by the RNAdependent RNA polymerase (NS5B) of hepatitis C virus. J. Virol. 74:851–863. Mackenzie, J. (2005). Wrapping things up about virus RNA replication. Traffic 6:967–977. Mackenzie, J. M., Khromykh, A. A., Jones, M. K., and Westaway, E. G. (1998). Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A. Virology 245:203–215. Mackenzie, J. S., Gubler, D. J., and Petersen, L. R. (2004). Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat. Med. 10:S98–S109. Mackenzie, J. M., Kenney, M. T., and Westaway, E. G. (2007). West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis. J. Gen. Virol. 88:1163–1168. Malet, H., Egloff, M. P., Selisko, B., Butcher, R. E., Wright, P. J., Roberts, M., Gruez, A., Sulzenbacher, G., Vonrhein, C., Bricogne, G., Mackenzie, J. M., Khromykh, A. A., et al. (2007). Crystal structure of the RNA polymerase domain of the West Nile virus non-structural protein 5. J. Biol. Chem. 282:10678–10689. Malet, H., Masse, N., Selisko, B., Romette, J. L., Alvarez, K., Guillemot, J. C., Tolou, H., Yap, T. L., Vasudevan, S. G., Lescar, J., and Canard, B. (2008). The flavivirus polymerase as a target for drug discovery. Antiviral Res. 80:23–35. Malone, T., Blumenthal, R. M., and Cheng, X. (1995). Structure-guided analysis reveals nine sequence motifs conserved among DNA amino-methyltransferases, and suggests a catalytic mechanism for these enzymes. J. Mol. Biol. 253:618–632. Markoff, L. (2003). 50 - and 30 -noncoding regions in flavivirus RNA. Adv. Virus Res. 59:177–228. Martin, J. L., and McMillan, F. M. (2002). SAM (dependent) I AM: The Sadenosylmethionine-dependent methyltransferase fold. Curr. Opin. Struct. Biol. 12:783–793.

98

Andrew D. Davidson

Mastrangelo, E., Bollati, M., Milani, M., de Lamballerie, X., Brisbarre, N., Dalle, K., Lantez, V., Egloff, M. P., Coutard, B., Canard, B., Gould, E., Forrester, N., et al. (2006). Preliminary characterization of (nucleoside-20 -O-)-methyltransferase crystals from Meaban and Yokose flaviviruses. Acta. Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62:768–770. Mastrangelo, E., Bollati, M., Milani, M., Selisko, B., Peyrane, F., Canard, B., Grard, G., de Lamballerie, X., and Bolognesi, M. (2007). Structural bases for substrate recognition and activity in Meaban virus nucleoside-20 -O-methyltransferase. Protein Sci. 16:1133–1145. Mazzon, M., Jones, M., Davidson, A., Chain, B., and Jacobs, M. (2009). Dengue virus NS5 inhibits interferon-a signalling by blocking STAT2 phosphorylation. J. Infect. Dis. 200:(in press). Medin, C. L., Fitzgerald, K. A., and Rothman, A. L. (2005). Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion. J. Virol. 79:11053–11061. Miller, S., Sparacio, S., and Bartenschlager, R. (2006). Subcellular localization and membrane topology of the Dengue virus type 2 non-structural protein 4B. J. Biol. Chem. 281:8854–8863. Moreno-Altamirano, M. M., Romano, M., Legorreta-Herrera, M., SanchezGarcia, F. J., and Colston, M. J. (2004). Gene expression in human macrophages infected with dengue virus serotype-2. Scand. J. Immunol. 60:631–638. Morozova, O. V., Tsekhanovskaya, N. A., Maksimova, T. G., Bachvalova, V. N., Matveeva, V. A., and Kit, Y. (1997). Phosphorylation of tick-borne encephalitis virus NS5 protein. Virus Res. 49:9–15. Ng, K. K., Arnold, J. J., and Cameron, C. E. (2008). Structure–function relationships among RNA-dependent RNA polymerases. Curr. Top. Microbiol. Immunol. 320:137–156. Nomaguchi, M., Ackermann, M., Yon, C., You, S., and Padmanabhan, R. (2003). De novo synthesis of negative-strand RNA by Dengue virus RNA-dependent RNA polymerase in vitro: Nucleotide, primer, and template parameters. J. Virol. 77:8831–8842. Nomaguchi, M., Teramoto, T., Yu, L., Markoff, L., and Padmanabhan, R. (2004). Requirements for West Nile virus ()- and (þ)-strand subgenomic RNA synthesis in vitro by the viral RNA-dependent RNA polymerase expressed in Escherichia coli. J. Biol. Chem. 279:12141–12151. Ogino, T., Kobayashi, M., Iwama, M., and Mizumoto, K. (2005). Sendai virus RNA-dependent RNA polymerase L protein catalyzes cap methylation of virus-specific mRNA. J. Biol. Chem. 280:4429–4435. Park, G. S., Morris, K. L., Hallett, R. G., Bloom, M. E., and Best, S. M. (2007). Identification of residues critical for the interferon antagonist function of Langat virus NS5 reveals a role for the RNA-dependent RNA polymerase domain. J. Virol. 81:6936–6946. Peyrane, F., Selisko, B., Decroly, E., Vasseur, J. J., Benarroch, D., Canard, B., and Alvarez, K. (2007). High-yield production of short GpppA- and 7MeGpppAcapped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-20 O positions. Nucleic Acids Res. 35:e26. Platanias, L. C. (2005). Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol. 5:375–386. Poch, O., Sauvaget, I., Delarue, M., and Tordo, N. (1989). Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 8:3867–3874.

