Hepatitis C Viruses

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Contributors Glen N. Barber* Room 511 Papanicolau Building 1550 NW 10th Ave [M710] University of Miami School of Medicine Miami FL 33136 USA

Jean Dubuisson* CNRS-UPR2511 Institut de Biologie de Lille - Institut Pasteur de Lille Lille France David N. Frick* Department of Biochemistry and Molecular Biology New York Medical College Valhalla NY 10595 USA

Keril J. Blight* Department of Molecular Microbiology Center for Infectious Disease Research Washington University School of Medicine 660 South Euclid Ave Campus Box 8230 St. Louis MO 63110-1093 USA

Jeffrey S. Glenn* Division of Gastroenterology and Hepatology Stanford University School of Medicine CCSR Building Room 3115 269 Campus Drive Palo Alto CA 94305-5187 USA

D. Spencer Carney Department of Digestive and Liver Diseases University of Texas Southwestern Medical Center 5323 Harry Hines Blvd. Dallas TX 75390-9048 USA

Anne Goffard CNRS-UPR2511 Institut de Biologie de Lille - Institut Pasteur de Lille Lille France

Stéphane Chevaliez Department of Virology INSERM U635 Henri Mondor Hospital University of Paris 12 Créteil France

David R. Gretch* Laboratory Medicine Virology Box 359690 325 9th Ave Seattle WA 98104-2499 USA

Linda B. Couto Benitec LLC 2375 Garcia Ave Mountain View CA 94043 USA

Xiao-Song He* VA Medical Center 154C Building 101 Room C4-171 3801 Miranda Ave. Palo Alto CA94304 USA

Michael Gale Jr.* Department of Microbiology University of Texas Southwestern Medical Center 5323 Harry Hines Blvd. Dallas TX 75390-9048 USA

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Chao Lin* Department of Infectious Diseases Vertex Pharmaceuticals Incorporated 130 Waverly Street Cambridge Massachusetts 02139 USA

Yupeng He Abbott Laboratories Abbott Park IL 60064 USA Cheng Kao Department of Biochemistry and Biophysics Texas A&M University College Station TX 77843 USA

Jaisri R. Lingappa Department of Pathobiology University of Washington Seattle WA USA

Takanobu Kato Liver Diseases Branch NIDDK National Institute of Health Bethesda Maryland 20892 USA

Ayaz M. Majid Department of Microbiology and Immunology University of Miami School of Medicine and Sylvester Comprenhensive Cancer Center Miami Florida USA

Kevin C. Klein Department of Pathobiology University of Washington Seattle WA USA

Tatsuo Miyamura Department of Virology II National Institute of Infectious Diseases 1-23-1 Toyama Shinjuku-ku Tokyo 162-8640 Japan

Alexander A. Kolykhalov* Benitec LLC 2375 Garcia Ave Mountain View CA 94043 USA

Elizabeth A. Norgard Department of Molecular Microbiology Center for Infectious Disease Research Washington University School of Medicine 660 South Euclid Ave Campus Box 8230 St. Louis MO 63110-1093 USA

Michael M. C. Lai* University of Southern California Keck School of Medicine 2011 Zonal Avenue Los Angeles CA 90033 USA

Arnim Pause* McGill Cancer Center and Department of Biochemistry McGill University Montreal Quebec H3G 1Y6 Canada

Muriel Lavie CNRS-UPR2511 Institut de Biologie de Lille Institut Pasteur de Lille Lille France

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Ella H. Sklan Division of Gastroenterology and Hepatology Stanford University School of Medicine CCSR Building Room 3115 269 Campus Drive Palo Alto CA 94305-5187 USA

Jean-Michel Pawlotsky* Department of Virology INSERM U635 Henri Mondor Hospital University of Paris 12 Créteil France Stephen J. Polyak* Department of Laboratory Medicine Virology Division Box 359690 325 9th Ave. Seattle WA 98104-2499 USA

Kirk A. Staschke Lilly Research Laboratories Indianapolis IN 46285 USA Seng-Lai Tan* Lilly Research Laboratories Indianapolis IN 46285 USA

C. T. Ranjith-Kumar* Department of Biochemistry and Biophysics Texas A&M University College Station TX 77843 USA

Takaji Wakita* Department of Microbiology Tokyo Metropolitan Institute for Neuroscience 2-6 Musashidai Fuchu Tokyo 183-8526 Japan

Stephanie T. Shi Department of Virology Pfizer Inc. San Diego CA 92121 USA

Sarah Welbourn McGill Cancer Center and Department of Biochemistry McGill University Montreal Quebec H3G 1Y6 Canada

Ikuo Shoji Department of Virology II National Institute of Infectious Diseases 1-23-1 Toyama Shinjuku-ku Tokyo 162-8640 Japan

* Corresponding author

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Preface Chronic hepatitis C is a serious public health problem and a disease burden in many parts of the world. The discovery of the causative agent, hepatitis C virus (HCV), in 1989 has initiated an almost unparalleled research activity in academic and pharmaceutical-industry laboratories over the ensuing years. This book aims to provide a state-of-the-art account of recent advances in the molecular and cellular biology, immunology and pathogenesis of HCV. It also aspires to discuss new strategies as well as outstanding issues for future research. Hepatitis C has been dubbed the "silent epidemic" because it is generally asymptomatic for decades after infection; its victims often are unaware of the infection until it is too late for therapy. What is the genetic makeup and molecular features that make HCV such a "silent" yet deadly assassin? This question, in fact, is the premise by which this monograph was prepared – it was an attempt to decode the secrets of HCV, one viral gene at a time. To that end, we assembled a team of highly regarded experts from different disciplines who have prepared 16 chapters on various aspects of HCV, including the HCV genome and the role(s) of each viral gene product within the context of the viral life cycle, host interactions, and regulation of host antiviral defense and adaptive immunity. This book can be divided into six main sections. The Introduction sets the stage by providing an overview of the history and the significant hallmarks in the discovery, diagnosis and initial treatments of HCV infection. In the first section, the authors provide an overview of our present understanding of the HCV genome, the structure and replication of these viruses (Chapter 1) and the role of the non-coding regions of HCV in regulating HCV gene expression and RNA replication (Chapter 2). The next two sections include in-depth reviews of the structural (Chapters 3 and 4) and nonstructural (Chapters 5-10) proteins of HCV. A major drawback in the past has been the lack of a robust cell-culture and small-animal model system for HCV infection and replication. However, substantial scientific progress has been made in recent years (Chapters 11-12). Armed with these tools, we are beginning to dissect the molecular mechanisms by which the virus disrupts the host innate and adaptive immune response (Chapters 13 and 14), yielding novel insights into the pathogenicity of HCV. The final section covers the development of infectious HCV-like particle systems (Chapter 15) and the recently developed robust in vitro HCV infection systems based on the JFH-1strain (Chapter 16), which should greatly expedite our study of the full viral life cycle, and our efforts to construct anti-viral strategies and to develop effective immunization strategies with prophylactic and therapeutic

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potential. Needless to say, this is the Holy Grail of HCV research considering that there is no vaccine available and current treatments fail in about half of HCVinfected patients. In the meantime, biotechnology and pharmaceutical companies are making exciting progress in discovery and development of new drugs for HCV therapeutic intervention. These have been the subject of many excellent recent reviews and thus will not be covered in this book. Although it is likely to be several years before any new drug candidate will be available as an anti-HCV agent, the clinical pipeline for hepatitis C is starting to show promise for safer and more effective therapies. Seng-Lai Tan, Ph.D. Indianapolis, May 2006

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Introduction

Introduction David R. Gretch

When the term emerging infectious diseases is loosely applied, then chronic hepatitis C is recognized as one of the most important new diseases afflicting man. The term paradigm is useful when describing this disease, since the discovery, diagnosis and initial treatments of hepatitis C virus infection are all perfect examples of the increasing impact molecular biology is currently having on disease management throughout the globe. The discovery of HCV in the late 1980s occurred without the aid of conventional tissue culture or classical virological methods other than the essential reliance of the chimpanzee model for propagation and initial definition of the infectious agent as an enveloped RNA virus. Reverse transcription and PCR amplification of a subgenomic fraction of the HCV genome not only lead to the initial genetic characterization of HCV as a putative member of the Pestivirus family. It also paved the way for development of the first diagnostic test, an enzyme linked immunoassay that utilized recombinant HCV protein fragments to capture HCV antibodies from patient serum and thus provide serological evidence of infection. This critical step was a major accomplishment for molecular medicine since it provided the first opportunity to positively identify individuals with this highly prevalent yet clinically silent disease. Even though it was well established from epidemiological studies that non-A, non-B (NANB) hepatitis was efficiently transmitted by blood transfusion, and that screening blood products for anti-hepatitis B core antibody and ALT significantly reduced the incidence of post-transfusion NANB hepatitis, development of the first generation HCV antibody screening assay had an impact far greater than many medical scientists in the field had anticipated. Results of early studies indicated that up to 10% of all units of blood transfused in the U.S. prior to the discovery of HCV had lead to transmission of the infectious agent to recipients, accounting for the vast majority of cases of post-transfusion NANB hepatitis, a fact that may have been as surprising as it was fortuitous. However, this was not the whole iceberg; world wide population-based studies revealed a global seroprevalence of well over 100 million individuals, with current estimates being frequently quoted as 170 million HCV infections today. Initial studies reported that approximately 40% of HCV infections in the U.S. were "community acquired", with no known risk factors for acquisition. Subsequent epidemiological studies have suggested that many of

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these cases were actually associated with the most important risk factor for HCV acquisition today, namely intravenous drug use. Such studies have also led to the identification of other previously unknown risk factors, so the term community acquired hepatitis C is no longer in vogue. Thus, cloning of a portion of the HCV RNA genome and development of an effective diagnostic test for HCV antibodies unveiled the insidious disease that is so heavily researched today; this would never have occurred without the use of molecular tools. A second major accomplishment of molecular medicine was development and standardization of tests that efficiently detect and characterize HCV nucleic acids in patient blood. Use of the reverse transcription polymerase chain reaction (RT-PCR) assay in epidemiological studies revealed that of all patients acquiring post-transfusion hepatitis C, over 80% became chronically infected with persistent viremia for decades if not for life. Being able to define HCV viremia in a patient with a risk factor for infection or an asymptomatic individual with serological evidence of exposure to HCV has become a hallmark tool for hepatitis C management in the clinical setting from several perspectives. Confirmation of viremia equals confirmation of active disease. Since most patients with hepatitis C are asymptomatic, a large percentage has normal ALT levels in the blood, and up to 20% of infections spontaneously resolve, knowing HCV infection status is critical for defining subsequent management. Determining whether the disease is mild, moderate or severe still requires a liver biopsy unless the patient has clinical evidence of liver decompensation. The ability to detect, quantify and genetically characterize HCV RNA in patients had an irreplaceable impact on our understanding of hepatitis C disease long before the molecular studies described in the following chapters began to unravel the complex mysteries associated with this truly unique virus-host relationship. Studies of HCV molecular epidemiology indicated that six distinct genotypes have evolved over centuries throughout the world. From clinical studies we learned that HCV persistently replicates in humans for decades, maintaining remarkably constant serum titers that often exceed 1 million viral genomes per milliliter of serum. Pharmacodynamic studies indicated that the HCV production rate exceeds one trillion new virions per day in the face of active immune responses, which is remarkable because this level of virus production is often without overt detriment to the infected host. However, HCV continuously evolves within the host as a pool of genetic variants termed viral quasispecies, presumably as an adaptation to host pressure. How host pressures shape these viral quasispecies without causing significant perturbations in HCV RNA titers is also a mystery, as is the mechanistic relationship between host pressure, viral evolution and disease progression. Again, development of HCV nucleic acid-based assays was an essential contribution of molecular medicine in terms of furthering our understanding of the fundamental

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Introduction

virology of HCV infection in humans, and defining the mysteries of viral pathogenesis that may never be approachable for study by in vitro models. An additional point to touch on with regards to the contribution of molecular medicine to hepatitis C disease pertains to recombinant human interferon, a drug that was engineered from the human genome many decades ago as new wonder drug for the treatment of cancer. Although the utility of interferon in treating cancer should not be understated, it was the astute observations of clinical investigators in the pre-hepatitis C era that interferon lead to normalization of serum ALT levels in about 50% of subjects treated for NANB hepatitis, a remarkable finding even before the discovery of the etiological agent. Long-term studies indicated that although many of these patients relapsed after completion of interferon treatment, several patients continue to have durable and sustained responses with long-term clearance of HCV RNA from blood. Thus, it is not unreasonable to assume that exogenous interferon alone can lead to a cure of this highly efficient virus from the infected host. We now know that HCV genotype and viral load are independent predictors of response to interferon, and other viral factors have also been implicated in influencing treatment outcome. Molecular testing also played an essential role in the optimization of therapy for hepatitis C. Sentinel studies of HCV RNA dynamics following acute interferon dosing not only revealed a rapid dose response effect that was not previously recognized, they also lead directly to the understanding that thrice weekly dosing of interferon was not optimal. At the same time came the serendipitous discovery that the more traditional antiviral agent ribavirin potentiates long-term response to interferon by greatly reducing post-therapy relapse. The end result: the development and licensing of a much more effective combination therapy for hepatitis C, including a pegylated interferon compound with extended half-life, plus ribavirin. Today combination therapy gives clinicians the ability to achieve sustained clearance of HCV and subsequent improvements in liver disease in over 50% of their treated patients. This is an outstanding accomplishment when one considers the relatively poor prognosis for durable sustained remissions in other insidious chronic diseases in humans. Optimization of therapy through traditional clinical trial research without the use of molecular analysis of HCV RNA may never have lead to such a dramatic improvement in hepatitis C treatment outcome. It is at this point that present research takes over with the clear goal of developing new therapies capable of improving long-term response rates in those patients who remain resistant to the best available conventional therapies. It is this problem combined with the perplexing molecular clinical biology of chronic hepatitis C that has fueled the enormous surge in basic research that is the topic of the following chapters. Over a decade of research in the chimpanzee model has

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provided much relevant information with respect to HCV infection and immunity in the host, and small animal models have been developed which should become important tools for further characterizing HCV biology in the near future. Aside from the ever growing body of knowledge related to basic HCV virology, several key interactions between HCV proteins and the host cell regulatory pathways have now been described, including some which have exciting potential in terms of designing new approaches to therapy. Development of the HCV replicon provided for the first time a highly efficient system for studying HCV protein function during viral replication and the effects of experimental drugs on specific aspects of the viral life cycle. However, one important limitation of the HCV replicon is the fact that infectious virus is not produced; thus it falls short of the ideal. Although the lack of a robust tissue culture system has been a major impediment to HCV research in the past, productive infection of culture cells by a unique HCV isolate has very recently been reported. It is the hope of investigators that this system will now provide the opportunity to study for the first time several essential steps in the HCV life cycle. However, it is also essential that more flexible and even more robust infection models be continuously developed. In summary, both the intensity and breadth of HCV research are growing at a remarkable pace, and exciting new discoveries are becoming almost commonplace in the literature. The following chapters were written to provide in-depth reviews of several of the most critical areas of HCV molecular research today. However, it is the goal of this Introduction to remind readers and investigators that hepatitis C disease is highly complex and very likely involves multiple poorly defined viralhost interactions that still cannot be and may never be recapitulated in any animal model or in vitro system. For this reason, molecular research into other Pestivirus animal disease models should be pursued with renewed vigor. Finally, continuous research in the human disease model is essential for defining the most important questions for in vitro study, as is the continuous development of new molecular tools for dissecting the intriguing biopathogenesis of chronic hepatitis C in man. Just as the progress on this disease to date has been phenomenal, so too will be the future progress in furthering our understanding of HCV infection, replication, and molecular biology, and in improving the treatment of hepatitis C. The present state of progress and unanswered questions currently facing molecular investigators are both very well summarized in the following chapters. As for molecular medicine, hepatitis C may long remain the essential paradigm of how new technologies can impact in a very real manner existing problems afflicting man.

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Genome and Life Cycle

Chapter 1

HCV Genome and Life Cycle Stéphane Chevaliez and Jean-Michel Pawlotsky

ABSTRACT Hepatitis C virus (HCV) infection afflicts more than 170 million people worldwide, with the great majority of patients with acute hepatitis C developing chronic HCV infection. It can ultimately result in liver cirrhosis, hepatic failure or hepatocellular carcinoma, which are responsible for hundreds of thousands of deaths each year. Despite the discovery of HCV over 15 years ago, our knowledge of the HCV lifecycle has been limited by our inability to grow the virus in cell culture, as well as by the lack of small-animal models of HCV infection. Nevertheless, data accumulated through the use of multiple in vitro and in vivo study systems have provided a general picture of the biology of HCV, although sometimes with contradictory results. Herein, we summarize our current understanding of the HCV genome and how its structure and encoded gene products, in a complex interplay with host cell factors, might orchestrate a productive viral lifecycle while evading the scrutiny of the host immune system. The recently developed robust in vitro HCV infection systems should help fill in some of the gaps in understanding the HCV lifecycle in the next few years.