Flavivirus NS5

99

Potterton, E., McNicholas, S., Krissinel, E., Cowtan, K., and Noble, M. (2002). The CCP4 molecular-graphics project. Acta Cryst. D58:1955–1957. Pryor, M. J., Rawlinson, S. M., Wright, P. J., and Jans, D. A. (2006). CRM1dependent nuclear export of dengue virus type 2 NS5. Novartis Found. Symp. 277:149–161. Pryor, M. J., Rawlinson, S. M., Butcher, R. E., Barton, C. L., Waterhouse, T. A., Vasudevan, S. G., Bardin, P. G., Wright, P. J., Jans, D. A., and Davidson, A. D. (2007). Nuclear localization of dengue virus nonstructural protein 5 through its importin alpha/beta-recognized nuclear localization sequences is integral to viral infection. Traffic 8:795–807. Quintavalle, M., Sambucini, S., Summa, V., Orsatti, L., Talamo, F., De Francesco, R., and Neddermann, P. (2007). Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J. Biol. Chem. 282:5536–5544. Raghupathy, R., Chaturvedi, U. C., Al-Sayer, H., Elbishbishi, E. A., Agarwal, R., Nagar, R., Kapoor, S., Misra, A., Mathur, A., Nusrat, H., Azizieh, F., Khan, M. A., et al. (1998). Elevated levels of IL-8 in dengue hemorrhagic fever. J. Med. Virol. 56:280–285. Ramadevi, N., Burroughs, N. J., Mertens, P. P., Jones, I. M., and Roy, P. (1998). Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus. Proc. Natl. Acad. Sci. USA 95:13537–13542. Randall, R. E., and Goodbourn, S. (2008). Interferons and viruses: An interplay between induction, signalling, antiviral responses and virus countermeasures. J. Gen. Virol. 89:1–47. Ranjith-Kumar, C. T., Gutshall, L., Kim, M. J., Sarisky, R. T., and Kao, C. C. (2002). Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases. J. Virol. 76:12526–12536. Rawlinson, S. M., Pryor, M. J., Wright, P. J., and Jans, D. A. (2006). Dengue virus RNA polymerase NS5: A potential therapeutic target? Curr. Drug Targets 7:1623–1638. Rawlinson, S. M., Pryor, M. J., Wright, P. J., and Jans, D. A. (2009). CRM1-mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production. J. Biol. Chem. 284:15589–15597. Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., Deas, T. S., Zhou, Y., Li, H., and Shi, P. Y. (2006). West Nile virus 50 -cap structure is formed by sequential guanine N-7 and ribose 20 -O methylations by nonstructural protein 5. J. Virol. 80:8362–8370. Reed, K. E., Gorbalenya, A. E., and Rice, C. M. (1998). The NS5A/NS5 proteins of viruses from three genera of the family flaviviridae are phosphorylated by associated serine/threonine kinases. J. Virol. 72:6199–6206. Reinisch, K. M., Nibert, M. L., and Harrison, S. C. (2000). Structure of the reovirus ˚ resolution. Nature 404:960–967. core at 3.6 A Rice, C. M., Lenches, E. M., Eddy, S. R., Shin, S. J., Sheets, R. L., and Strauss, J. H. (1985). Nucleotide sequence of yellow fever virus: Implications for flavivirus gene expression and evolution. Science 229:726–733. Santoni, M. J., Pontarotti, P., Birnbaum, D., and Borg, J. P. (2002). The LAP family: A phylogenetic point of view. Trends Genet. 18:494–497. Selisko, B., Dutartre, H., Guillemot, J. C., Debarnot, C., Benarroch, D., Khromykh, A., Despres, P., Egloff, M. P., and Canard, B. (2006). Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases. Virology 351:145–158.