HCV GENOME ORGANIZATION AND FUNCTION The Flaviviridae family is divided into three genera: flavivirus, pestivirus, and hepacivirus. Flaviviruses include yellow fever virus, dengue fever virus, Japanese encephalitis virus, and Tick-borne encephalitis virus. Pestiviruses include bovine viral diarrhea virus, classical swine fever virus and Border disease virus. HCV, with at least 6 genotypes and numerous subtypes, is a member of the hepacivirus genus, which includes tamarin virus and GB virus B (GBV-B) and is closely related to human virus GB virus C (GBV-C) (Lindenbach and Rice, 2001). The members of the Flaviviridae family share a number of basic structural and virological characteristics. They are all enveloped in a lipid bilayer in which two or more envelope proteins (E) are anchored. The envelope surrounds the nucleocapsid, which is composed of multiple copies of a small basic protein (core or C), and contains the RNA genome. The Flaviviridae genome is a positive-strand RNA molecule ranging in size from 9.6 to 12.3 thousand nucleotides (nt), with an open reading frame (ORF) encoding a polyprotein of 3000 amino acids (aa) or more. 5

Chevaliez and Pawlotsky

The structural proteins are encoded by the N-terminal part of the ORF, whereas the remaining portion of the ORF codes for the nonstructural proteins (Fig. 1). Sequence motif-conserved RNA protease-helicase and RNA-dependant RNA polymerase (RdRp) are found at similar locations in the polyproteins of all of the Flaviviridae (Miller and Purcell, 1990). In addition, all Flaviviridae share similar polyprotein hydropathic profile, with flaviviruses and hepaciviruses being closer to each other than to pestiviruses (Choo et al., 1991). The ORF is flanked in 5' and 3' by untranslated regions (UTR) of 95-555 and 114-624 nt in length, respectively, which play an important role in polyprotein translation and RNA replication (Fig. 1) (Thurner et al., 2004). Flaviviridae bind to one or more cellular receptors organized as a receptor complex and appear to trigger receptor-mediated endocytosis. Fusion of the virion envelope with cellular membranes delivers the nucleocapsid to the cytoplasm. After decapsidation, translation of the viral genome occurs in the cytoplasm, leading to the production of a precursor polyprotein, which is then cleaved by both cellular and viral proteases into structural and nonstructural proteins. Replication of the viral genome is carried out by the viral replication complex which is associated with cellular membranes and resistant to actinomycin D. Viral replication occurs in the cytoplasm via the synthesis of full-length negative-strand RNA intermediates. Progeny virions are assembled from cytoplasmic vesicles formed by budding

Fig. 1. Organization of Flaviviridae genomes. The figure shows, from top to bottom, the genomes of HCV (hepacivirus), pestivirus, and yellow fever virus (flavivirus). NS: non structural.

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through intracellular membranes. Finally, mature virions are released into the extracellular milieu by exocytosis. Despite the above-mentioned similarities with the members of other Flaviviridae genera, HCV does exhibit a number of virological, epidemiological as well as pathophysiological differences. Flavivirus translation is cap-dependent, i.e. mediated by a type I cap structure located in the 5'UTR (m7GpppAmp), followed by the conserved AG sequence and a relatively short stretch upstream of the polyprotein coding region (Brinton and Dispoto, 1988). In contrast, the HCV 5'UTR is not capped and, like that of pestiviruses and GB viruses, folds into a complex secondary RNA structure forming, together with a portion of the core-coding domain, an internal ribosome entry site (IRES) that mediates direct binding of ribosomal subunits and cellular factors and subsequent translation (see Chapter 2). Whereas the flavivirus 3' UTR is highly structured, the HCV 3'UTR is relatively short, less structured and contains a poly-uridyl tract that varies in length. HCV has a narrow host specificity and tissue tropism. HCV is transmitted exclusively through direct blood-to-blood contacts between humans. Flaviviruses are principally vectored by mosquitoes or ticks and can infect a broad range of vertebrate animals, with humans being a dead-end host that does not participate in the perpetuation of virus transmission. No known pestivirus can infect humans and no known insect vector has been identified. Infections caused by flaviviruses are acute-limited in vertebrate animals, whereas HCV has a high chronicity rate in humans (50%-80%, depending on the age at infection). Strong and adapted humoral and cellular immune responses have been shown to be involved in flavivirus and pestivirus infection recovery and protection. However, HCV infection induces an immune response that fails to prevent chronicity in most cases and does not confer protection against reinfection with homologous and heterologous strains in the chimpanzee model (Farci et al., 1997).

HCV GENOME STRUCTURE AND ORGANIZATION The structural organization of HCV genome is schematically depicted in Fig. 2. 5' UNTRANSLATED REGION

The HCV 5'UTR contains 341 nt located upstream of the ORF translation initiation codon. It is the most conserved region of the genome (nt sequence identity is 60% with GBV-B and approximately 50% with pestiviruses (Choo et al., 1991; Han et al., 1991). The 5'UTR contains four highly structured domains, numbered I to IV, containing numerous stem-loops and a pseudoknot (Brown et al., 1992; Wang et al., 1995). Domains II, III and IV together with the first 12 to 30 nt of the corecoding region constitute the IRES (Honda et al., 1996). Structural characterization by electron microscopy (EM) indicated that domains II, III and IV form distinct

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Fig. 2. HCV genome organization (top) and polyprotein processing (bottom). The 5'UTR consists of four highly structured domains and contains the IRES. The 3'UTR consists of stable stem-loop structures and an internal poly(U)-poly(U/C) tract. The central 9.6-kb ORF codes for a polyprotein of slightly more than 3000 aa depending on the HCV genotype. S and NS correspond to regions coding for structural and nonstructural proteins, respectively. The polyprotein processing and the location of the 10 HCV proteins relative to the ER membrane are schematically represented. Scissors indicate ER signal peptidase cleavage sites; cyclic arrow, autocatalytic cleavage of the NS2-NS3 junction; black arrows, NS3-NS4A protease complex cleavage sites; intramembranous arrow, cleavage by the signal peptide peptidase. The transmembrane domains of E1 and E2 are shown after signal-peptidase cleavage and reorientation of the respective C-terminus hydrophobic stretches (dotted rectangles). Spots denote glycosylation sites of the E1 and E2 envelope proteins. Reproduced from Penin et al., 2004b with permission.

regions within the molecule, with a flexible hinge between domains II and III (Beales et al., 2001). The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors, an event that likely constitutes the first step of HCV polyprotein translation. Several reports suggested a tissue compartmentalization of IRES sequences (Laskus et al., 2000; Lerat et al., 2000; Nakajima et al., 1996; Shimizu et al., 1997). Infection of lymphoid cell lines with HCV genotype 1a H77 strain led to the selection of a quasispecies with nucleotide substitutions within the 5'UTR relative to the inoculum that conferred a 2- to 2.5-fold increase in translation efficiency in human lymphoid cell lines relative to monocyte, granulocyte or monocyte cell lines (Lerat et al., 2000). Furthermore, different translation efficiencies of HCV quasispecies variants isolated from different cell types in the same patient were observed, suggesting cell type-specific IRES interactions with cellular factors may also modulate polyprotein translation (Forton et al., 2004; Laporte et al., 2000; Lerat et al., 2000). 8

Genome and Life Cycle 3' UNTRANLATED REGION

The 3'UTR contains approximately 225 nt. It is organized in three regions including, from 5' to 3', a variable region of approximately 30-40 nt, a long poly(U)-poly(U/ UC) tract, and a highly conserved 3'-terminal stretch of 98 nt (3'X region) that includes three stem-loop structures SL1, SL2 and SL3 (Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996). The 3'UTR interacts with the NS5B RdRp and with two of the four stable stem-loop structures located at the 3' end of the NS5B-coding sequence (Cheng et al., 1999; Lee et al., 2004). The 3'X region and the 52 upstream nt of the poly(U/C) tract were found to be essential for RNA replication, whereas the remaining sequence of the 3'UTR appears to enhance viral replication (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b).

CHARACTERISTICS AND FUNCTIONS OF HCV PROTEINS The HCV ORF contains 9024 to 9111 nt depending on the genotype. The ORF encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and E2), a small protein, p7, whose function has not yet been definitively defined, 6 nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), and the so-called "F" protein which results from a frameshift in the core coding region (Fig. 2; Table 1). The characteristics and functions of HCV proteins are extensively Table 1. HCV proteins and their functions in the viral life cycle. Adapted from Bartenschlager et al., 2004. HCV protein Function Apparent molecular weight (kDa) Core Nucleocapsid 23 (precursor) 21 (mature) 16-17 F/ARFa-protein ? E1 Envelope 33-35 Fusion domain? E2 Envelope 70-72 Receptor binding Fusion domain? p7 Calcium ion channel (viroporin) 7 NS2 NS2-3 autoprotease 21-23 69 NS3 Component of NS2-3 and NS3-4A proteinases NTPase/helicase 6 NS4A NS3-4A proteinase cofactor NS4B Membranous web induction 27 56 (basal form) NS5A RNA replication by formation of replication complexes 58 (hyperphosphorylated form) 68 NS5B RNA-dependant RNA polymerase a Frameshift/ alternate reading frame

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described elsewhere in this book. Here, we provide a brief overview of the viral gene products and their roles in the HCV lifecycle. STRUCTURAL PROTEINS CORE PROTEIN

The HCV core protein is a highly basic, RNA-binding protein, which presumably forms the viral capsid (see Chapter 3). The HCV core protein is released as a 191 aa precursor of 23-kDa (P23). Although proteins of various sizes (17 to 23 kDa) were detectable, the 21-kDa core protein (P21) appeared to be the predominant form (Yasui et al., 1998). The core protein contains three distinct predicted domains : an N-terminal hydrophilic domain of 120 aa (domain D1), a C-terminal hydrophobic domain of about 50 aa (domain D2), and the last 20 or so aa that serve as a signal peptide for the downstream envelope protein E1 (Grakoui et al., 1993c; Harada et al., 1991; Santolini et al., 1994). Domain D1 contains numerous positive charges. It is principally involved in RNA binding and nuclear localization, as suggested by the presence of three predicted nuclear localization signals (NLS) (Chang et al., 1994; Suzuki et al., 1995; Suzuki et al., 2005). Domain D2 is responsible for core protein association with endoplasmic reticulum (ER) membranes, outer mitochondria membranes and lipid droplets (Schwer et al., 2004; Suzuki et al., 2005). Both membrane-bound and membrane-free core proteins appear to exist as dimeric or multimeric forms. When expressed in various in vitro systems, including cell-free or mammalian, bacterial, insect or yeast cell culture models, the HCV core protein can form nucleocapsid-like particles (NLPs) (Baumert et al., 1998; Blanchard et al., 2002; Blanchard et al., 2003; Dash et al., 1997; Ezelle et al., 2002; Iacovacci et al., 1997; Klein et al., 2004; Mizuno et al., 1995; Pietschmann et al., 2002; Serafino et al., 1997; Shimizu et al., 1996). The region between aa 82 and 102 of hydrophilic domain D1 contains a tryptophan-rich sequence and has been suggested to allow the P21 core protein to interact with itself, a property not borne by the precursor P23 (Nolandt et al., 1997). On the other hand, the 75 N-terminal residues of the core protein appear sufficient for NLP assembly in a bacterial system (Majeau et al., 2004). Recently, two clusters of basic residues located in the 68 N-terminal nt were shown to play a critical role in capsid assembly in a cell-free system, whereas the region between aa 82 and 102 did not play a major role (Klein et al., 2005). The critical residues for capsid assembly remain to be precisely identified. In addition to its role in viral capsid formation, the core protein has been suggested to directly interact with a number of cellular proteins and pathways that may be important in the viral lifecycle (McLauchlan, 2000). The HCV core protein has pro- and anti-apoptotic functions (Chou et al., 2005; Kountouras et al., 2003; Meyer et al., 2005), stimulates hepatocyte growth in Huh-7 cell line by transcriptional upregulation of growth-related genes (Fukutomi et al., 2005), and has been 10


implicated in tissue injury and fibrosis progression (Nunez et al., 2004). The HCV core protein could also regulate the activity of cellular genes, including c-myc and c-fos, and alter the transcription of other viral promoters (Ray et al., 1995; Shih et al., 1993). It induces hepatocellular carcinoma when expressed in transgenic mice (Moriya et al., 1998; Moriya et al., 1997). It could also induce the formation of lipid droplets and may play a direct role in steatosis formation (Barba et al., 1997; Moriya et al., 1998; Moriya et al., 1997). E1 AND E2 ENVELOPE GLYCOPROTEINS

The two envelope glycoproteins, E1 and E2, are essential components of the HCV virion envelope and necessary for viral entry and fusion (Bartosch et al., 2003a; Nielsen et al., 2004) (see Chapter 4). E1 and E2 have molecular weights of 33-35 and 70-72 kDa, respectively, and assemble as noncovalent heterodimers (Deleersnyder et al., 1997). E1 and E2 are type I transmembrane glycoproteins, with N-terminal ectodomains of 160 and 334 aa, respectively, and a short C-terminal transmembrane domain of approximately 30 aa. The E1 and E2 transmembrane domains are composed of two stretches of hydrophobic aa separated by a short polar region containing fully conserved charged residues. They have numerous functions, including membrane anchoring, ER localization and heterodimer assembly (Cocquerel et al., 1998; Cocquerel et al., 2000). The ectodomains of E1 and E2 contain numerous proline and cysteine residues, but intramolecular disulfide bonds have not been observed (Matsuura et al., 1994). E1 and E2 are highly glycosylated, containing up to 5 and 11 glycosylation sites, respectively. In addition, E2 contains hypervariable regions with aa sequences differing up to 80% between HCV genotypes and between subtypes of the same genotype (Weiner et al., 1991). Hypervariable region 1 (HVR1) contains 27 aa and is a major (but not the only) HCV neutralizing epitope (Farci et al., 1996; Zibert et al., 1997). Despite the HVR1 sequence variability, the physicochemical properties of the residues at each position and the overall conformation of HVR1 are highly conserved among all known HCV genotypes, suggesting an important role in the virus lifecycle (Penin et al., 2001). E2 plays a crucial role in the early steps of infection. Viral attachment is thought to be initiated via E2 interaction with one or several components of the receptor complex (Flint and McKeating, 2000; Rosa et al., 1996). Because HVR1 is a basic region with positively charged residues located at specific sequence positions, it can theoretically interact with negatively charged molecules at the cell surface. This interaction could play a role in host cell recognition and attachment, as well as in cell or tissue compartmentalization (Barth et al., 2003; Bartosch et al., 2003b). In addition, it was recently shown that human serum facilitated infection of Huh7 cells by HCV pseudoparticles, apparently mediated through an interplay between serum high-density lipoproteins (HDL), HVR1 and the scavenger receptor B type I (SR-BI) (Bartosch et al., 2005; Voisset et al., 2005). Less is known about E1, but 11


it is thought to be involved in intra-cytoplasmic virus-membrane fusion (Flint and McKeating, 2000; Rosa et al., 1996). FRAMESHIFT PROTEIN

The F (frameshift) protein or ARFP (alternate reading frame protein) is generated as a result of a -2/+1 ribosomal frameshift in the N-terminal core-encoding region of the HCV polyprotein. Antibodies to peptides from the F protein were detected in chronically infected patients, suggesting that the protein is produced during infection (Walewski et al., 2001). However, the exact translational mechanisms governing the frequency and yield of the F protein during the various phases of HCV infection are completely unknown. Thus, the role of F protein in the HCV lifecycle remains enigmatic but it was proposed to be involved in viral persistence (Baril and Brakier-Gingras, 2005). NONSTRUCTURAL PROTEINS P7

p7 is a small, 63 aa polypeptide, that has been shown to be an integral membrane protein (Carrere-Kremer et al., 2002). It comprises two transmembrane domains organized in α-helices, connected by a cytoplasmic loop. p7 appears to be essential, because mutations or deletions in its cytoplasmic loop suppressed infectivity of intra-liver transfection of HCV cDNA in chimpanzees (Sakai et al., 2003). In vitro studies suggested that p7 belongs to the viroporin family and could act as a calcium ion channel (Gonzalez and Carrasco, 2003). However, these results remain to be confirmed in vivo. NS2

NS2 is a non-glycosylated transmembrane protein of 21-23 kDa (see Chapter 5). It contains two internal signal sequences at aa positions 839-883 and 928-960, which are responsible for ER membrane association (Santolini et al., 1995; Yamaga and Ou, 2002). NS2, together with the amino-terminal domain of the NS3 protein, the NS2-3 protease, constitutes a zinc-dependent metalloprotease that cleaves the site between NS2 and NS3 (Grakoui et al., 1993b; Grakoui et al., 1993c; Hijikata et al., 1993). NS2 is a short-lived protein that looses its protease activity after self-cleavage from NS3 and is degraded by the proteasome in a phosphorylation-dependent manner by means of protein kinase casein kinase 2 (Franck et al., 2005). In addition to its protease activity, NS2 could interact with host cell proteins, such as the liverspecific pro-apoptotic cell death-inducing DFF45-like effector (CIDE-B), and affect reporter genes controlled by liver and non-liver-specific promoters and enhancers (Dumoulin et al., 2003; Erdtmann et al., 2003). However, the consequences of such interactions within the context of the HCV lifecyle are not clear.

12

Genome and Life Cycle NS3-NS4A

NS3 is a multi-functional viral protein containing a serine protease domain in its Nterminal third and a helicase/NTPase domain in its C-terminal two-thirds. NS4A is a cofactor of NS3 protease activity. NS3-4A also bears additional properties through its interaction with host cell pathways and proteins that may be important in the lifecycle and pathogenesis of infection (see Chapters 6 and 13). Not surprisingly, the NS3-NS4A protease is one of the most popular viral targets for anti-HCV therapeutics (Pawlotsky and McHutchison, 2004; Pawlotsky, 2006). NS3-NS4A PROTEASE

The NS3-NS4A protease is essential for the HCV lifecycle. It catalyzes HCV polyprotein cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/ NS5B junctions. The 3D structure of the NS3 serine protease domain complexed with NS4A has been determined (Kim et al., 1996; Love et al., 1996; Yan et al., 1998). The catalytic triad is formed by residues His 57, Asp 81 and Ser 139 (Bartenschlager et al., 1993; Grakoui et al., 1993a; Tomei et al., 1993). The central region of NS4A (aa 21–30) acts as a cofactor of NS3 serine protease activity, allowing its stabilization, localization at the ER membrane as well as cleaveagedependent activation, particularly at the NS4B/NS5A junction (Bartenschlager et al., 1995; Lin et al., 1995; Tanji et al., 1995). Recently, HCV NS3-NS4A was shown in vitro to antagonize the dsRNA-dependent interferon regulatory factor 3 (IRF-3) pathway, an important mediator of interferon induction in response to a viral infection (Foy et al., 2003). NS3-NS4A also appears to prevent dsRNA signaling via the toll-like receptor 3 upstream of IRF-3 (Li et al., 2005). One potential mechanism includes a blockade of the intracellular doublestranded RNA sensor protein (RIG-I) pathway by NS3-NS4A (Sumpter et al., 2005). Thus, HCV could utilize NS3-4A protease to circumvent the innate immune response at the early stages of infection. In addition, NS3 was also reported to induce malignant transformation of NIH3T3 cells (Sakamuro et al., 1995), suppress actinomycin D-induced apoptosis in murine cell lines (Fujita et al., 1996), and to be involved in hepatocarcinogenesis events (Borowski et al., 1996; Hassan et al., 2005), although the exact mechanisms are not clear. NS3 HELICASE-NTPASE

The NS3 helicase-NTPase domain consisting of the 442 C-terminal aa of the NS3 protein is a member of the helicase superfamily-2 (see Chapter 7). Its threedimentional structure has also been determined (Cho et al., 1998; Kim et al., 1998; Yao et al., 1997). The NS3 helicase-NTPase has several functions, including RNA-stimulated NTPase activity, RNA binding, and unwinding of RNA regions of extensive secondary structure by coupling unwinding and NTP hydrolysis (Gwack et al., 1997; Tai et al., 1996). During RNA replication, the NS3 helicase has been suggested to translocate along the nucleic acid substrate by changing protein 13


conformation, utilizing the energy of NTP hydrolysis. A recent study proposed that the helicase directional movement step is fueled by single-stranded RNA binding energy, while NTP binding allows for a brief period of random movement that prepares the helicase for the next cycle (Levin et al., 2005). In addition, NS3 helicase activity appears to be modulated by the NS3 protease domain and the NS5B RdRp (Zhang et al., 2005). NS4B

NS4B is an integral membrane protein of 261 aa with an ER or ER-derived membrane localization (Hugle et al., 2001; Lundin et al., 2003). NS4B is predicted to harbor at least four transmembrane domains and an N-terminal amphipathic helix that are responsible for membrane association (Elazar et al., 2004; Hugle et al., 2001; Lundin et al., 2003). One of the functions of NS4B is to serve as a membrane anchor for the replication complex (see Chapter 8) (Egger et al., 2002; Elazar et al., 2004; Gretton et al., 2005). Additional putative properties include inhibition of cellular syntheses (Florese et al., 2002; Kato et al., 2002), modulation of HCV NS5B RdRp activity (Piccininni et al., 2002), transformation of NIH3T3 cell lines (Park et al., 2000), and induction of interleukin 8 (Kadoya et al., 2005). NS5A

NS5A is a 56-58 kDa phosphorylated zinc-metalloprotein that probably plays an important role in virus replication and regulation of cellular pathways (see Chapter 9). The N-terminal region of NS5A (aa 1-30) contains an amphipathic α-helix that is necessary and sufficient for membrane localization in perinuclear membranes as well as for assembly of the replication complex (Brass et al., 2002; Elazar et al., 2003; Penin et al., 2004a). Downstream of this motif, the NS5A protein was predicted to contain three domains, numbered I to III. Domain I, located at the Nterminus, contains an unconventional zinc-binding motif formed by four cysteine residues conserved among the hepacivirus and pestivirus genera (Tellinghuisen et al., 2004). HCV replicon RNA replication was inhibited by mutations in the NS5A sequence (Elazar et al., 2003; Penin et al., 2004b) and abolished by alterations of the zinc-binding site (Tellinghuisen et al., 2004). The recently determined 3-D structure of Domain I suggested the presence of protein, RNA and membrane interaction sites (Moradpour et al., 2005; Tellinghuisen et al., 2005). The mechanisms by which NS5A regulate HCV replication are not entirely clear. NS5A associates with lipid rafts derived from intracellular membranes through its binding to the C-terminal region of a vesicle-associated membrane-associated protein of 33 kDa (hVAP-33) (Shi et al., 2003; Tu et al., 1999). This interaction appears to be crucial for the formation of the HCV replication complex in connection with lipid rafts (Gao et al., 2004). A recent study in the replicon system proposed a model in which NS5A hyperphosphorylation disrupts the interaction with hVAP33 and negatively regulates viral RNA replication (Evans et al., 2004). Another 14


report suggested that the level of NS5A phosphorylation plays an important role in the viral lifecycle by regulating a switch from replication to assembly, whereby hyperphosphorylated forms function to maintain the replication complex in an assembly-incompetent state (Appel et al., 2005). Furthermore, NS5A can interact directly with NS5B, but the mechanism by which NS5A modulates the RdRp activity has not been elucidated (Shimakami et al., 2004). In addition, NS5A was reported to interact with a geranylgeranylated cellular protein (Wang et al., 2005a). This is potentially significant considering that assembly of the viral replication complex has been shown to require geranylgeranylation of one or more host cell proteins (Ye et al., 2003). Multiple functions have been assigned to NS5A based on its interactions with cellular proteins (Tellinghuisen and Rice, 2002) (see Chapter 9). For instance, NS5A appears to play a role in interferon resistance by binding to and inhibiting PKR, an antiviral effector of interferon-α (Gale et al., 1998). NS5A also bears transcriptional activation functions (Pellerin et al., 2004; Polyak et al., 2001) and appears to be involved in the regulation of cell growth and cellular signaling pathways (Tan and Katze, 2001; Tellinghuisen and Rice, 2002). However, these observations remain to be confirmed in vivo. NS5B RNA-DEPENDENT RNA POLYMERASE

NS5B belongs to a class of membrane proteins termed tail-anchored proteins (Ivashkina et al., 2002; Schmidt-Mende et al., 2001) (see Chapter 10). Its C-terminal region (21 residues) forms an α-helical transmembrane domain responsible for post-translational targeting to the cytosolic side of the ER, where the functional protein domain is exposed (Moradpour et al., 2004; Schmidt-Mende et al., 2001). The crystal structure of NS5B revealed that the RdRp has a classical "fingers, palm and thumb" structure formed by its 530 N-terminal aa (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). Interactions between the fingers and thumb subdomains result in a completely encircled catalytic site that ensures synthesis of positive- and negative-strand HCV RNAs (Lesburg et al., 1999). The RdRp is another important target for the development of anti-HCV drugs (Di Marco et al., 2005; Ma et al., 2005; Pawlotsky and McHutchison, 2004; Pawlotsky, 2006). Interactions between NS5B and cellular components have also been reported. The C-terminus of NS5B can interact with the N-terminus of hVAP-33, and the interaction may play an important role in the formation of the HCV replication complex (Gao et al., 2004; Schmidt-Mende et al., 2001). More recently, NS5B was reported to bind cyclophilin B, a cellular peptidyl-prolyl cis-trans isomerase that apparently regulates HCV replication through modulation of the RNA binding capacity of NS5B (Watashi et al., 2005).

15


THE HCV LIFECYCLE CELLULAR ATTACHMENT OF HCV VIRIONS AND ENTRY

Many efforts have been made to develop models to identify candidate HCV receptors and study viral binding and entry into target cells. Various cellular and in vivo systems utilizing infected blood samples, virus-like particles produced by expression of structural HCV proteins in insect or mammalian cells, liposomes containing E1-E2, as well as pseudotype particles have yielded a considerable amount of data, although they are not always easy to reconcile. Fig. 3 summarizes the hypothetical HCV lifestyle. HCV RECEPTORS

Several cell surface molecules have been proposed to mediate HCV binding or HCV binding and internalization. CD81

Among all putative HCV receptor molecules, CD81 has been the most extensively studied (Pileri et al., 1998). Human CD81 (target of antiproliferative antibody 1, TAPA-1) is a 25-kDa molecule belonging to the tetraspanin or transmembrane 4 superfamily. It is found at the surface of numerous cell types, where it is thought to assemble as homo- and/or heterodimers by means of a conserved hydrophobic interface. CD81 contains four hydrophobic transmembrane regions (TM1 to TM4) and two extracellular loop domains of 28 and 80 aa, respectively: the small extracellular loop (SEL) and the large extracellular loop (LEL). The LEL is located between TM3 and TM4. It is composed of five α-helices and contains four cysteine residues (Kitadokoro et al., 2001). The SEL is needed for optimal surface expression of the LEL (Masciopinto et al., 2001). The intracellular and transmembrane domains of CD81 are highly conserved among different species. In contrast, the LEL is variable, except between humans and chimpanzees, the only two species permissive to HCV infection (Major et al., 2004; Walker, 1997). The CD81 LEL has been shown to mediate binding of HCV through its envelope glycoprotein E2 (Pileri et al., 1998). The integrity of two disulfide bridges is necessary for the CD81-HCV interaction to occur (Petracca et al., 2000), and the site of interaction appears to involve CD81 residues 163, 186, 188 and 196 (Flint et al., 1999; Meola et al., 2000). The E2 domains involved in CD81 binding remain controversial. Early studies suggested the involvement of aa 480-493 and 544-551 in the truncated soluble form of E2 (Flint et al., 1999), whereas a more recent study pointed to a role for two other domains, including aa 613-618 and a second domain spanning the two HVRs (aa 384-410 and 476-480) (Roccasecca et al., 2003). Several studies argue that cellular factors other than CD81 are required for HCV infection. The expression of human CD81 in a CD81-deficient human hepatoma cell line restored permissiveness to infection with HCV pseudo-particles, but a 16


murine fibroblast cell line expressing human CD81 remained resistant to HCV entry (Cormier et al., 2004). In addition, expression of human CD81 in transgenic mice did not confer susceptibility to HCV infection (Masciopinto et al., 2002). It is possible that the CD81 molecule could act as a post-attachment entry co-receptor and that other cellular factors act together with CD81 to mediate HCV binding and entry into hepatocytes (Cormier et al., 2004). SR-BI

The scavenger receptor B type I (SR-BI) has been proposed as another candidate receptor for HCV (Scarselli et al., 2002). SR-BI is a 509-aa glycoprotein with a large extracellular loop anchored to the plasma membrane at both N- and Ctermini by means of transmembrane domains with short cytoplasmic extensions (Krieger, 2001). SR-BI is a fatty acylated protein located in lipid raft domains. It is expressed at high levels in hepatocytes and steroidogenic cells (Babitt et al., 1997; Krieger, 2001). The natural ligand of SR-BI is high density lipoproteins (HDL). HDLs are internalized through a non-clathrin-dependent endocytosis process that mediates cholesterol uptake and recycling of HDL apoprotein (Silver et al., 2001). HCV genotypes 1a and 1b recombinant E2 envelope glycoproteins were shown to bind HepG2 cells (a human hepatoma cell line that does not express CD81) by interacting with an 82 kDa glycosylated SR-BI molecule (Scarselli et al., 2002). Binding appeared to be highly specific: tranfection of rodent cells with human or tupaia SR-BI (88 % aa identity with human SR-BI) resulted in E2 binding, whereas neither mouse SR-BI (80 % aa identity) nor the closely related human scavenger receptor CD36 (60 % aa identity) bound E2. The SR-BI LEL appeared to be responsible for HCV binding, and HVR1 was recently suggested to be the E2 envelope region involved in the interaction, which was facilitated by serum HDLs (Bartosch et al., 2003b; Scarselli et al., 2002; Voisset et al., 2005). However, the fact that antibodies directed against SR-BI resulted only in a partial blockade of binding suggests that SR-BI is not the only cell surface molecule involved in HCV binding to hepatocytes (Barth et al., 2005). DC-SIGN AND L-SIGN

The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN or CD209) and the liver/lymph node-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing integrin (L-SIGN or CD209L) have been proposed as tissue-specific capture receptors for HCV present in various cell types that could play a critical role in viral pathogenesis and tissue tropism (Gardner et al., 2003; Lozach et al., 2004; Lozach et al., 2003; Pohlmann et al., 2003). DC-SIGN is a 44-kDa homotetrameric type II integral membrane protein with a short aminoterminal cytoplasmic domain and a carboxy-terminal C-type (calcium-dependent) lectin domain. DC-SIGN is expressed at a high level on myeloid-lineage dendritic cells. Its interaction with ICAM-3 activates T cells (Geijtenbeek et al., 2000). L-SIGN is abundantly expressed at the surface of endothelial cells of the liver 17


and lymph nodes and shares 77% aa sequence identity with DC-SIGN (Bashirova et al., 2001). A rapid internalization of virus-like particles upon capture of HCV pseudo-particles by both DC-SIGN and L-SIGN, presumably via E2 binding, was reported (Ludwig et al., 2004), although this was not observed in another study (Lozach et al., 2004). LDL-R

The low-density lipoprotein (LDL) receptor (LDL-R) is an endocytic receptor that transports lipoproteins, mainly the cholesterol-rich LDLs, into cells through receptormediated endocytosis (Chung and Wasan, 2004). Virus-like particles complexed with LDLs have been reported to enter into cells via the LDL receptor (Agnello et al., 1999; Monazahian et al., 1999). In support of this view, binding of low-density HCV particles recovered from plasma by sucrose gradient sedimentation correlated with the density of LDL receptors at the surface of MOLT-4 cells and fibroblasts, and the binding was inhibited by LDL but not by soluble CD81 (Wunschmann et al., 2000). ASIALOGLYCOPROTEIN RECEPTOR

The asialoglycoprotein receptor (ASGP-R) has been reported to mediate binding and internalization of structural HCV proteins (C-E1-E2±p7) expressed in a baculovirus system. Cotransfection of a non-permissive mouse fibroblast cell line with cDNAs of both ASGP-R subunits (H1 and H2) restored permissiveness (Saunier et al., 2003). GLYCOSAMINOGLYCANS

Conservation of positively charged residues in the N-terminus of E2 is in keeping with a possible interaction with heparan sulfate proteoglycans (HSPG) (Barth et al., 2003). E2, in particular its HVR-1, has been shown to bind HSPG with a stronger affinity than other viral envelope glycoproteins, such as human herpes virus 8 or dengue virus envelope proteins. However, glycosaminoglycans are ubiquitously expressed as cell surface molecules. It is conceivable that HSPG could serve as the initial docking site for HCV attachment and the virus is subsequently transferred to another high-affinity receptor (or receptor complex) triggering entry (Barth et al., 2003). MECHANISMS OF CELL ENTRY AND FUSION

After attachment, the nucleocapsid of enveloped viruses is released into the cell cytoplasm as a result of a fusion process between viral and cellular membranes. Fusion is mediated by specialized viral proteins and takes place either directly at the plasma membrane or following internalization of the particle into endosomes. The entry process is controlled by viral surface glycoproteins that trigger the changes required for mediating fusion. At least two different classes of fusion proteins (I and II) can be distinguished (Lescar et al., 2001). The flaviviruses enter target cells 18


by receptor-mediated endocytosis and use class II fusion proteins (Lindenbach and Rice, 2001). By analogy, HCV envelope glycoproteins are believed to belong to class II fusion proteins (Yagnik et al., 2000). However, in contrast with other class II fusion proteins, HCV envelope glycoproteins do not appear to require cellular protease cleavage during their transport through the secretory pathway (Op De Beeck et al., 2004). HCV entry into cells is pH-dependent and endocytosisdependent (Agnello et al., 1999; Bartosch et al., 2003b; Hsu et al., 2003), but the identity of the HCV fusion peptide remains controversial. E1 appeared as a good candidate because sequence analysis suggested the presence of a fusion peptide in its ectodomain (Flint and McKeating, 2000; Rosa et al., 1996). Nevertheless, E2 was shown to share structural homology with class II fusion proteins (Lescar et al., 2001; Yagnik et al., 2000). Crystallographic 3D structure determination and cryoEM-based studies of both envelope glycoproteins are needed to better understand the mechanisms of HCV fusion. RNA TRANSLATION AND POST-TRANSLATIONAL PROCESSING POLYPROTEIN SYNTHESIS

Decapsidation of viral nucleocapsids liberates free positive-strand genomic RNAs into the cell cytoplasm, where they serve, together with newly synthesized RNAs, as messenger RNAs for synthesis of the HCV polyprotein. HCV genome translation is under the control of the IRES, spanning domains II to IV of the 5'UTR and the first nucleotides of the core-coding region. IRES domain I is not part of the IRES but plays an important role by modulating IRES-dependent translation (Friebe et al., 2001; Luo et al., 2003). The IRES mediates cap-independent internal initiation of HCV polyprotein translation by recruiting both cellular proteins, including eukaryotic initiation factors (eIF) 2 and 3 and viral proteins (Ji et al., 2004; Lukavsky et al., 2000; Otto and Puglisi, 2004). Three distinct translation initiation complexes (40S, 48S and 80S) are generated, as shown by in vitro translation experiments in HeLa S10 cells and rabbit reticulocyte lysates and by ex vivo experiments in mammalian cells (Kong and Sarnow, 2002). The HCV IRES has the capacity to form a stable pre-initiation complex by directly binding the 40S ribosomal subunit without the need of canonical translation initiation factors (Otto et al., 2002; Spahn et al., 2001). The 40S subunit assembles with eIF3 and this ternary complex joins with eIF2, GTP, and the initiator tRNA to form a 48S particle in which the tRNA is positioned in the P site of the 40S subunit, base-paired to the start codon of the mRNA. Upon hydrolysis of GTP, eIF2 releases the initiator tRNA and dissociates from the complex. A second GTP hydrolysis step involving initiation factor eIF5B then enables the 60S ribosomal subunit to associate, forming a functional 80S ribosome that initiates viral protein synthesis (Ji et al., 2004; Kieft et al., 2001; Otto and Puglisi, 2004; Sizova et al., 1998).