100

Andrew D. Davidson

Shuman, S. (1995). Capping enzyme in eukaryotic mRNA synthesis. Prog. Nucleic Acid Res. Mol. Biol. 50:101–129. Shuman, S. (2001). Structure, mechanism, and evolution of the mRNA capping apparatus. Prog. Nucleic Acid Res. Mol. Biol. 66:1–40. Steffens, S., Thiel, H. J., and Behrens, S. E. (1999). The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro. J. Gen. Virol. 80:2583–2590. Sumiyoshi, H., Mori, C., Fuke, I., Morita, K., Kuhara, S., Kondou, J., Kikuchi, Y., Nagamatu, H., and Igarashi, A. (1987). Complete nucleotide sequence of the Japanese encephalitis virus genome RNA. Virology 161:497–510. Sutton, G., Grimes, J. M., Stuart, D. I., and Roy, P. (2007). Bluetongue virus VP4 is an RNA-capping assembly line. Nat. Struct. Mol. Biol. 14:449–451. Takhampunya, R., Padmanabhan, R., and Ubol, S. (2006). Antiviral action of nitric oxide on dengue virus type 2 replication. J. Gen. Virol. 87:3003–3011. Tan, B. H., Fu, J., Sugrue, R. J., Yap, E. H., Chan, Y. C., and Tan, Y. H. (1996). Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. Virology 216:317–325. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673–4680. Ubol, S., Chareonsirisuthigul, T., Kasisith, J., and Klungthong, C. (2008). Clinical isolates of dengue virus with distinctive susceptibility to nitric oxide radical induce differential gene responses in THP-1 cells. Virology 376:290–296. Uchil, P. D., and Satchidanandam, V. (2003). Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. J. Biol. Chem. 278:24388–24398. Uchil, P. D., and Satchidanandam, V. (2003). Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus. Virology 307:358–371. Uchil, P. D., Kumar, A. V., and Satchidanandam, V. (2006). Nuclear localization of flavivirus RNA synthesis in infected cells. J. Virol. 80:5451–5464. van Dijk, A. A., Makeyev, E. V., and Bamford, D. H. (2004). Initiation of viral RNA-dependent RNA polymerization. J. Gen. Virol. 85:1077–1093. Villordo, S. M., and Gamarnik, A. V. (2009). Genome cyclization as strategy for flavivirus RNA replication. Virus Res. 139:130–139. Vo, N. V., Tuler, J. R., and Lai, M. M. (2004). Enzymatic characterization of the full-length and C-terminally truncated hepatitis C virus RNA polymerases: Function of the last 21 amino acids of the C terminus in template binding and RNA synthesis. Biochemistry 43:10579–10591. Wengler, G. (1993). The NS3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology 197:265–273. Wengler, G., Wengler, G., and Gross, H. J. (1978). Studies on virus-specific nucleic acids synthesized in vertebrate and mosquito cells infected with flaviviruses. Virology 89:423–437. Werme, K., Wigerius, M., and Johansson, M. (2008). Tick-borne encephalitis virus NS5 associates with membrane protein scribble and impairs interferon-stimulated JAK–STAT signalling. Cell. Microbiol. 10:696–712. Westaway, E. G., Mackenzie, J. M., Kenney, M. T., Jones, M. K., and Khromykh, A. A. (1997). Ultrastructure of Kunjin virus-infected cells: Colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J. Virol. 71:6650–6661.