19


M ER N

Fig. 3. Hypothetical HCV replication cycle. HCV particles bind to the host cells via a specific interaction between the HCV envelope glycoproteins and a yet unknown cellular receptor. Bound particles are probably internalized by receptor-mediated endocytosis. After the viral genome is liberated from the nucleocapsid (uncoating) and translated at the rough ER, NS4B (perhaps in conjunction with other viral or cellular factors) induces the formation of membranous vesicles (referred to as the membranous web; EM in the lower right). These membranes are supposed to serve as scaffolds for the viral replication complex. After genome amplification and HCV protein expression, progeny virions are assembled. The site of virus particle formation has not yet been identified. It may take

20


A number of cellular proteins were reported to interact with the 5'UTR including the polypyrimidine tract-binding protein (PTB) (Ali and Siddiqui, 1995), heterogeneous nuclear ribonucleoprotein L (hnRNP L) (Hahm et al., 1998), La autoantigen (Ali and Siddiqui, 1997), the poly(rC)-binding protein 2 (PCP2) (Spangberg and Schwartz, 1999) and NS1-associated protein 1 (NSAP1) (Kim et al., 2004). The biological significance of these protein-RNA interactions remains unknown. In addition, HCV proteins may affect IRES translational efficiency, including the core protein (Zhang et al., 2002) and non-structural proteins NS4A and NS5B (Kato et al., 2002). The HCV 3'UTR may also modulate IRES-dependent translation, but this remains controversial (Imbert et al., 2003; Wang et al., 2005b). POST-TRANSLATIONAL PROCESSING

HCV genome translation generates a large precursor polyprotein, which is targeted to the ER membrane for translocation of the E1 ectodomain into the ER lumen, a process mediated by the internal signal sequence located between the core and E1 sequences. Cleavage of the signal sequence by the host signal peptidase yields the immature form of the core protein (P23) (McLauchlan et al., 2002). The signal peptide is further processed by a host signal peptide peptidase (SPP, a presenilintype aspartic protease that resides in the ER membrane) to yield the mature form of the core protein (P21) (Fig. 3) (Penin et al., 2004b). The host signal peptidase also ensures cleavage at the E1-E2 junction in the ER lumen. Additional signal peptidase cleavages at the C-terminal end of E2 and between p7 and NS2 give rise to p7 (Fig. 3). An incomplete cleavage may lead to the production of non-cleaved E2-p7 proteins, the role of which is unknown. E1 and E2 subsequently undergo several maturation steps, including N-glycosylation, conformation and assembly of E1E2 heterodimers (Penin et al., 2004b). Heterogeneous E1E2 aggregates are also produced, but their role in viral particle formation is not known. The zinc-dependent NS2-3 auto-protease ensures cis-cleavage of NS3 from NS2 (Fig. 2). NS3 needs to assemble with its cofactor NS4A to catalyze cis-cleavage at the NS3-NS4A junction and trans-cleavage at all downstream junctions including NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B (Fig. 2) (Bartenschlager and Lohmann, 2000; Lindenbach and Rice, 2005). The cleavage sites recognized by the

place at intracellular membranes derived from the ER or the Golgi compartment. Newly produced virus particles may leave the host cell by the constitutive secretory pathway. The upper right panel of the figure shows a schematic representation of an HCV particle. The middle panel shows a model for the synthesis of negative-stranded (-) and positive stranded (+) progeny RNA via a doublestranded replicative form (RF) and a replicative intermediate (RI). The bottom panel shows an electron micrograph of a membranous web (arrow heads) in Huh7 cell containing subgenomic HCV replicons. The web is composed of small vesicles embedded in a membrane matrix. Bar: 500 nm; N: nucleus; ER: endoplasmic reticulum; M: mitochondria. Reproduced from Bartenschlager et al., 2004 with permission.

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NS3-NS4A protease have in common the following sequence: Asp/GluXXXXCys/ Thr-Ser/Ala, with trans cleavages occurring downstream of a cysteine residue and the cis cleavage occurring downstream of a threonine residue. HCV REPLICATION THE HCV REPLICATION COMPLEX

Infection with a positive-strand RNA virus leads to rearrangements of intracellular membranes, a prerequisite to the formation of a replication complex that associates viral proteins, cellular components and nascent RNA strands. The HCV NS4B protein seems to be sufficient to induce the formation of a membranous web or membrane-associated foci (Egger et al., 2002; Gretton et al., 2005). It is not known whether NS4B recruits cellular proteins responsible for vesicle formation or induces vesicle formation by itself. The membranous web is derived from ER membranes (Bartenschlager et al., 2004). It is rich in cholesterol and fatty acids, the degree of saturation of which (that influences membrane fluidity) modulates HCV replication (Kapadia and Chisari, 2005). HCV replication was shown to occur in detergentresistant membranes that co-localize with caveolin-2, an essential component of lipid raft domains (Shi et al., 2003). Indeed, lipid rafts are involved in the formation of the replication complex, through protein-protein interactions between hVAP-33 and both NS5A and NS5B HCV proteins (Gao et al., 2004; Shi et al., 2003; Tu et al., 1999). Overall, the membranous web consists of small vesicles embedded in a membranous matrix, forming a membrane-associated multiprotein complex that contains all of the nonstructural HCV proteins (Egger et al., 2002). MECHANISM OF HCV REPLICATION

The precise mechanisms of HCV replication are still poorly understood. By analogy with other positive-strand RNA viruses, HCV replication is thought to be semi-conservative and asymmetric with two steps, both of which are catalyzed by the NS5B RdRp. The positive-strand genome RNA serves as a template for the synthesis of a negative-strand intermediate of replication during the first step. In the second step, negative-strand RNA serves as a template to produce numerous strands of positive polarity that will subsequently be used for polyprotein translation, synthesis of new intermediates of replication or packaging into new virus particles (Bartenschlager et al., 2004). The positive-strand RNA progeny is transcribed in a five to ten fold in excess compared to negative-strand RNA. NS5B RpRd was initially thought to catalyze primer-dependent initiation of RNA synthesis, either through elongation of a primer hybridized to the RNA template or through a copyback mechanism (Behrens et al., 1996). More recently, the HCV RdRp was shown to be capable of initiating de novo RNA synthesis under certain experimental conditions (Zhong et al., 2000).

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Initiation of RNA strand synthesis at the 3'-end of the plus and minus strands involves domain I of the 5'UTR, which can form a G/C-rich stem-loop, the 3'UTR and a cis-acting replication element (5BSL3.2) consisting of 50 bases located in a large predicted cruciform structure at the 3' end of the HCV NS5B-coding region (You et al., 2004). Initiation of RNA replication is triggered by an interaction between proteins of the replication complex, the 3'X region of the 3'UTR, and 5BSL3.2 that forms a pseudoknot structure with a stem-loop in the 3'UTR (AstierGin et al., 2005; Friebe et al., 2005; You et al., 2004). A phosphorylated form of PTB was found in the replication complex and PTB was shown to interact with two conserved stem-loop structures of the 3'UTR, an interaction thought to modulate RNA replication (Chang and Luo, 2005; Luo, 1999; Luo, 2004). Importantly, inhibition of PTB expression by means of small interfering RNAs reduced the amount of HCV proteins and RNA in HCV replicon-harboring Huh7 cells (Chang and Luo, 2005). VIRUS ASSEMBLY AND RELEASE

Little is known about HCV assembly and release due to the lack of appropriate study models. Different variants of the HCV core protein, which can exist as dimeric, and probably multimeric forms as well, have been shown to be capable of self assembly in yeast in the absence of viral RNA, generating virus-like particles with an average diameter of 35 nm (Acosta-Rivero et al., 2004a; Acosta-Rivero et al., 2004b). Recent reports suggested that the N-terminal portion of the core protein is sufficient for capsid assembly, in particular the two clusters of basic residues (Klein et al., 2005; Klein et al., 2004; Kunkel et al., 2001; Lorenzo et al., 2001; Majeau et al., 2004). In bacterial systems, HCV core proteins efficiently self-assembled to yield nucleocapsid-like particles with a spherical morphology and a diameter of 60 nm, but the presence of a nucleic acid was required (Kunkel et al., 2001). Overall, particle formation is probably initiated by the interaction of the core protein with genomic RNA; HCV core can indeed bind positive-strand RNA in vitro through stem-loop domains I and III and nt 23-41 (Shimoike et al., 1999; Tanaka et al., 2000). It is tempting to speculate that the core-RNA interaction may play a role in the switch from replication to packaging. Virus-like particles were produced in mammalian cells by using a chimeric virus replicon allowing high-level expression of HCV structural proteins in BHK-21 cell lines (Blanchard et al., 2002). Budding of virus-like particles of 50 nm in diameter in the dilated ER lumen was observed (Blanchard et al., 2003). Transfection of full-length HCV RNA in HeLa G and HepG2 cell lines led to the formation of virus-like particles with a diameter of 45 to 60 nm, which were synthesized and assembled in the cytoplasm and budded into the ER cisternae to form coated particles (Dash et al., 1997; Mizuno et al., 1995). Indeed, the HCV envelope glycoproteins E1 and E2 associate with ER membranes through their transmembrane domains

23


(Cocquerel et al., 1998), suggesting that virus assembly occurs in the ER. Structural proteins have been detected both in the ER and the Golgi apparatus, suggesting that both compartments are involved in later maturation steps (Serafino et al., 2003). Moreover, the presence of N-glycan residues at the surface of HCV particles is also in keeping with a transit via the Golgi apparatus. The mechanisms underlying exportation of mature virions in the pericellular space have yet to be understood. Newly produced virus particles may leave the host cell by the constitutive secretory pathway.

STRUCTURE OF HCV VIRIONS HCV is thought to adopt a classical icosahedral scaffold in which glycoproteins E1 and E2 are anchored to the host cell-derived double-layer lipid envelope. Within the envelope is the nucleocapsid which is likely composed of multiple copies of the core protein, forming an internal icosahedral viral coat that encapsidates the viral genomic positive-strand RNA. EM and immuno-EM (IEM) studies of bona fide HCV particles have been hampered by the low amount of viruses in blood and tissues, the failure to efficiently propagate HCV in cell culture, the poor sensitivity of these methods, and antibody cross-reactivity. Visualization of HCV virions or virus-like particles was therefore made essentially from in vitro or non-human in vivo models. Infection of primary cells or stable cell lines of hepatic or lymphoid origin with sera from HCV-infected patients revealed the presence of spherical virus-like particles (Lacovacci et al., 1997; Serafino et al., 1997; Shimizu et al., 1996). Transfection of Huh7 cells with full-length HCV genomes did not lead to virion production (Pietschmann et al., 2002), but virus-like particles were generated after transfection of HepG2 or Hela G cells (Dash et al., 1997; Mizuno et al., 1995). HCV virus-like particles could also be produced in mammalian cells, by means of recombinant Semliki Forest virus (SFV) or vesicular stomatitis virus (VSV) replicons expressing genes encoding the structural HCV proteins (Blanchard et al., 2003; Ezelle et al., 2002), and in insect cells infected with a recombinant baculovirus expressing HCV structural proteins (Baumert et al., 1998; Luckow and Summers, 1988; Maillard et al., 2001). MORPHOLOGY OF HCV PARTICLES

Early filtration studies performed in sera from chimpanzees with non-A, non-B hepatitis suggested that the diameter of the causal agent was in the order of 30-60 nm (He et al., 1987; Yuasa et al., 1991). EM and IEM analysis of particles recovered from the blood and liver of infected chimpanzees and patients revealed the presence of spherical particles of 33-70 nm (Bosman et al., 1998; Ishida et al., 2001; Jacob et al., 1990; Kaito et al., 1994; Li et al., 1995; Petit et al., 2003). Detergent treatment of infectious sera yielded 30-40 nm icosahedron-shaped particles containing both

24


the HCV core protein and HCV RNA (Takahashi et al., 1992). Virus-like particles of 45-60 nm was observed in the supernatant of primary cells or stable cell cultures treated with infectious sera and of cell lines transfected with the full-length HCV ORF (Dash et al., 1997; Iacovacci et al., 1997; Mizuno et al., 1995; Serafino et al., 1997; Shimizu et al., 1996). HCV-like particles of 20-60 nm in diameter were also produced by the expression of HCV structural proteins in cell-free systems (Klein et al., 2004), SFV replicons (Blanchard et al., 2002; Blanchard et al., 2003), VSV vectors in rodent BHK-21 cells (Ezelle et al., 2002), bacterial systems (Kunkel et al., 2001; Lorenzo et al., 2001), baculovirus vectors in insect cells (Baumert et al., 1998; Xiang et al., 2002) and yeast expression vectors (Acosta-Rivero et al., 2001; Acosta-Rivero et al., 2004b; Falcon et al., 1999). The recently developed cell culture system is capable of producing large amounts of infectious HCV virions (Lindenbach et al., 2005b; Wakita et al., 2005; Zhong et al., 2005). Two types of viral particles could be visualized in IEM: particles of 30-35 nm in diameter likely to correspond to the viral nucleocapsids, and particles of 50-60 nm in diameter likely to be the infectious virions (Fig. 4) (Wakita et al., 2005).

Fig. 4. HCV viral particle produced in a tissue culture system from a cloned viral genome (Wakita et al., 2005). Viral particles were generated after transfection of the human hepatoma cell line Huh7 by HCV replicons of the JFH1 genotype 2a strain cloned from a Japanese patient with fulminant hepatitis (see Chapter 16). HCV particles had a density of 1.15-1.17 g/ml and a spherical morphology with an average diameter of approximately 55 nm. They were infectious for chimpanzees (Wakita et al., 2005). The photograph is a courtesy of Ralf Bartenschlager.

25

Chevaliez and Pawlotsky CIRCULATING FORMS OF HCV VIRIONS PLASMA COMPARTMENTALIZATION OF HCV PARTICLES

HCV was initially reported to have a lower buoyant density than other members of the Flaviviridae family on 20-60% isopycnic sucrose density gradients (1.05 to 1.07 g/ml vs 1.15 to 1.25 g/ml, respectively) (Lindenbach and Rice, 2001; Trestard et al., 1998; Yoshikura et al., 1996). Ultracentrifugation of sera from patients with acute and chronic HCV infection revealed the presence of two populations of HCV particles with a broad range of densities, from 1.06 to 1.25 g/ml. Low-density HCV particles were shown to be principally associated with lipids and lipoproteins and to contain the infectious virus, whereas high-density HCV particles were largely associated with immunoglobulins in the form of immune complexes and supposedly less infectious (Aiyama et al., 1996; Andre et al., 2002; Dienstag et al., 1979; Hijikata et al., 1993; Thomssen et al., 1992). Interestingly, the respective proportions of high- and low-density fractions in infected patients' blood were reported to fluctuate over the course of infection and according to the stage of liver disease (Choo et al., 1995; Hijikata et al., 1993; Kanto et al., 1994; Kanto et al., 1995; Petit et al., 2003). NON-ENVELOPED NUCLEOCAPSIDS

The existence of non-enveloped HCV nucleocapsids during natural infection and their role in the pathophysiology of HCV infection has been debated. Lipo-viroparticles (LVPs) rich in HCV RNA, HCV core protein, triglycerides and apoproteins (especially apoB and apoE) were recently described as large spherical particles of 100 nm, the delipidation of which yielded capsid-like structures (Andre et al., 2002). Non-enveloped nucleocapsids were detected in the serum of infected patients and in hepatocytes from patients and experimentally infected chimpanzees (Falcon et al., 2003a; Falcon et al., 2003b; Maillard et al., 2001). Non-enveloped HCV particles recovered from the plasma of infected individuals had a buoyant density of 1.27 to 1.34 g/ml (Maillard et al., 2001). They were heterogeneous in size, with a diameter of 38-62 nm in EM, and were recently shown to exhibit Fcγ receptor-like activity and bind non-immune IgG (Maillard et al., 2001; Maillard et al., 2004). Whether or not non-enveloped nucleocapsids are infectious remains to be established.

CONCLUSION The development of novel anti-HCV therapeutic agents has been stymied by the lack of an efficient in vitro viral infection system and a suitable animal model. Although significant progress has been made through genetic and biochemical approaches in dissecting the molecular processes of HCV replication, our understanding of the viral entry and virion production steps remains rudimentary. Furthermore, HCV exists as "quasispecies" in patients due to its high mutation rate and thus viral resistance will likely be a problem for the emerging small-molecule HCV inhibitors (Pawlotsky, 2003; Pawlotsky, 2006). The recent development of a robust cell culture 26


system for HCV infection may unravel new aspects of HCV replication, which in turn will facilitate the development of specific antivirals that target each stage in the virus life cycle.

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HCV 5' and 3'UTR

Chapter 2

HCV 5' and 3'UTR: When Translation Meets Replication Stephanie T. Shi and Michael M. C. Lai

ABSTRACT Similar to other positive-strand RNA viruses, the non-coding regions of HCV RNA, referred herein as 5' and 3' untranslated regions (5'UTR and 3'UTR), contain important sequence and structural elements critical for HCV translation and RNA replication. The 5'UTR harbors an internal ribosome entry site (IRES) that directs viral protein translation via a cap-independent mechanism. As the initiation sites for RNA synthesis, both 5'UTR and 3'UTR contain signals that are indispensable for and regulate viral RNA replication. Additional structural elements involved in translation or RNA replication are also present in both ends of the protein (core and NS5B)-coding regions. These RNA elements interact with each other either directly or through the binding of viral and cellular proteins that are most likely involved in the regulation of translation and RNA replication processes. Since RNA replication and translation occur on the same RNA molecule, mechanisms must exist to regulate and separate these two processes. This chapter details the current understanding of the roles of the UTRs and other structural components in the viral RNA as well as their binding proteins in HCV translation and RNA replication and speculate on the possible mechanisms regulating these two different processes.