Flavivirus NS5

101

Westaway, E. G., Mackenzie, J. M., and Khromykh, A. A. (2002). Replication and gene function in Kunjin virus. Curr. Top. Microbiol. Immunol. 267:323–351. Westaway, E. G., Mackenzie, J. M., and Khromykh, A. A. (2003). Kunjin RNA replication and applications of Kunjin replicons. Adv. Virus Res. 59:99–140. Yap, T. L., Chen, Y. L., Xu, T., Wen, D., Vasudevan, S. G., and Lescar, J. (2007). A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution. Acta Crystallograph. Sect F Struct. Biol. Cryst. Commun. 63:78–83. Yap, T. L., Xu, T., Chen, Y. L., Malet, H., Egloff, M. P., Canard, B., Vasudevan, S. G., and Lescar, J. (2007). Crystal structure of the dengue virus RNA-dependent RNA polymerase catalytic domain at 1.85-angstrom resolution. J. Virol. 81:4753–4765. Yocupicio-Monroy, R. M., Medina, F., Reyes-del Valle, J., and del Angel, R. M. (2003). Cellular proteins from human monocytes bind to dengue 4 virus minusstrand 30 untranslated region RNA. J. Virol. 77(5):3067–3076. Yocupicio-Monroy, M., Padmanabhan, R., Medina, F., and del Angel, R. M. (2007). Mosquito La protein binds to the 30 untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells. Virology 357:29–40. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V. K., Murthy, K. H., and Padmanabhan, R. (2005). Modulation of the nucleoside triphosphatase/RNA helicase and 50 -RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase. J. Biol. Chem. 280:27412–27419. You, S., and Padmanabhan, R. (1999). A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 30 -end of exogenous viral RNA templates requires 50 - and 30 -terminal complementary sequence motifs of the viral RNA. J. Biol. Chem. 274:33714–33722. You, S., Falgout, B., Markoff, L., and Padmanabhan, R. (2001). In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 50 - and 30 -terminal regions that influence RNA structure. J. Biol. Chem. 276:15581–15591. Yu, F., Hasebe, F., Inoue, S., Mathenge, E. G., and Morita, K. (2007). Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein. Arch. Virol. 152:1859–1869. Zhang, B., Dong, H., Zhou, Y., and Shi, P. Y. (2008). Genetic interactions among the West Nile virus methyltransferase, the RNA-dependent RNA polymerase, and the 50 stem-loop of genomic RNA. J. Virol. 82:7047–7058. Zheng, S., Hausmann, S., Liu, Q., Ghosh, A., Schwer, B., Lima, C. D., and Shuman, S. (2006). Mutational analysis of Encephalitozoon cuniculi mRNA cap (guanine-N7) methyltransferase, structure of the enzyme bound to sinefungin, and evidence that cap methyltransferase is the target of sinefungin’s antifungal activity. J. Biol. Chem. 281:35904–35913. Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., Guo, Y., Bernard, K. A., Shi, P. Y., and Li, H. (2007). Structure and function of flavivirus NS5 methyltransferase. J. Virol. 81:3891–3903.