INTRODUCTION HCV is a typical flavivirus containing a single-stranded, positive-sense RNA of 9.7 kb in length (Choo et al., 1991). The viral RNA contains a single large open reading frame (ORF) flanked by an untranslated region (UTR) at each end, a genomic organization conserved among members of the Flaviviridae family. One of the most important features of HCV RNA is its high degree of genetic variability as a result of mutations that occur during viral replication. However, the mutation rate varies significantly in the different regions of the HCV genome, of which the 5'UTR and the extreme end of the 3'UTR have the lowest sequence diversity among various genotypes and subtypes (Choo et al., 1991; Miller and Purcell, 1990; Muerhoff et al., 1995). The relatively conserved nature of these regions signifies their functional importance in the viral life cycle.

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A combination of phylogenetic analysis, computer modeling, and chemical and enzymatic probing has enabled the identification of structural elements in the 5' and 3' UTRs of HCV RNA. The viral RNA elements (internal ribosome entry site, IRES) critically involved in the cap-independent translation of HCV RNA have been analyzed extensively. In contrast, the study of the mechanism of HCV RNA replication was more limited due to the lack of efficient cell culture or small animal models. The generation of consensus cDNA clones that are infectious in chimpanzees provided the first tools for molecular genetic analysis of HCV RNA replication (Beard et al., 1999; Choo et al., 1989; Kolykhalov et al., 1997; Yanagi et al., 1997; Yanagi et al., 1999a; Yanagi et al., 1998). Using this approach, the regions in the 3'UTR that are required for viral replication have been identified (Kolykhalov et al., 1997; Yanagi et al., 1999b). More recently, the development of and advances in the cell-based subgenomic replicon system have identified additional RNA elements of the UTRs and other cis-acting replication elements (CREs) that are involved in RNA replication and translation (Friebe et al., 2005; Friebe et al., 2001; Lee et al., 2004a; You et al., 2004). A number of viral and cellular proteins have been shown to interact with the essential structural elements in the non-coding and coding regions of HCV RNA and are presumably involved in the regulation of the viral translation and/or RNA replication processes. The precise functional roles of most of these proteins have not been established. The recent development of cell-free HCV RNA replication systems (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003) provides an additional tool for studying the viral and host proteins involved in the translation and replication of HCV RNA, thus identifying novel targets for the development of more effective antiviral therapies.

STRUCTURAL AND FUNCTIONAL COMPONENTS OF THE HCV RNA The 5'UTR and the extreme end of the 3'UTR are the most conserved regions of HCV RNA in terms of primary sequence and secondary structures. Together with the fact that these structured domains are located at the 5' and 3' ends of the genome, it stands to reason that they play important roles in viral RNA translation and/or replication. 5'UTR

The 5'UTR of the HCV genome is 341-nt long in most viral isolates. There is more than 90% sequence identity among different HCV genotypes, with some segments nearly identical among different strains (Bukh et al., 1992). The secondary and tertiary structures of this region are also largely conserved (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a). The 5'UTRs of HCV, GBV-B (Muerhoff et al., 1995), and pestiviruses, such as bovine viral diarrhea virus (BVDV) and 50

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classical swine fever virus, share extensive homology in primary sequence and secondary structure (Brown et al., 1992; Han et al., 1991; Honda et al., 1996a; Simons et al., 1995), signifying GBV-B and pestiviruses as the closest relatives to HCV (Ohba et al., 1996). A combination of computational, phylogenetic, and mutational analyses of the HCV 5'UTR has identified four major structural domains (domains I-IV) (Fig. 1), most of which are also conserved among HCV genotypes, GBV-B, and pestiviruses (Brown et al., 1992; Honda et al., 1999a; Honda et al., 1996a; Smith et al., 1995). Common features include a large stem-loop III and a pseudoknot (psk). The 5'UTR sequences of HCV and GBV-B have two smaller stem-loops, stem-loop Ia near the extreme 5' end and stem-loop IV containing the translation initiation codon (Honda et al., 1996a).

Fig. 1. The structures of the 5'UTR (Rijnbrand and Lemon, 2000) and 3'UTR (Ito and Lai, 1997; Kolykhalov et al., 1996) of HCV RNA (represented by the HCV-H strain). The structural diagram of the 5'UTR was kindly provided by Drs. René Rijnbrand and Stanley Lemon. psk, pseudoknot. The start codon (nt 342) and stop codon are indicated by boldface characters. The shaded boxes in 5'- and 3'-UTR and are RNA elements putatively involved in RNA replication. The enclosed RNA sequences in 5'-UTR are the reported elements required for efficient IRES-dependent translation.

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The first 40 nt of the 5'UTR constitutes domain I, which is involved in RNA replication but not essential for translation; therefore, the function of this region is distinct from the rest of the 5'UTR, which is critical for translation (Friebe et al., 2001; Luo et al., 2003). The remaining domains II-IV constitute an IRES (Fig. 1) (Brown et al., 1992), which mediates the cap-independent translation of the HCV ORF (Tsukiyama-Kohara et al., 1992). Domains II and III are relatively more complex than domain IV and contain multiple stems and loops (Honda et al., 1999a; Lemon and Honda, 1997). Several electron microscopy (Beales et al., 2001; Spahn et al., 2001) and NMR studies (Lukavsky et al., 2000) have provided detailed structural information on the main domains of the IRES. Domains IIIa–IIIc and II extend in opposite directions from a small central domain that includes stem loops and junctions IIIe–IIIf (Spahn et al., 2001). The hairpin loop of the small IIIe subdomain forms a novel tetraloop fold with three exposed Watson–Crick faces that may be involved in 40S ribosome binding (Lukavsky et al., 2000). The stem of subdomain IIId forms a loop E motif similar to those observed in prokaryotic and eukaryotic ribosomal RNA, and a six-nucleotide hairpin loop containing an S-turn motif (Klinck et al., 2000; Lukavsky et al., 2000). The sequences of the hairpin loops of subdomains IIIe and IIId are conserved among all HCV isolates and play an important role in translation initiation. The base of domain III forms a highly conserved pseudoknot, which is critical for IRES activity (Wang et al., 1995). Similar pseudoknots with almost identical primary sequences also exist in the pestiviral and GBV-B IRES elements (Lemon and Honda, 1997). The pseudoknot is part of the binding site for the 40S ribosome subunit (Kolupaeva et al., 2000). Another tertiary structural element in domain II, identified by RNA-RNA crosslinking, may also be involved in ribosome binding (Lyons et al., 2001). Domain IV is composed of a small stem-loop (stem-loop IV) in which the initiator codon AUG is located within the single-stranded loop region (Honda et al., 1996a). Stem-loop IV is not required for internal entry of ribosomes. In fact, the stability of this stem-loop structure is negatively correlated with the translation efficiency of the viral RNA (Honda et al., 1996a). According to a structure-based classification scheme originally designed for picornaviral IRES elements (Wimmer et al., 1993), the HCV IRES, together with the IRES elements of the closely related pestiviruses and GBV-B, is classified into type 3 of four existing types (Lemon and Honda, 1997). The picornaviral and flaviviral IRES elements are significantly different in a number of aspects, suggesting distinct mechanisms of translation initiation for these two virus families (Rijnbrand and Lemon, 2000). The picornaviral IRES elements have been shown to be more efficient than the HCV IRES in directing translation (Borman et al., 1995). In contrast, viruses in the genus Flavivirus (e.g. yellow fever virus) have significantly shorter 5' UTRs with a cap structure, m7GpppN1mpN2 (Westaway, 1987). 52

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The 3'UTR of HCV varies between 200 and 235 nt long, which typically consists of three distinct regions, in the 5' to 3' direction, a variable region, a poly(U/UC) stretch, and a highly conserved 98-nt X region (Blight and Rice, 1997; Kolykhalov et al., 1996; Tanaka et al., 1995; Tanaka et al., 1996; Yamada et al., 1996). The variable region follows immediately the termination codon of the HCV polyprotein, and is variable in length (ranging from 27 to 70 nt) and composition among different genotypes. However, it is highly conserved among viral strains of the same genotype (Kolykhalov et al., 1996; Yanagi et al., 1997; Yanagi et al., 1998). Computer analysis has identified two possible stem-loop structures in the variable region, with the first stem-loop extending into the 3' end of the NS5B-coding sequence (Han and Houghton, 1992; Kolykhalov et al., 1996). The poly(U/UC) tract consists of a poly(U) stretch and a C(U)n-repeat region (referred to as the transitional region) and varies greatly in length and slightly in sequence among different viral isolates (Tanaka et al., 1996). The transitional regions of genotypes 2a, 3a, and 3b have several conserved A residues, which are not present in genotypes 1b and 2b (Tanaka et al., 1996; Yamada et al., 1996; Yanagi et al., 1999a). The presence of the polypyrimidine tract within the 3'UTR is unique to HCV and GBVB (Simons et al., 1995) among flaviviruses. The length of this region has been correlated with the replication capability of HCV RNA (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). The X region forms three stable stem-loop structures that are highly conserved across all genotypes (Blight and Rice, 1997; Ito and Lai, 1997; Kolykhalov et al., 1996) (Fig. 1). A recent study of the structure of the X region by chemical and enzyme probing has confirmed the presence of SL1 and SL3, but proposed that the region between the two stem-loops folds into two hairpins instead of one and may further form a hypothetical pseudoknot (Dutkiewicz and Ciesiolka, 2005). On the other hand, the complementary sequence of the X region in this region forms a 3-stemloop structure (Dutkiewicz and Ciesiolka, 2005). There is no poly(A) sequence in the 3'UTR. Instead, the 3'UTR sequence, particularly the X region, is involved in the regulation of translation, much in the same way as the poly(A) sequence in the mRNAs of other RNA viruses. Conceivably, these sequences are involved in the replication, stabilization and also packaging of viral RNA. As a result of the stem-loop formation in the X region, the HCV genome is predicted to end with a double-stranded stem. Examination of the 3'-terminal sequences of the HCV genome in sera from infected patients revealed that most HCV RNAs contain identical 3' ends with no extra sequence downstream of the X tail (Tanaka et al., 1996). However, one particular cDNA clone derived from a patient's serum did contain 2 additional nt (UU), thus generating a single-stranded tail (Yamada et al., 1996). The structure of the exact 3'-end will have implications for the initiation of RNA replication.

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Bioinformatic analysis has revealed the possible presence of additional secondary structures in other parts of the HCV genome (Hofacker et al., 1998). These include possible secondary structures in the core- and NS5B-coding regions (Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002). Consistent with the importance of the predicted secondary structures, it has been shown that synonymous nucleotide mutations are suppressed in the core- and NS5B-coding regions and that compensatory mutations are frequently observed within the predicted stems (Ina et al., 1994; Smith and Simmonds, 1997). The predicted secondary structures within the core-coding region encompass the first 14 nts of the core gene, which form part of the IRES stem-loop IV (Lemon and Honda, 1997). There are two more stem-loops between nt 47 and 167 of the core-coding sequence (nt 391-511 of the genome), which is conserved among all six HCV genotypes (Smith and Simmonds, 1997). This region, corresponding roughly to nt 408-929, has been shown to interact with the 5'UTR, resulting in the reduction of HCV IRES-mediated translation (Honda et al., 1999b). In the NS5Bcoding region (Hofacker et al., 1998; Rijnbrand et al., 2001; Smith and Simmonds, 1997; Tuplin et al., 2002; You et al., 2004), six potential stem-loop structures have been predicted based on computer modeling (You et al., 2004). The functional significance of five of these structures in RNA replication has been implicated from mutational analysis and RNA structure probing in the context of the subgenomic replicon. Of particular interest is a cruciform structure (5BSL3) at the 3' terminus of NS5B, which contains three major stem-loop structures, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Its involvement in RNA replication will be discussed in a later section.

HCV TRANSLATION Translation of the polyprotein from the HCV RNA genome is the first macromolecular synthetic event after the viral RNA is released into the cytoplasm of host cells. It is carried out by a cap-independent mechanism mediated by the highly structured HCV IRES. The HCV genomic RNA serves as an mRNA for the translation of a single polyprotein, which is processed by cellular and viral proteases into at least 10 structural and nonstructural proteins (De Francesco et al., 2000). STRUCTURAL COMPONENTS REQUIRED FOR TRANSLATION

The first 40 nts of the HCV RNA genome, including the first stem-loop domain (domain I), are not required for translation (Honda et al., 1996b; Rijnbrand et al., 1995). Instead, deletion of this domain resulted in a stimulation of translation of a heterologous reporter RNA (Yoo et al., 1992). However, in the context of the HCV subgenomic replicon, deletion of this domain reduced protein expression by 3 to 5 fold (Luo et al., 2003). In addition, a dinucleotide sequence at nt 34-35 has been shown to contribute to the differential translation efficiencies between genotype 54

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Fig. 2. Cis-acting RNA replication regulatory elements in the NS5B-coding region that interact with the 3'UTR (represented by the HCV-Con1 strain). (A) The cruciform structure formed at the end of the NS5B-coding sequence contains 5BSL3.1, 5BSL3.2, and 5BSL3.3, among which 5BSL3.2 is required for HCV RNA replication (You et al., 2004). (B) Kissing-loop interaction between the loop sequences of 5BSL3.2 and SL2 of the X region (Friebe et al., 2005).

1a and 1b isolates (Honda et al., 1999b). It is, therefore, possible that domain I is also involved in the regulation of HCV translation in some fashions. The primary element of the IRES starts at nt 44, which coincides with the 5' border of domain II (Honda et al., 1999a; Honda et al., 1996b; Reynolds et al., 1995; Rijnbrand et al., 1995). However, the precise 3' border of the IRES is controversial. The stem-loop IV of the 5'UTR is predicted to extend into the coding region to include the first 10 nts (nt 345-354) of the core-encoding gene. Indeed, several studies have reported the requirement for a short sequence (up to 30 nt) in the corecoding region for optimal IRES function (Honda et al., 1996a; Hwang et al., 1998; Lu and Wimmer, 1996; Reynolds et al., 1996). However, efficient translation has also been observed with certain reporter genes fused immediately after the start codon, without the core protein-coding sequences (Tsukiyama-Kohara et al., 1992; Wang et al., 1993). The differences in the conclusions may have been due to the assay systems and heterologous reporters employed. It has been found that expression of the reporter gene secretory alkaline phosphatase, but not that of chloramphenicol acetyltransferase, depends on the presence of downstream core-coding sequences (Rijnbrand et al., 2001). Conceivably, the core-coding region may contribute to IRES function by preventing undesirable base pairing of the IRES with other inhibitory sequences or by promoting favorable protein binding to the IRES. This core-coding 55

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region contains an adenosine-rich stretch, which has been shown to recruit a cellular protein that enhances the HCV IRES activity (Kim et al., 2004a; Reynolds et al., 1995). So far, nt 354 is generally regarded as the consensus 3' boundary of the IRES (Honda et al., 1999a), but the sequence immediately downstream of the IRES (up to nt 371) may have a stimulating effect on IRES-directed translation. Interestingly, the core-coding sequences further downstream (near the C-terminal portion) have been shown to play a negative-regulatory role in HCV translation (Ito and Lai, 1999; Kim et al., 2003; Wang et al., 2000). Besides the 5'UTR, the 3'UTR sequences, particularly the X region, may also play a role in HCV RNA translation. It has been shown that HCV RNA containing the X region was translated 3- to 5-fold more efficiently than the corresponding RNAs without this region (Ito et al., 1998). The enhancement of IRES-dependent translation by 3'UTR may be mediated by polypyrimidine tract-binding protein (PTB), which binds to both the 5' and 3'UTR (Ali and Siddiqui, 1995; Ito and Lai, 1997; Tsuchihara et al., 1997). Since PTB can interact with itself, it can potentially mediate circularization of HCV RNA, thereby enhancing translation. The role of the 3'UTR in translation is reminiscent of the poly(A) tail and the poly(A)-binding protein in the translation of poly(A)-containing mRNAs (Kahvejian et al., 2001). However, a different study reported that deletion of the poly(U/UC) tract or the stem-loop 3 of the X region resulted in an enhancement of translation efficiency; the increase in translation was not mediated by PTB (Murakami et al., 2001). Additional studies are required to understand the role of the 3'UTR in IRESmediated translation of HCV proteins. THE HCV TRANSLATION MACHINERY

The HCV IRES is responsible for directing the 40S ribosomal subunit in close contact with the start codon for translation initiation (Lemon and Honda, 1997; Wang et al., 1993). Enzymatic and chemical footprinting and domain-deletion experiments have identified domain II and the basal part of domain III, excluding domain IIIb, as the binding site for the 40S ribosome subunits (Kieft et al., 2001; Kolupaeva et al., 2000; Lukavsky et al., 2000; Pestova et al., 1998). Although the HCV IRES with or without domain II recruits the 40S ribosome subunit with comparable efficiency (Otto et al., 2002), interaction of domain II with the 40S subunit induces or stabilizes the conformational changes within the ribosome and facilitates the 3´ end of the coding RNA to thread into the mRNA entry channel (Spahn et al., 2001). The GGG triplet (nt 266-268) of the hexanucleotide (UUGGGU) apical loop of stem-loop IIId and the pseudoknot are essential for ribosome binding (Kolupaeva et al., 2000). Mutagenesis studies have also confirmed that the GGG triplet is essential for IRES activity both in vitro and in vivo (Jubin et al., 2000).