CHAPTER

3 Replication of the Hepatitis Delta Virus RNA Genome John M. Taylor

104 106 108 110 112 113 115 116 116 117 117

Contents

I. Background II. Polymerase(s) III. Promoters and Priming IV. Pausing and Switching V. Replication in the Nucleus VI. Role(s) of the Delta Antigen VII. Host Factors VIII. Viroid Analogy IX. Conclusions and Outlook Acknowledgments References

Abstract

Hepatitis delta virus (HDV) is a subviral agent dependent upon hepatitis B virus (HBV). HDV uses the envelope proteins of HBV to achieve assembly and infection of target cells. Otherwise, the replication of the RNA genome of HDV is totally different from that of its helper virus, and involves redirection of host polymerase activity. This chapter is concerned with recent developments in our understanding of the genome replication process.

Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA Advances in Virus Research, Volume 74 ISSN 0065-3527, DOI: 10.1016/S0065-3527(09)74003-5

#


103

104

John M. Taylor

I. BACKGROUND Hepatitis delta virus (HDV) was discovered in 1977 in studies of patients with a more severe form of hepatitis B virus (HBV) infection (Rizzetto et al., 1977). Much has since been learnt about its molecular biology and for detailed information the reader is directed to recent reviews (Lai, 2005; Taylor et al., 2007) and monographs (Casey, 2006a; Handa and Yamaguchi, 2006). The focus of this chapter is on recent developments in our understanding of the genome replication process. But before considering the recent studies, a brief introduction is needed. HDV is a subviral agent that depends upon HBV as a helper virus. In cells infected with both HDV and HBV, HBV shares its envelope proteins so as to allow the assembly and release of particles containing the HDV genome. These HDV particles can then infect new susceptible hepatocytes, presumably in very much the same manner as HBV infects such cells (Sureau, 2006). Subsequent rounds of HDV replication continue to require HBV to provide the necessary envelope proteins. The HDV genome is a small 1700-nucleotide (nt) long single-stranded RNA. As represented in Fig. 1, this RNA is circular in conformation and is predicted to make about 74% intramolecular base pairing so as to form an unbranched rod-like structure. During replication a second circular RNA, exactly complementary to the genome is produced. This antigenome contains the coding region for the one protein of HDV, the small delta Genome

Antigenome

mRNA

δ Ag ORF

3⬘

5⬘

3⬘ Ribozyme cleavage sites

Poly(A) signal

5⬘

FIGURE 1 Representation of three processed RNAs detected during HDV genome replication. The model shown is reproduced with permission (Taylor, 2006). The antigenome is an exact complement of the genome. The circular genomic and antigenomic RNAs have significant intramolecular pairing and form unbranched rod-like folding. They each contain a sequence that acts in vitro, as a ribozyme that produces a site-specific cleavage. The upper and lower ends of these RNA foldings are referred to here, as the top and bottom, respectively. The mRNA, which is of antigenomic polarity, contains the open reading frame (ORF) for the delta antigen (dAg). Processing of this RNA depends upon a poly(A) signal.