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The viral 5'UTR forms a binary complex with the 40S ribosomal subunit in the absence of any canonical or non-canonical initiation factors (Pestova et al., 1998). A ribosomal protein S5, in particular, is important for the efficient translation initiation of HCV RNA (Fukushi et al., 1997; Fukushi et al., 2001b; Pestova et al., 1998). Blocking of the S5 binding to HCV IRES interfered with efficient ribosome assembly at the translation initiation site (Ray and Das, 2004). These features suggest that HCV IRES uses the prokaryotic mode for forming the mRNA-40S ribosome complex (Pestova et al., 1998). Several basal translation initiation factors have been reported to be involved in the HCV IRES-mediated translation. The eukaryotic initiation factor-3 (eIF3), alone or together with the 40S ribosome subunit and the eIF2-GTP-initiator tRNA complex, can specifically interact with the HCV IRES stem-loop IIIb in the absence of eIF4A, eIF4B and eIF4F, which are required for ribosomal binding during cap- or EMCV IRES-dependent translation (Kieft et al., 2001; Kolupaeva et al., 2000; Pestova et al., 1998; Sizova et al., 1998). eIF3 binding is not necessary for 40S-HCV IRES assembly, but is essential for the joining of 60S subunit to form the active 80S ribosomal complexes (Pestova et al., 1998). These findings suggest that HCV employs a modified mechanism of IRES-dependent translation. Rabbit reticulocyte lysates depleted of certain translation factors, such as eIF4G, cannot support foot-and-mouth-disease virus IRES-, but still can support HCV IRESdependent translation (Stassinopoulos and Belsham, 2001). eIF2Bγ and eIF2γ have also been identified as cofactors of HCV IRES-mediated translation by a functional genomics approach (Krüger et al., 2000), although their roles in translation have not been established. These findings combined indicate that HCV IRES-dependent translation employs a prokaryotic mode for assembling RNA-ribosome complex and requires only a minimum set of canonical translation factors.

HCV RNA REPLICATION By analogy with other members of the Flaviviridae, HCV is presumed to replicate its genome through the production of a full-length negative-strand RNA. Positivestrand RNAs are then synthesized from the negative-strand template in five- to ten-fold molar excess over the negative-strand RNA (Lohmann et al., 1999) to be used in translation, replication, and packaging into progeny viruses. Since RNA replication has to initiate from the 3'- end of the RNA template of both strands, the corresponding 5' and 3' UTR of HCV RNA genome likely contains the sequences required for the initiation and/or regulation of RNA replication. STRUCTURAL COMPONENTS REQUIRED FOR RNA REPLICATION

Since the 5'UTR is involved in the initiation of both translation and RNA replication, any possible effects of this region on translation will impact RNA replication indirectly and vice versa. Therefore, the direct role of 5'UTR in RNA replication

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is difficult to assess. Separation of RNA replication and translation was initially achieved by inserting the IRES elements of poliovirus or classical swine fever virus between the serially deleted 5'UTR of HCV and the ORF (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). The deletions introduced into the 5'-terminal 40 nt upstream of the IRES region abolished RNA replication but only moderately affected translation. The first 125 nt of the HCV genome, which includes domain I and II of the 5'UTR, was shown to be essential and sufficient for RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003). This region overlaps with the 5'end of the IRES. The replication efficiency of RNA was tremendously increased by the inclusion of the complete 5'UTR (Friebe et al., 2001). Compared with its close relative BVDV, the requirements for RNA sequences or structures within the 5'UTR of HCV appear to be more complex because much longer sequences or particular structures within the IRES are necessary for efficient RNA replication (Frolov et al., 1998; Wilhelm Grassmann et al., 2005). However, further studies are required to show whether the sequences downstream are directly involved in RNA replication or merely contribute to the preservation of the structural and functional integrity of the minimal replication signal. Consistent with a role for the 3' terminal nt of the viral RNA in the initiation of negative-strand RNA, the 3'UTR sequences have been shown to play an essential role in HCV RNA replication in vitro (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a) and in vivo (Kolykhalov et al., 2000; Yanagi et al., 1999b). The 3'UTR sequences were first shown to be required for the replication of HCV RNA when deletion of the 3' terminal sequences destroyed the ability of otherwise infectious synthetic genome-length HCV RNA to initiate infection in intrahepatically inoculated chimpanzees (Kolykhalov et al., 2000; Yanagi et al., 1999b). Using a subgenomic HCV replicon, the 3' terminal RNA signals required for HCV RNA replication were determined to be approximately 225 nt from the 3' end of the genome (Yi and Lemon, 2003a). The 3'-most 150 nt of the genome, which includes the 98-nt X region and the 52 nt of the poly(U/UC) tract, are essential for replication of HCV RNA, while the remaining upstream region of the 3'UTR plays a facilitating role (Friebe and Bartenschlager, 2002; Ito and Lai, 1997; Yi and Lemon, 2003a; Yi and Lemon, 2003b). These results suggest an interesting symmetry in the 5'- and 3'- terminal RNA replication signals since the 5'-most domains I and II of the 5'UTR are essential for replication, while sequences lying further downstream within the 5'UTR help to facilitate replication but are not absolutely required (Friebe et al., 2001; Kim et al., 2002b). The X region interacts with the recombinant HCV RNA polymerase (Cheng et al., 1999; Oh et al., 2000), although other parts of the 3' end of HCV genome may contain additional NS5B-binding sites (Cheng et al., 1999). The NS5B-binding domain within the X region has been mapped to stem II and the single-stranded region connecting stem-loops I and II (Oh et al., 2000). Truncation of 40 nts or more from the 3' end of the X region abolished its template

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activity in vitro (Oh et al., 1999; Oh et al., 2000). A more extensive mutational analysis of the 3'-end 46 nt that form the terminal hairpin (stem-loop I) in the HCV replicon provided strong functional evidence for the existence of the structure and for an essential role of the structure in the replication of HCV RNA (Yi and Lemon, 2003b). It is interesting that the X region is also necessary for efficient translation of HCV protein (Ito et al., 1998); thus, the same set of sequences are involved in both RNA replication and translation. The poly(U/UC) tract is required for HCV RNA replication (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). It is possible that this region assists in circularizing the viral genome, which has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). This sequence binds several cellular proteins (e.g. PTB), which may mediate RNA-RNA interaction (Ito and Lai, 1999) and/or the binding of the replicase complex to RNA. The length of the poly(U/UC) region may influence viral replication as HCV RNA with a longer poly(U/UC) region had a replicative advantage in chimpanzees (Kolykhalov et al., 1997; Yanagi et al., 1999b) than the one with a shorter poly(U/UC). Similar observation was made in the subgenomic replicon RNAs (Friebe and Bartenschlager, 2002; Yi and Lemon, 2003a). Conversely, the poly(U/UC)-rich sequence may serve as a modulator of RNA replication under some conditions, as shown in an in vitro RNA polymerase reaction, in which HCV RNA polymerase stutters at this region (Oh et al., 1999). The sequences within the variable region of the 3'UTR are not essential for RNA replication (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b; Yi and Lemon, 2003a), a finding similar to those of other flaviviruses (Khromykh and Westaway, 1997; Mandl et al., 1998; Men et al., 1996). Interruption of sequence integrity within this region by insertion of the extraneous sequences in this region did not interfere with the replication of the HCV RNA or replicons (Friebe and Bartenschlager, 2002; Yanagi et al., 1999b). Nevertheless, deletions in this region impaired the efficiency of amplification of subgenomic replicons (Yi and Lemon, 2003a). Some of the conserved RNA elements identified in the NS5B-coding region may serve as recognition sites for the HCV replicase complex since partially purified NS5B specifically binds to the coding sequences of NS5B RNA (Cheng et al., 1999), but their involvement in RNA replication has not been established until recently. The NS5B-coding region contains a predicted cruciform structure (5BSL3) consisting of three stem-loops, 5BSL3.1, 5BSL3.2, and 5BSL3.3 (Fig. 2). Mutations disrupting the 5BSL3.2 blocked RNA replication, whereas 5BSL3.1 and 5BSL3.3 were shown not to be required for RNA replication (Friebe et al., 2005; You et al., 2004). Insertion of 5BSL3.2 alone into the variable region of the 3'UTR was sufficient to rescue RNA replication of a replicon in which all three 5BSLs in the

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NS5B-coding region were disrupted, indicating that 5BSL3.2 can act as a cis-acting RNA replication element. This insertion allowed the analysis of individual elements within 5BSL3.2 in more detail without the complication of introducing amino acid changes in the NS5B-coding region (Friebe et al., 2005). 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop; the stem structures, but not their primary sequences, are required for RNA replication (You et al., 2004). In addition, a kissing-loop interaction between a 7-nt-long complementary sequence in 5BSL3.2 and SL2 in the X region has been proven essential for RNA replication (Friebe et al., 2005). In the upper loop of 5BSL3.2, a CACAGC sequence motif is found to be virtually invariant among HCV genotypes and is also present in cis-acting RNA sequences of distantly related flaviviruses, such as Kunjin virus, West Nile virus, or Dengue virus (Markoff, 2003). Given the high genetic conservation in this particular region of the genome, it may be speculated that certain ubiquitously expressed and evolutionarily conserved host cell proteins are involved in the formation of a replication complex that interacts with the 3' end of the flavivirus genome. INITIATION OF NEGATIVE- AND POSITIVE-STRAND RNA SYNTHESIS

Recombinant NS5B proteins are capable of primer-independent initiation of RNA synthesis on a variety of virus-specific and nonspecific RNA templates in vitro (Ferrari et al., 1999; Lohmann et al., 1998; Oh et al., 1999). However, there are conflicting descriptions of the precise initiation site of negative-strand RNA transcription on the HCV-specific templates (Hong et al., 2001; Kim et al., 2002a; Oh et al., 2000; Shim et al., 2002). Oh et al. reported that the transcription of the negative-strand RNA was initiated within the loop sequence of the 3'X stem-loop I, at approximately 21 nt from the 3' end of the RNA (Oh et al., 2000). Kim et al. reported that transcription initiated further downstream, within the 3' stem sequence of SL1 (Kim et al., 2002a). Shim et al. has shown that transcription can be initiated by a recombinant NS5B polymerase in vitro at the 3' end of short oligonucleotide templates representing the 3' terminus of the positive-strand genomic RNA (Shim et al., 2002). The terminal U is preferred as the initiation nt (Shim et al., 2002), which is confirmed by the study of a subgenomic replicon (Yi and Lemon, 2003b). Hong et al. proposed that a unique β-hairpin within the thumb domain of the NS5B polymerase positions the terminal sequences of the genome so as to initiate de novo transcription from the 3' terminal nucleotides (Hong et al., 2001). It was proposed that the β-hairpin ensures the initiation of de novo RNA synthesis at the 3' terminus by preventing movement of the 3' end of the single-stranded RNA template into the active site of the enzyme. Conceivably, the presence of other viral and cellular proteins may affect the selection of the initiation point of RNA replication in vivo.

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The initiation of the positive-strand RNA synthesis has not been as well studied. Conceivably, the 3' terminus of the negative-strand RNA is essential for positivestrand RNA replication. In vitro replication studies using recombinant NS5B showed that the minimal RNA fragment required for efficient replication of the negative-strand RNA spans nt –239 to –1 (Oh et al., 1999), which is complementary to domains I to III of the 5'UTR. Various site-specific mutation studies on the 5'UTR of the HCV replicons have revealed the importance of these regions on HCV genome replication. However, these studies did not distinguish their effects on either positive- or negative-strand RNA synthesis (Friebe et al., 2001; Kim et al., 2002b). The predicted secondary structures of positive- or negative-strand RNA of 5'UTR are slightly different (Schuster et al., 2002). In in vitro RNA synthesis using the full-length HCV RNA as the template, the NS5B polymerase is capable of positive-strand RNA synthesis, continuing from the 3' end of the full-length negative-strand RNA product, resulting in a dimeric hairpin HCV RNA (Oh et al., 1999). The significance of such a product is not clear. THE HCV RNA REPLICATION MACHINERY

HCV RNA replication is believed to occur in the cytoplasm of virus-infected cells based on the cytoplasmic localization of viral RNA (Gowans, 2000) and polymerase (Hwang et al., 1997; Selby et al., 1993). RNA is synthesized by a membrane-associated replication complex that includes the HCV RNA-dependent RNA polymerase (RdRP) NS5B, most of the other viral NS proteins (NS3, NS4A, NS4B, and NS5A), and possibly cellular proteins (Asabe et al., 1997; Bartenschlager et al., 1995; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Among the viral NS proteins, NS4B protein by itself induces membranous alterations that morphologically resemble the membranous webs found in replicon cells where viral RNA replication takes place (Egger et al., 2002; Gosert et al., 2003; Shi et al., 2003). A variety of biochemical evidence suggests that NS4B anchors the formation of the RNA replication complex (Gao et al., 2004), which is formed on the detergent-resistant membrane structures containing cholesterol-rich lipid rafts (Aizaki et al., 2004; Shi et al., 2003). Interestingly, all the nonstructural proteins, except NS5A, have to be translated in cis from the ORF of the very RNA molecule in order for RNA replication to occur (Appel et al., 2005). This finding suggests that the viral proteins are assembled in an ordered and sequential way into the replication complex soon after translation. The only trans-acting protein NS5A may enter the replication complex by binding to a cellular protein, VAP-33 (see below). From the replicon studies, it appears that the RNA replication requires all the HCV nonstructural proteins except NS2. The NS3 is directly involved in RNA synthesis probably through its helicase function. The RNA helicase function is presumed to be necessary for unwinding the secondary structures of RNA template and to separate

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the positive- and negative-strand HCV RNA during replication. The HCV helicase lies within the C-terminal half of NS3, which has been shown to possess NTPase, single-stranded (ss) polynucleotide binding, and duplex-unwinding activities (Kim et al., 1995; Tai et al., 1996). NS3 alone has only a weak RNA unwinding activity, which can be significantly enhanced by the presence of NS4A (Pang et al., 2002). The resolution of the crystal structure of NS3 either alone or complexed with deoxyuridine octamer has provided additional insights into the mechanism of the HCV NS3 helicase function (Cho et al., 1998; Kim et al., 1998; Yao et al., 1999). NS5B is a membrane-associated phosphoprotein (Hwang et al., 1997), which contains signature motifs, such as the GDD, shared by other viral RdRps (Koonin, 1991). The C-terminal 21 aa of NS5B plays a role in anchoring the protein to the membrane (Yamashita et al., 1998) but also plays a direct role in RNA synthesis (Lee et al., 2004b; Vo et al., 2004). NS5B also interacts with a SNARE-like cellular membrane protein, human vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (hVAP-33), which may directly or indirectly target the polymerase to the RNA replication site (Gao et al., 2004; Tu et al., 1999). Reduction of hVAP-33 expression either by dominant-negative mutants or small interfering RNA (siRNA) of hVAP-33 blocked the association of NS5B with detergent-resistant membranes and led to an inhibition of HCV RNA replication (Gao et al., 2004; Zhang et al., 2004). Although multiple potential phosphorylation sites exist within the NS5B aa sequence, no site is conserved among all HCV isolates examined (Altschul et al., 1997), suggesting that phosphorylation of NS5B may vary among different isolates. Screening of a phage-display library with HCV NS5B protein as bait has identified one peptide with amino acid sequences homologous to protein kinase C-related kinase 2 (PRK2) (Kim et al., 2004b). In vitro analysis has revealed that PRK2 binds and phosphorylates the N-terminal region of NS5B. Further studies in the subgenomic replicon system have indicated that phosphorylation of NS5B by PRK2 is involved in the regulation of HCV RNA replication. It is not clear whether this phosphorylation has an effect on the NS5B polymerase activity and whether it is conserved among different isolates. The crystal structure of NS5B shares significant similarity to those of other polymerases, but also displays certain striking differences (Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., 1999). The domain organization in NS5B can be subdivided into the fingers, palm and thumb, similar to other polymerases. However, as other polymerases, such as the poliovirus 3D polymerase, are distinctly U-shaped, the fingers and the thumb domains of NS5B exhibit extensive contacts between each other, resulting in a globular-shaped molecule. The encircled active site is relatively inflexible and can accommodate only a template:primer duplex

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without global conformational changes. The C terminus of NS5B (excluding the hydrophobic tail) is present in the active site of the protein and has been hypothesized to play a role in the regulation of RdRp activity and template discrimination (Ago et al., 1999). In in vitro RdRP assays, NS5B often uses the 3' end of the template RNA or an artificial oligonucleotide as a primer. (Al et al., 1998; Behrens et al., 1996; De Francesco et al., 1996; Ferrari et al., 1999; Lohmann et al., 1997; Yamashita et al., 1998; Yuan et al., 1997). However, it can also initiate de novo RNA synthesis in a primer-independent manner (Luo et al., 2000; Oh et al., 1999; Sun et al., 2000; Zhong et al., 2000). NS5B binds in vitro preferentially to several regions in the 3'-end of HCV RNA, including the 3'-coding region of NS5B, the U/UC-rich sequence, and part of the X region (in the stem I and II) (Fig. 1) (Cheng et al., 1999; Oh et al., 2000). Partial deletion of the 3'UTR of HCV RNA abolished the template activity of the RNA (Cheng et al., 1999; Oh et al., 2000). Thus, it appears that NS5B recognizes some specific sequence or structural elements at the 3' end of HCV RNA (Cheng et al., 1999; Oh et al., 2000). Once it binds the stem structure of the 3'UTR, however, NS5B initiates RNA synthesis only from the single-stranded RNA region closest to the 3' end of the template (Oh et al., 2000). This conclusion is supported by another study showing that the RdRp reaction mediated by NS5B requires a stable secondary structure and a single-stranded sequence with at least one 3'-end cytidylate in the RNA template (Kao et al., 2000). Since the 3' end of HCV RNA ends with a near-perfect double-stranded stem (stem I) (Fig. 1), then how does HCV RNA synthesis initiate in vivo, if the in vitro mechanism reflects the mechanism of RNA synthesis in vivo? There are several potential mechanisms whereby the 3' end sequence of the viral RNA is retained during RNA replication: (1) The 3' end of HCV RNA may be extended by a terminal transferase so that there is a single-stranded tail at the 3' end to allow NS5B to initiate from the precise 3'-end. Indeed, an HCV cDNA clone containing two additional nt (UU) at the 3'-end of HCV RNA has been detected (Yamada et al., 1996). (2) RNA helicase or unwinding proteins may be present in the HCV replicative complex to unwind the 3'-end stem structure into the single-stranded region. (3) RNA synthesis may initiate internally in the single-stranded region within the 3'UTR; the 3'-end sequence may be recovered during the positive-strand RNA synthesis since the complementary sequence can be made by fold-back RNA synthesis. (4) The presence of other viral or cellular proteins may alter the choice of the initiation site of RNA replication. HCV RdRp activity has been detected in the crude replication complexes prepared from lysates of cells carrying HCV replicons. This lysate can synthesize RNA from the endogenous template, but not exogenously added templates, and requires both