Replication of the Hepatitis Delta Virus RNA Genome

105

antigen, dAg-S. This antigen is translated from a third and less abundant RNA, a mRNA of about 900 nt in length and like cellular mRNAs, in that it possesses a 50 -cap structure and a 30 -poly(A) tail (Gudima et al., 2000). The only cells that are susceptible to HDV infection are hepatocytes. However, many different cell types can support HDV genome replication; initiation in other cell types requires either delivery into the cells of HDV RNA or expression from DNA of the HDV RNA sequences. As discussed further in Section VI, HDV genome replication is somehow dependent upon a source of dAg-S (Chao et al., 1990). The genome, antigenome, and mRNA are all processed RNA transcripts. The processing to make the genomic and antigenomic RNA circles is mediated by two site-specific ribozymes (Kuo et al., 1988b; Sharmeen et al., 1988) that are thought to release unit-length linear RNAs from multimeric transcripts produced in what is referred to as rolling-circle replication, as has been previously used to describe the replication of plant viroid RNAs (Branch and Robertson, 1984). Such an adapted double rolling-circle model is presented in Fig. 2. In this particular model, all the transcription is considered to take place in the nucleus and all transcription is mediated by the redirection of host RNA polymerase II, pol II (Taylor, 2006). Genomic RNA in the nucleus is transcribed to make RNAs that are either processed to become mRNA, steps 1–2, or undergo cleavage and ligation to become new antigenomic RNAs, steps 3–6. The new antigenomic RNA is transcribed to make RNAs that are processed to become new genomic RNAs, steps 6–8. Sections II–VII consider in more detail this and alternative replication schemes. dAg-S binds to both the genomic and antigenomic rod-like RNAs, but only the genome is assembled into new virus particles (Sureau, 2006). As represented in Fig. 3, dAg-S is 195 amino acids in length. During HDV genome replication some of the nascent antigenomic RNA transcripts undergo a site-specific editing by an adenosine deaminase (Casey, 2006a). This leads to a change in the amber termination codon of the mRNA, allowing the translation of a somewhat longer protein, the large delta antigen (dA-L). dAg-L does not support genome replication, and can be a dominant negative inhibitor (Chao et al., 1990). However, its unique C-terminus undergoes modification by farnesylation (Glenn et al., 1992), producing a protein that has an essential role in the assembly of new particles as mediated by the HBV envelope proteins (Chang et al., 1991). The two forms of the dAg have shared sequences and not surprisingly shared features, as represented in Fig. 3 and discussed further in Section VI. Questions still arise as to in what ways, if any, does the dAg-S contribute to the HDV RNA-directed RNA transcription process. Unlike for HBV, HDV has no DNA intermediates. All HDV replication is via RNA-directed RNA transcription. This chapter will focus on progress relating to how we consider the HDV RNA genome is replicated,

106

John M. Taylor

4

5

3 Genome

mRNA 2

Antigenome

1

8

6 7

FIGURE 2 A double rolling-circle model of HDV genome transcription and processing. This is an extension of models proposed earlier for plant viroids RNAs (Branch et al., 1990). The model shown is reproduced with permission (Taylor, 2006). Implied is that all transcription takes place using one enzyme, pol II, and that there are alternative processing pathways based on the two ribozymes and the polyadenylation signal. A more complicated model has been proposed by Li et al. that incorporates the concept that transcription of genomic RNA to make new antigenomes can occur with pol I and in the nucleolus (Li et al., 2006).

along with some discussion of associated questions not yet resolved. The reader is directed elsewhere for reviews of other important topics, such as RNA editing (Casey, 2006b; Linnstaedt et al., 2009), the two ribozymes (Doudna and Lorsch, 2005; Perrotta and Been, 2007), the process of infection of susceptible cells (Urban, 2008), the assembly of new virus particles using HBV envelope proteins (Sureau, 2006), and the multiple genotypes of HDV (Deny, 2006; Radjef et al., 2004).

II. POLYMERASE(S) For some time, it was asked which host polymerase is used for the RNA-directed transcription of HDV RNAs. Several different approaches were used that indicated host RNA polymerase II (pol II) was necessary. (i) The mRNA possessed a 50 -cap structure consistent with pol II


δ Ag-S 1 12 60 66 75 97

107

δ Ag-L Coiled-coil dimerization domain NLS

RNA-binding mofif

146 195 214

Farnesylation site

FIGURE 3 Features on the two forms of the delta antigen. CCD at positions 12–60 is the coiled-coil dimerization domain based upon the structural study of Zuccola et al. (1998). However, more recent studies using phylogenetics and structural predictions suggest a shorter region (Deny, 2006; Enomoto et al., 2006). See also Fig. 5. NLS at positions 66–75 is the nuclear localization signal based upon Alves et al. (2008) whereas earlier studies advocate an additional facilitating domain (Lai, 2006). RBD at positions 97–146 is drawn as a single RNA-binding domain, although studies have shown that some of the central sequences are not needed (Lai, 2006); that is, the RNA-binding domain can be considered as bipartite. Near the C-terminus of the large delta, antigen is a cysteine that becomes farnesylated.

transcription (Gudima et al., 1999; Nie et al., 2004). (ii) The mRNA also contained a 30 -poly(A) as directed by a AAUAAA poly(A)-signal, features typical of processed host mRNAs transcribed by pol II. (iii) In cells undergoing HDV replication inhibition could be achieved with concentrations of amanatin consistent with inhibition of host pol II (Chang et al., 2006; Moraleda and Taylor, 2001). (iv) In nuclear run-on reactions, the synthesis of genomic RNA was again sensitive to pol II inhibition (Chang et al., 2006; Moraleda and Taylor, 2001). (v) Studies with nuclear extracts under conditions optimized for DNA-directed transcription by pol II indicated that exogenous HDV RNAs could be transcribed into full-length transcripts (Fu and Taylor, 1993); however, it has subsequently not been possible to repeat such results (unpublished). Nevertheless, other studies have detected short transcripts (less than 100 nt) and as discussed in Section III, such were primer-dependent transcripts. (vi) Studies with purified pol II or with nuclear extracts have indicated that pol II will bind HDV RNA sequences (Greco-Stewart et al., 2007). However, a recent extension of such in vitro studies has shown that pol I and pol III will also bind such RNAs (Greco-Stewart et al., 2009).

108

John M. Taylor

Along the way, the story has become more complicated with reports from the laboratory of Michael Lai (Li et al., 2006; Macnaughton et al., 2002; Modahl et al., 2000; Tseng et al., 2008). It was reported that the transcription of new antigenomic RNA in nuclear run-on was resistant to amanitin. This suggested a role for an enzyme like pol I, the enzyme which acts in the nucleolus in the transcription of ribosomal RNA precursors. Actually, there is no direct evidence that pol I will transcribe HDV RNAs whereas there is evidence to the contrary, using nuclear runon with an endogenous template, that the synthesis of new antigenomic RNAs, like that of genomic RNAs, is sensitive to low doses of amanitin, consistent with pol II transcription (Chang et al., 2008). Nevertheless, as discussed further in Section III, such contrary findings provoke more objectivity, in that the observed mechanism of HDV RNA-directed transcription might actually vary according to the experimental situation that is used to study such transcription.

III. PROMOTERS AND PRIMING In terms of DNA-directed RNA transcription, a promoter is typically defined as the specific sequences on the DNA template that are recognized by transcription factors which recruit RNA polymerase at the site. Thus, it seems reasonable to ask whether some form of promoters can be defined for HDV RNA-directed transcription? In an early study, cells were transfected with a reporter construct containing as promoter, double-stranded cDNA of sequences corresponding to the top of the HDV genomic RNA (Macnaughton et al., 1993). In this way, bidirectional initiation of transcription of genomic and antigenomic RNAs was detected from a region corresponding to the top of the rod-like fold. This transcription was from DNA; thus, this study did not directly address the question of whether some sequence or structure on the RNAs functions as a promoter. Already there was the suspicion of at least one such promoter because the HDV mRNA species has a unique initiation site (Chen et al., 1986; Gudima et al., 1999, 2000). As indicated in Fig. 1, this site corresponds to initiation at sequences near to the top of the rod-like folding of the genomic RNA. Furthermore, mutagenesis that alters the sequence and/or structure of the stem-loop region at and around this site was found to interfere with HDV replication ability (Gudima et al., 1999; Wu et al., 1997). Greco-Stewart et al. (2007) have since shown that both the top and bottom stem-loop structures of the genomic and antigenomic RNAs are capable of pol II binding. Only in one case, at the top of the genomic RNA, did they report initiation in vitro (Abrahem and Pelchat, 2008), and this