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NS5B and NS3 (Ali et al., 2002; Hardy et al., 2003; Lai et al., 2003). The whole complex is localized on the detergent-resistant membrane and contains all the nonstructural proteins of HCV. The viral RNA is enclosed within the membrane complex and shielded from outside. All the nonstructural proteins are probably anchored on the membrane structures by a series of protein-protein interactions between them and with a cellular protein hVAP-33 (Tu et al., 1999). It has been shown that most of the HCV NS proteins, including NS3, NS4A, NS4B, NS5A, and NS5B, can interact with each other either directly or indirectly (Asabe et al., 1997; Bartenschlager et al., 1995; Gao et al., 2004; Ishido et al., 1998; Lin et al., 1997; Tu et al., 1999). Interestingly, while NS5B interacts with the N terminus of hVAP-33, NS5A binds the C terminus of hVAP-33. The importance of NS5A in HCV replication has been further suggested by the detection of a number of adaptive mutations clustered in a defined region of NS5A in a subgenomic HCV replicon (Blight et al., 2000). It is conceivable that this region may mediate the interaction of NS5A with a cellular protein that inhibits HCV replication. Further evidence supporting the existence of a replication complex consisting of multiple HCV NS proteins came from an analysis of the adaptive mutations derived from a subgenomic HCV replicon (Lohmann et al., 2001). An adaptive mutation in NS5B was found incompatible with those in NS5A or NS4B when introduced back into the same replicon. These mutations may affect contact sites between these proteins in the replication complex, resulting in a dramatic reduction in replication efficiency.

REGULATION OF HCV TRANSLATION AND REPLICATION The 5' and 3' UTRs are clearly the sites of important events leading to the onset of translation and replication of HCV RNA. The binding of viral or cellular proteins to the UTRs may modulate the secondary and/or tertiary structure of the viral RNA to facilitate its recognition by the translation machinery and/or the replicase complex. These proteins may recruit additional cellular factors and mediate longrange cross-talks between the ends of HCV RNA. REGULATION OF TRANSLATION BY VIRAL AND CELLULAR PROTEINS

The HCV IRES-mediated translation is relatively inefficient as compared to that of other viruses (Borman et al., 1995). It has been suggested that HCV has a selfmodulating mechanism to maintain a low level of replication and translation that may promote viral persistence. In this regard, it was speculated that domain IV of the IRES may be stabilized by interaction with the viral core protein, resulting in translation inhibition (Honda et al., 1996a). It has indeed been shown that the core protein binds to several sites within HCV IRES, thereby inhibiting translation (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). However, other studies have suggested that the core-coding sequence, rather than the core protein itself, is responsible for the suppression of IRES-mediated translation, possibly through long-range RNA-RNA interactions with the 5'UTR (Kim et al., 2003; Wang et al.,

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2000). The sites of RNA-RNA interaction have been mapped to nt 24-38 within the 5'UTR and nt 428-442 of the core-coding sequence (Kim et al., 2003), which is part of a stem-loop structure (Wang et al., 2000). The stem-loop IV of the IRES may be one of the candidates for feedback control, since the stabilization of this structure can reduce IRES activity and the primary sequence within this stemloop is conserved in nearly all HCV strains (Honda et al., 1996a). However, these conflicting reports may have been due to the different reporter RNA constructs used in the different studies since the stable RNA structure assumed by some heterologous sequences fused directly at the initiation codon may be detrimental to translation directed by IRES (Rijnbrand et al., 2001). Furthermore, a cellular protein PTB binds to the 3'-end of the core-coding region and negatively regulates HCV translation (Ito and Lai, 1999). Thus, translation can be regulated by multiple RNA segments and viral proteins. Several other HCV proteins, E2 (Taylor et al., 1999) and NS5A (Gale et al., 1997; He et al., 2003), may have an indirect effect on HCV translation by inhibiting PKR, but the biological significance of this effect is not clear. Besides the canonical translation factors, such as the 40S ribosomal subunit and eIF3, the HCV IRES also recruits noncanonical cellular translation factors, such as La autoantigen (Ali and Siddiqui, 1997) and PTB (Ali and Siddiqui, 1995), which may regulate translation (Fig. 3). The La antigen is an RNA-binding protein belonging to the RNA recognition motif (RRM) superfamily (Gottlieb and Steitz, 1989). It has been implicated in various cellular processes (Ford et al., 2001; Gottlieb and Steitz, 1989) and the translation initiation of picornaviruses and flaviviruses (Ray and Das, 2002; Wolin and Cedervall, 2002). The La antigen recognizes the intact HCV IRES structure and significantly augments the IRES-directed translation in vitro (Ali and Siddiqui, 1997; Costa-Mattioli et al., 2004; Pudi et al., 2003; Pudi et al., 2004). Inhibition of HCV IRES activity caused by sequestration of La protein can be rescued by the addition of purified La protein (Das et al., 1998; Izumi et al., 2004). La protein binds to the GCAC motif near the initiator AUG within stem-loop IV (Pudi et al., 2003). Mutations in the GCAC, which alter the primary sequence while retaining the overall secondary structure, affect the binding of La protein to HCV IRES and significantly inhibit IRES-mediated translation both in vitro and in vivo (Pudi et al., 2004). It has been suggested that the nucleic aciddependent ATPase activity of La may promote the transformation of stem-loop IV into single-stranded conformation, which is favorable for 40S ribosome binding and the formation of active initiation complex (Lemon and Honda, 1997; Pudi et al., 2004). In addition, La protein may enhance the binding of the ribosomal protein S5 to HCV IRES, which, in turn, facilitates the formation of the IRES-40S complex (Pudi et al., 2004). A recent study suggests that La antigen may also be involved in HCV RNA replication (Domitrovich et al., 2005).

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Fig. 3. Cellular proteins that interact with HCV RNA. The 5'UTR interacts with a basal translation factor (eIF3), noncanonical translation factors (PTB and La), and other cellular proteins that may regulate translation (hnRNP L and PCBP). The numbers in parentheses represent the nt sequence in the HCV genome, where the proteins bind. PTB has three distinct binding sites in the 5'UTR, whereas hnRNP L interacts with a region immediately downstream of the AUG codon. Both La autoantigen and PCBP recognize the entire 5'UTR. There is a PTB-binding site in the core-coding region, which plays a negative regulatory role in HCV translation. The 3'UTR is bound by a variety of proteins, all of which interact with the poly(U/UC) region. PTB also binds the X region. The length of poly(U/UC) affects the replication efficiency (Friebe and Bartenschlager, 2002; Kolykhalov et al., 1997; Yanagi et al., 1999b; Yi and Lemon, 2003a). These 5'UTR- and 3'UTR-binding proteins may affect viral replication (HuR, hnRNP C and GAPDH), translation (PTB), or RNA stability (La). VR, variable region.

PTB interacts with three distinct pyrimidine-rich sequences within the HCV IRES (Ali and Siddiqui, 1995) (Fig. 3). The interaction of PTB with domain III of the IRES has been confirmed by electron microscopy analysis (Beales et al., 2001). Immunodepletion of PTB results in the loss of IRES-directed translation, which, however, cannot be restored by the addition of purified PTB, suggesting that additional factors tightly associated with PTB are also required to enhance IRES activity (Ali and Siddiqui, 1995). In addition to the IRES, PTB has also been shown to interact with the 3' X region (Ito and Lai, 1997; Tsuchihara et al., 1997) and to enhance HCV IRES-mediated translation (Ito et al., 1998). This long-range effect suggests that the HCV 5' and 3'UTR may interact with each other through PTB or other viral or cellular proteins. Furthermore, the presence of RNA aptamers of PTB inhibited HCV IRES translation (Anwar et al., 2000). In contrast, results obtained in a study of the subgenomic replicon system do not support a significant role of PTB in HCV replication (Tischendorf et al., 2004). However, PTB has been found in the detergent-resistant membrane complex in cells harboring the HCV subgenomic replicon, while it is in the detergent-sensitive membrane in 66

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the control cells, indicating the recruitment of PTB to the HCV RNA replication complex; knockdown of PTB inhibited HCV RNA replication (Domitrovich et al., 2005)(Aizaki and Lai, unpublished). Besides PTB, also known as heterogeneous nuclear ribonucleoprotein I (hnRNP I), several other proteins of the hnRNP family have been shown to interact with HCV IRES. hnRNP L specifically interact with the 3' border of the HCV IRES in the core-coding sequence; the binding correlates with the translation efficiency from the IRES (Hahm et al., 1998). The mouse minute virus nonstructural protein NS1associated protein 1 (NSAP1) (Harris et al., 1999), a homolog of hnRNP R, also known as SYNCRIP (Synaptotagmin-binding cytoplasmic RNA-interacting protein) (Hassfeld et al., 1998), was recently shown to enhance IRES-dependent translation through the interaction with an adenosine-rich region in the 5'-proximal region of the core-coding sequence (Kim et al., 2004a; Reynolds et al., 1995). This protein appears to be involved in RNA replication as well (Choi and Lai, unpublished observation). Poly(rC)-binding protein (PCBP), which is also known as hnRNP E and involved in the expression regulation of numerous cellular and viral RNAs (Ostareck-Lederer et al., 1998), interacts with the HCV 5'UTR (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). PCBP has been implicated in the regulation of poliovirus IRES activity by binding to the 5'UTR of the viral genome (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). However, the specific interaction of PCBP-2 with the 5' terminal domain I of HCV RNA has no effect on IRES-mediated translation (Fukushi et al., 2001a). Consistent with the role of domain I in RNA replication (Friebe et al., 2001; Kim et al., 2002b; Reusken et al., 2003), PCBP-2 may be involved in the replication rather than translation of HCV RNA. Using a functional genomics approach, the proteasome α-subunit PSMA7 has been shown to be involved in IRES-mediated translation, but it is unknown whether the protein acts directly on IRES or indirectly through the regulation of other cellular proteins (Kruger et al., 2001). In summary, multiple cellular proteins binding to the 5' or 3'UTR can regulate HCV translation; some of them regulate both translation and RNA replication. REGULATION OF RNA REPLICATION BY VIRAL AND CELLULAR PROTEINS

All the viral nonstructural proteins except NS2 are required for replication, but the modes of their participation are not clear. Adaptive mutations in the HCV replicons that allowed the replicons to enhance replication efficiencies have been detected in all of the viral NS proteins, particularly NS3 and NS5A, indicating that every viral nonstructural protein (except NS2) contributes to RNA replication. The purified recombinant HCV NS3 protein or its helicase domain alone can interact efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal 67

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RNA sequences (Banerjee and Dasgupta, 2001). Specific interaction of NS3 with the 3'-terminal sequences of the positive-strand RNA appears to require the entire 3'UTR. A predicted stem-loop structure present at the 3' terminus (nt 5 to 20 from the 3' end) of the negative-strand RNA, particularly the three G-C pairs within the stem, appears to be important for NS3 binding to the negative-strand UTR. This interaction may anchor RNA-protein complexes to the cytoplasmic membrane where viral replication complexes are formed. The poly(U/UC)-rich region of the 3'UTR is a hot spot in the HCV genome for binding cellular proteins (Fig. 3), two of which are the Drosophila melanogaster embryonic lethal, abnormal visual system (ELAV)-like RNA-binding protein, HuR, and hnRNP C (Gontarek et al., 1999; Spångberg et al., 2000). Both HuR and hnRNP C interact with the 3' ends of both the positive- and negative-strand HCV RNA. Due to its pyrimidine-rich nature, it is not surprising that the poly(U/UC)-rich region has been identified to interact with PTB (Gontarek et al., 1999; Luo, 1999). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) also interacts with the poly(U/UC) tract (Petrik et al., 1999), but the functional relevance of this interaction has yet to be determined. Based on studies of hepatitis A virus (HAV), the binding of GAPDH to the 5'UTR of HAV may directly influence IRES-dependent translation and/or replication of viral RNA by destabilizing the folded structure of the stemloop IIIa of HAV IRES and competing with PTB for the binding to this structure (Schultz et al., 1996; Yi et al., 2000). The 3'UTR has also been shown to bind La autoantigen, which protects the HCV RNA from rapid degradation (Spångberg et al., 2001). Although the role of these proteins in HCV RNA replication has not be characterized, a group of host factors that bind to the 3'UTR of the closely related pestivirus BVDV has been shown to be required for viral RNA replication (Isken et al., 2003). It is conceivable that these cellular proteins are involved in not only RNA replication but translation as well, possibly through the 5' and 3' UTR interaction, causing the circularization of the viral RNA. In addition to viral and cellular proteins, a liver-specific cellular microRNA, miR122, was suggested by a recent study to regulate HCV RNA replication by directly interacting with the 5'UTR (Jopling et al., 2005). A 7-nt sequence (ACACUCC) complementary to the seed sequence of miR-122 was found in both the 5' and 3' UTRs and predicted to be potential binding sites for miR-122. Disruption of sequence complementarity between the 5'UTR, but not the 3'UTR, and miR-122 reduced HCV RNA replication without affecting RNA stability or translation. It is speculated that miR-122 may aid in RNA folding or RNA sequestration in replication complexes. Since miR-122 is expressed in Huh7 but not HepG2 cells, it may also play a role in host-range determination.

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Translation of vast majority of eukaryotic mRNAs, which are capped at the 5' end and polyadenylated at the 3' end, has been shown to adopt a closed-loop mechanism, in which the mRNAs are circularized via a 5'-3' interaction mediated by the capbinding proteins, eIF4F and eIF4G, and the poly(A)-binding protein, PABP. The eIF4G-PABP interaction has also been shown to be required for poly(A)-mediated stimulation of picornaviral IRES-dependent translation, indicating that the 5'-3' crosstalk is mechanistically conserved between classical eukaryotic mRNAs and picornaviral RNA (Herold and Andino, 2001; Michel et al., 2001; Michel et al., 2000). Circularization has been shown to be important for efficient RNA replication of other flaviviruses (Khromykh et al., 2001). Even in the absence of a poly(A) tail in HCV RNA, the closed-loop model may still be preserved in HCV IRES-mediated translation by the presence of RNA sequences and proteins that can functionally replace the poly(A) tail and PABP (Ito and Lai, 1999). Indeed, the X region of the 3'UTR has been shown to bind PTB and enhance translation of HCV RNA (Ito et al., 1998), suggesting that the functions of the X region may be similar to that of poly(A) in eukaryotic mRNA translation (Kahvejian et al., 2001). Since PTB also interacts with the 5'UTR, it may mediate crosstalk between the 5'- and 3'-ends of HCV RNA. Thus, the mechanism of translation enhancement by PTB may be similar to that of eIF4G-PABP in the translation of cellular and viral RNAs that contain a poly(A) tail. SWITCH BETWEEN TRANSLATION AND RNA REPLICATION

Since RNA replication and translation occur on the same RNA molecules, the question arises how these two processes are coordinated. For some RNA viruses, there is evidence of coupling between RNA replication and translation. For example, poliovirus defective-interfering RNA without a translatable ORF can not replicate; the nature of the protein product is not critical, but the translatability is essential (Collis et al., 1992; Hagino-Yamagishi and Nomoto, 1989; Novak and Kirkegaard, 1994). This requirement has been demonstrated for several other viral RNAs, such as clover yellow mosaic virus RNA (White et al., 1992), Kunjin virus (Khromykh et al., 2000), and rubella virus (Liang and Gillam, 2001). In coronavirus, the cisacting protein appears to confer a replication advantage to the RNA; the longer the ORF, the more robust the RNA replication is (de Groot et al., 1992; Kim et al., 1993; Liao and Lai, 1995). The mechanism of the coupling of these two processes is not yet clear. However, there are also viral RNAs (e.g., vesicular stomatitis virus, influenza virus, Sindbis virus) whose replication does not depend on translation of the ORF on the same RNA. In any case, translation and replication must be separated since translation goes in the 5' to 3' direction, whereas negative-strand RNA synthesis goes from 3' to 5' on the same positive-strand RNA template. When the translation machinery meets the replication complex in opposite direction, there must be a mechanism to prevent confrontation. The situation is akin to the separation of transcription and replication of cellular DNA. 69

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In HCV, the 5' and 3'UTR sequences are involved in the regulation of both translation and RNA replication. There is substantial overlap in the UTR regions required for translation and RNA replication. Nevertheless, the structural and sequence requirement for these two processes may be different. It is conceivable that the structural changes involved in translation and RNA replication may be effected by the viral or cellular proteins binding to these regions. Indeed, several cellular proteins binding to the 5' and 3'UTR of HCV have been shown to affect both translation and replication. In poliovirus, a switch between translation and RNA replication has been proposed to be controlled by PCBP, which enhances translation by binding to the 5'-terminal cloverleaf structure of the poliovirus RNA, and the viral 3CD polymerase, which promotes negative-strand RNA synthesis by binding to the same RNA structure, possibly by altering the structure of this region (Gamarnik and Andino, 1998; Gamarnik and Andino, 2000). Interestingly, PCBP-1 and 2 have also been shown to interact with the HCV 5'UTR, with PCBP-2 binding particularly to stem-loop I, suggesting a possibly similar role of these proteins in regulating a switch between HCV RNA replication and translation (Fukushi et al., 2001a; Spångberg and Schwartz, 1999). In addition, the HCV core protein may also be involved in the switch by down-regulating IRES-dependent translation as a regulatory mechanism required for the initiation of RNA replication (Li et al., 2003; Shimoike et al., 1999; Zhang et al., 2002). Since many of the cellular proteins binding to the 5' and 3'UTR of HCV have been reported to regulate both translation and replication, it is conceivable that the relative ratios of the different proteins may control the switch between translation and replication. Furthermore, the HCV RNA elements required for translation and those for replication partially overlap. So, the key question in this regard is how the structures of these elements are altered by RNA-RNA or protein-RNA interactions so that the RNA can be properly directed to be used for translation or replication. Alternatively, an entirely different mechanism may operate to regulate translation and RNA replication of HCV. The RNA replication complex has been shown to reside in the cholesterol-rich, detergent-resistant membrane complex (Aizaki et al., 2004; Shi et al., 2003), whereas translation occurs on the detergent-sensitive, endoplasmic reticulum membrane. Thus, there may be separate machineries in different subcellular compartments for these two processes. The different viral and cellular proteins may bind to RNA molecules differentially in these two different compartments. The key question in this regard is how the RNA is transported from the replication complex to the site of translation or vice versa so that these two functions can be separated.