109

was at and around the observed site for the 50 -end of in vivo mRNA (Gudima et al., 2000). Maybe the other three sites are capable of polymerase binding but do not lead to initiation. And, as mentioned earlier, Greco-Stewart et al. (2009) have since reported that pol I and pol III will also bind HDV stem-loop structures, but no data were presented for initiation. Implicit in the above discussion of promoters for HDV RNA-directed transcription is the assumption that some or all of the sites of initiation will be at specific locations and transcription will be initiated de novo, that is, without a primer. In this respect, the assumption is that RNA-directed transcription will resemble host DNA-directed RNA transcription, which is always unprimed. Contrary to this assumption, there is already evidence that some HDV RNA-directed transcription, as studied in vitro or even in vivo, can involve priming. Already two forms of RNA priming have been observed for in vitro transcription of HDV RNA sequences. The first is the process of addition to the 30 -end of the RNA templates. The additions have been achieved with purified pol II and/or extracts containing pol II (Beard et al., 1996; Filipovska and Konarska, 2000; Gudima et al., 2000). A second form of priming is one that is preceded by an endonucleolytic cleavage to produce a novel 30 -end. Filipovska et al. reported that for some in vitro transcription of HDV RNA there was initiation at a site somehow created by a prior cleavage. The RNA transcript was characterized and found to be quite short. In a subsequent study, it was shown that the length increased if dAg-S was present, but still the transcript was

Advances in Virus Research, Volume 74

Advances in Virus Research, Volume 46 (Advances in Virus Research)

Advances in Virus Research, Volume 47 (Advances in Virus Research)

Advances in Virus Research, Volume 58 (Advances in Virus Research)

Advances in Virus Research, Volume 54 (Advances in Virus Research)

Advances in Virus Research, Volume 47 (Advances in Virus Research)

Research Advances in Rabies (Advances in Virus Research, Volume 79)

Advances in Cancer Research Volume 74

Advances in Virus Research, Volume 77

Advances in Virus Research, Volume 71

Advances in Virus Research, Volume 38

Advances in Virus Research, Volume 73

Advances in Virus Research, Volume 78

Advances in Virus Research, Volume 38

Advances in Virus Research, Volume 72

Advances in Virus Research, Volume 29

Advances in Immunology Volume 74

Advances in Agronomy, Volume 74

Advances in Virus Research Vol33 (1987)

Advances in Virus Research: v. 3

Advances in Virus Research: v. 11

Advances in Virus Research Vol40 (1991)

Advances in Applied Microbiology, Volume 74

Insect Viruses, Volume 68: Biotechnological Applications (Advances in Virus Research, Volume 68)

Virus Structure, Volume 64 (Advances in Protein Chemistry)

Virus Structure, Volume 64 (Advances in Protein Chemistry)

Advances in Botanical Research, Volume 30

Advances in Psychology Research Volume 57

Advances in Botanical Research, Volume 41

Advances in Botanical Research, Volume 24

Advances in Food Research, Volume 17

Advances in Virus Research, Volume 74

Advances in Virus Research, Volume 46 (Advances in Virus Research)

Advances in Virus Research, Volume 47 (Advances in Virus Research)

Advances in Virus Research, Volume 58 (Advances in Virus Research)

Advances in Virus Research, Volume 54 (Advances in Virus Research)

Advances in Virus Research, Volume 47 (Advances in Virus Research)

Research Advances in Rabies (Advances in Virus Research, Volume 79)

Advances in Cancer Research Volume 74

Advances in Virus Research, Volume 77

Advances in Virus Research, Volume 71

Advances in Virus Research, Volume 38

Advances in Virus Research, Volume 73

Advances in Virus Research, Volume 78

Advances in Virus Research, Volume 38

Advances in Virus Research, Volume 72

Advances in Virus Research, Volume 29

Advances in Immunology Volume 74

Advances in Agronomy, Volume 74

Advances in Virus Research Vol33 (1987)

Advances in Virus Research: v. 3

Advances in Virus Research: v. 11

Advances in Virus Research Vol40 (1991)

Advances in Applied Microbiology, Volume 74

Insect Viruses, Volume 68: Biotechnological Applications (Advances in Virus Research, Volume 68)

Virus Structure, Volume 64 (Advances in Protein Chemistry)

Virus Structure, Volume 64 (Advances in Protein Chemistry)

Advances in Botanical Research, Volume 30

Advances in Psychology Research Volume 57

Advances in Botanical Research, Volume 41

Advances in Botanical Research, Volume 24

Advances in Food Research, Volume 17

Recommend Documents