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PERSPECTIVES The 5' and 3'UTR are the most conserved regions of HCV RNA and play key roles in regulating translation and RNA replication. The knowledge on these two processes is still rudimentary, but the development of subgenomic and genomic replicons and the infectious culture systems (Lohmann et al., 1999; Wakita et al., 2005) provides promises for the unraveling of these two processes in the near future. These two regions also offer promising targets for developing antiviral agents. Within the past two years, small molecule inhibitors of the NS3 protease and the RNA-dependent RNA polymerase have been shown in early clinical studies to be efficacious in both treatment-naïve patients and patients who failed interferon therapy. However, the extensive genetic heterogeneity of HCV RNA and the rapid evolution of quasispecies present a substantial challenge for these inhibitors to broad-spectrum activity. The high degree of sequence conservation in the 5'UTR and 3'UTR among different HCV genotypes makes these regions attractive targets for antiviral therapies, such as antisense oligonucleotides (Soler et al., 2004), ribozymes (Welch et al., 1996; Welch et al., 1998), and siRNAs (Kronke et al., 2004; Randall and Rice, 2004). The inhibition of HCV RNA translation or replication has been observed with these inhibitors that target the 5'UTR alone or together with the core-coding sequence of HCV (Hanecak et al., 1996; Kronke et al., 2004; Macejak et al., 2000; McCaffrey et al., 2003; Ohkawa et al., 1997; Sakamoto et al., 1996). Universal siRNAs targeting similar regions have been generated and proven to be effective against all known genotypes (Kronke et al., 2004; Yokota et al., 2003). Encouragingly, early clinical trials have demonstrated efficacy of some of these inhibitors in HCV-infected patients despite the limitations associated with RNA-based therapies and the inherent structures of the UTR sequences (Branch, 1998; Crooke and Bennett, 1996; Gomez et al., 2004). The interventions directing against conserved domains of viral RNAs may provide valuable alternatives to small molecule inhibitors that target HCV proteins.

REFERENCES 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 Fold Des 7, 1417-1426. Aizaki, H., Lee, K.J., Sung, V.M., Ishiko, H., and Lai, M.M. (2004). Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts. Virology 324, 450-461. Al, R.H., Xie, Y., Wang, Y., and Hagedorn, C.H. (1998). Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli. Virus Res 53, 141-149.

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(2000). Inhibition of hepatitis C virus (HCV)-RNA-dependent translation and replication of a chimeric HCV poliovirus using synthetic stabilized ribozymes. Hepatology 31, 769-776. Mandl, C.W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P.F., Allison, S.L., and Heinz, F.X. (1998). Spontaneous and engineered deletions in the 3' noncoding region of tick- borne encephalitis virus: construction of highly attenuated mutants of a flavivirus. J Virol 72, 2132-2140. Markoff, L. (2003). 5'- and 3'-noncoding regions in flavivirus RNA. Adv Virus Res 59, 177-228. McCaffrey, A.P., Meuse, L., Karimi, M., Contag, C.H., and Kay, M.A. (2003). A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice. Hepatology 38, 503-508. Men, R., Bray, M., Clark, D., Chanock, R.M., and Lai, C.J. (1996). Dengue type 4 virus mutants containing deletions in the 3' noncoding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70, 3930-3937. Michel, Y.M., Borman, A.M., Paulous, S., and Kean, K.M. (2001). Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation. Mol Cell Biol 21, 4097-4109. Michel, Y.M., Poncet, D., Piron, M., Kean, K.M., and Borman, A.M. (2000). Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation. J Biol Chem 275, 32268-32276. Miller, R.H., and Purcell, R.H. (1990). Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci USA 87, 2057-2061. Muerhoff, A.S., Leary, T.P., Simons, J.N., Pilot-Matias, T.J., Dawson, G.J., Erker, J.C., Chalmers, M.L., Schlauder, G.G., Desai, S.M., and Mushahwar, I.K. (1995). Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis. J Virol 69, 5621-5630. Murakami, K., Abe, M., Kageyama, T., Kamoshita, N., and Nomoto, A. (2001). Down-regulation of translation driven by hepatitis C virus internal ribosomal entry site by the 3' untranslated region of RNA. Arch Virol 146, 729-741. Novak, J.E., and Kirkegaard, K. (1994). Coupling between genome translation and replication in an RNA virus. Genes Dev 8, 1726-1737. Oh, J.W., Ito, T., and Lai, M.M. (1999). A recombinant hepatitis C virus RNAdependent RNA polymerase capable of copying the full-length viral RNA. J Virol 73, 7694-7702. Oh, J.W., Sheu, G.T., and Lai, M.M. (2000). Template requirement and initiation site selection by hepatitis C virus polymerase on a minimal viral RNA template. J Biol Chem 275, 17710-17717. 81

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Chapter 3

Assemble and Interact: Pleiotropic Functions of the HCV Core Protein Stephen J. Polyak, Kevin C. Klein, Ikuo Shoji, Tatsuo Miyamura and Jaisri R. Lingappa

ABSTRACT While surrogate capsid assembly model systems are currently the best tools for studying HCV core assembly, bona fide HCV culture systems are being developed. The time will soon come when HCV culture systems and small animal models will be the norm, rather than the exception (see Chapters 12 and 16). It is now clear that HCV core protein interacts with many cellular proteins and signal transduction pathways, that HCV quasispecies influence biologic responses, and HCV proteins such as core can have different effects depending on whether the protein is encountered inside or outside the cell. The studies discussed herein have enhanced the understanding of HCV capsid assembly and the role(s) of HCV core and host cell interactions in the establishment of persistent infection and the pathogenesis of HCV liver disease. Continued studies of this nature will also provide a basis for the rational design of vaccines and novel therapeutics against HCV infection in humans.

INTRODUCTION As covered elsewhere in this book, HCV infection is a serious global health problem, which accounts for billions of dollars in medical expenses in the US alone (Kim, 2002). Clinically, acute HCV infection is frequently anicteric and asymptomatic. The situation is compounded given the natural tendency for acute HCV infection to progress to chronic infection. Thus, more effective strategies to successfully cure patients of their infection are urgently needed. This chapter focuses on a key HCV molecule, the HCV core or nucleocapsid protein.

THE CORE OF THE PROBLEM The HCV core protein has been reported to have many functions. With respect to the virus, the main function of the core protein is to form the capsid shell that will house and protect the HCV genomic RNA while the virus passes from one cell to another, or from one person to another. However, the HCV core protein also modulates many different host pathways by interacting with a variety of cellular factors. In the following sections, we will highlight important new developments in HCV capsid assembly and HCV core-host interactions. 89

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THE ROLE OF HCV CORE IN CAPSID ASSEMBLY WHAT IS A CAPSID?

A viral capsid is the protein shell that encapsidates and protects the viral genome. Viral capsids can be composed of one or more virus-encoded proteins. In the case of enveloped viruses, after assembling and encapsidating the genomic RNA, a viral capsid then facilitates virion formation by interacting with the viral envelope glycoproteins and budding. The budding process is sometimes, but not always, mediated by the viral capsid. For example, the capsid proteins of Ebola and HIV contain domains that regulate budding, while in the case of tick borne encephalitis (TBE) virus, it is the envelope glycoproteins that mediate budding. These events (capsid assembly, encapsidation, and budding) are typically referred to as late events in the viral life cycle. For HCV, as will be discussed below, many details of the late events in the HCV life cycle are unclear. In the case of HCV, as is true for all members of the Flaviviridae, the core protein is the only viral protein present in the capsid. The final nucleocapsid contains genomic RNA, coated and protected by the capsid. HCV, being an enveloped virus, has a lipid envelope, containing the viral envelope glycoproteins as well as host membrane proteins, surrounding the nucleocapsid. The late events of the HCV life cycle, including capsid and virion assembly, are shown schematically in Fig. 1. In this section, we will focus on HCV core, its characteristics, what is known about its assembly into a bona fide HCV capsid, and the blocks to HCV capsid assembly that exist in mammalian cell culture systems. PROPERTIES OF HCV CORE

The HCV genomic RNA is approximately 9.6 kilobases in length and encodes a single, large polyprotein of about 3000 amino acids (aa). The polyprotein is cleaved by viral and cellular proteases to generate at least 10 viral proteins (Suzuki et al., 1999). The core protein represents the first protein in the polyprotein, followed by two glycoproteins, E1 and E2. The immature form of HCV core contains 191 aa. These 191 aa have been separated into three general domains (McLauchlan, 2000). The first domain (domain I), encompassing aa 1 - ~122, is highly basic and very hydrophilic. This domain is thought to be responsible for binding RNA and mediating capsid assembly, and has been reported to interact with many cellular proteins. The second domain encompasses the majority of the C-terminus of HCV core. In contrast to domain I, domain II is hydrophobic. Thus, domain II mediates interactions with lipids and membrane proteins and is not present in capsid proteins of most other viruses in the Flaviviridae. The final domain (III), which is very hydrophobic and is predicted to form an alpha helix, is at the extreme C-terminus of the immature core protein, and corresponds to the signal sequence for E1 (aa 175 - 191). This domain is cleaved soon after core is translated and is absent from the

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Fig. 1. Overview of capsid/virion assembly. Genomic RNA is translated by a host ribosome. HCV core is the first polypeptide encoded in the polyprotein. Just proximal to core is the membrane envelope glycoprotein E1. The signal sequence (SS) for E1 (distal to core) targets the polyprotein to the ER. Signal peptidase cleaves the immature form of core from the growing polypeptide. Signal peptide peptidase then cleaves the E1 SS releasing the mature form of core. Core then multimerizes and encapsidates HCV RNA at the cytoplasmic face of the ER. Capsids that are formed in the cytoplasm then interact with E1 and bud into the ER lumen. Enveloped virions are then released, presumably via the secretory pathway.

mature form of HCV core. Nevertheless, domain III appears to be very important in terms of HCV core stability, targeting, and function. Two major forms of core protein, corresponding to 21- and 23-kDa (p21 and p23), are generated in vitro and in cultured cells (Yasui et al., 1998), corresponding to the mature (signal cleaved) and immature (signal uncleaved) forms of the protein. HCV CORE BIOGENESIS

Synthesis of HCV core in the same polyprotein as the HCV envelope proteins creates an interesting predicament in that core, the capsid protein, needs to be soluble and cytoplasmic, while the envelope glycoproteins are transmembrane and anchored into the host membrane. Therefore, like other flaviviruses, HCV has evolved an internal signal sequence for E1, the first envelope glycoprotein (referred to as E1 SS or domain III, as described above). The E1SS is encoded between HCV core and E1. Thus, after core is translated, the nascent polyprotein is targeted to the ER translocation channel by the E1 SS (Fig. 1). A host enzyme located in the ER, signal peptidase, cleaves just proximal to the E1 SS, releasing the immature form of core from the polypeptide (Hijikata et al., 1991; Santolini et al., 1994). A different endoplasmic reticulum (ER) enzyme, signal peptide peptidase (SPP), subsequently cleaves just before the E1 SS liberating the mature form of HCV core at the cytoplasmic face of the ER (McLauchlan, 2000; McLauchlan et al., 2002). SPP is a presenilin-type aspartic protease that catalyses intramembrane proteolysis of signal sequences and membrane proteins within the ER (Weihofen et al., 2002). Precise mutational analyses have shown that intramembrane cleavage by SPP is abolished when helix-breaking and -bending residues in the C-terminal signal sequence are replaced by basic residues. Furthermore, the signal sequence itself and three hydrophobic aa Leu-139, Val-140, and Leu-144 of the core protein

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are required for SPP cleavage, although none of these residues are essential for cleavage at the core-E1 junction by signal peptidase, or for translocation of E1 into the ER (Okamoto et al., 2004). The exact cleavage site for producing mature core (p21) is still controversial, since Leu-179 (Hussy et al., 1996; McLauchlan et al., 2002), Leu-182 (Hussy et al., 1996), Ser-173 (Santolini et al., 1994), and Phe177 (Okamoto et al., 2004) have all been reported as potential sites of cleavage. After being cleaved into the mature form at the ER, core can undergo a number of possible fates, including assembly into capsids, targeting to other organelles, and interaction with host proteins resulting in modulation of various cellular processes, as will be discussed in more detail below. HCV CAPSID STRUCTURE AND PROPERTIES

The main role for HCV core in the viral life cycle is to form a nucleocapsid to protect the viral genome. Once cleaved from the polyprotein, the mature core protein presumably assembles into HCV capsids, most likely at the cytoplasmic face of the ER (Mizuno, 1995; Blanchard, 2002; Blanchard, 2003). Unfortunately, no cellular system robustly recapitulates late events in the viral life cycle, although there may be hope with the recent development of an infectious HCV system (see Chapter 16). For this reason, mechanistic details of this process are lacking. HCV replicon systems (see Chapter 11), first developed in 1999, represented a major breakthrough because they allowed replication of HCV RNA in mammalian cells (Blight et al., 2000; Lohmann et al., 1999). However, even when HCV core is synthesized to high levels, late events in the HCV life cycle do not occur in most replicon systems, as judged by electron microscopy (Pietschmann et al., 2002). Therefore a number of model systems have been developed to study the structure of HCV capsids and HCV capsid assembly. Knowledge of HCV capsid appearance in vivo has come from examining particles in serum or in infected liver biopsies. Non-enveloped capsids have been observed in the cytoplasm of liver cells, while enveloped particles have been seen in the cisternae of the ER, as judged by transmission electron microscopy (TEM) (Bosman et al., 1998; Shimizu et al., 1996). The presence of capsids at or in the ER by TEM in numerous studies implicates the ER as the site of HCV capsid assembly (Blanchard, 2002; Maillard, 2001; Mizuno, 1995; Shimizu, 1996). More recently, a careful TEM analysis of HCV virions and non-enveloped nucleocapsids from serum of HCV infected patients was performed (Maillard et al., 2001). This study revealed that non-enveloped HCV nucleocapids can be found in significant quantities in serum. These capsids, as well as those obtained by detergent treatment of enveloped virions, are spherical but heterogeneous in size, with a bimodal distribution of capsid diameters corresponding to ~38 - 43 nm and ~54 – 62 nm. It remains unclear what governs capsid size and whether the size differences are biologically significant. Unfortunately, unlike with other flaviviruses, visualization of HCV virions or capsids at atomic resolution has not yet been achieved. 92


Biochemical analyses have determined that enveloped HCV virions have a density 1.08 to 1.16 g/ml (Bradley et al., 1991; Kaito et al., 1994; Kanto et al., 1994; Miyamoto et al., 1992). Similar studies on non-enveloped HCV capsids have yielded conflicting results. HCV capsids with envelopes removed using detergent have densities of approximately 1.25 g/ml (Kaito et al., 1994; Kanto et al., 1994; Miyamoto et al., 1992) or 1.32 - 1.34 g/ml (Maillard et al., 2001; Shindo et al., 1994), with the electron microscopic appearance of capsids of both densities being otherwise very similar (Maillard et al., 2001). An explanation has been proposed to explain the finding of two different buoyant densities: capsids that band at the lower density (~1.25 g/ml) appear to be associated with fragments of membranes, while those banding at the higher density (~1.32 g/ml) appear to be free of membranes (Maillard et al., 2001). However, this hypothesis remains to be tested. Additionally, it appears that both the immature and mature form of core can assemble and be incorporated into capsids, although, not surprisingly, the mature form is the main species in virions (Yasui et al., 1998). MODEL SYSTEMS FOR CAPSID ASSEMBLY

While electron micrographs of infected serum and hepatocytes give a literal snapshot of what is occurring in vivo, at the other extreme are minimal systems that can be used as surrogates for understanding the process of capsid assembly. In these minimal systems, purified recombinant core is incubated with RNA in the absence of other cellular factors. In the presence of RNAs containing a high degree of secondary structure (e.g. tRNA or the HCV 5' untranslated region), Cterminal truncation mutants were found to assemble into regularly shaped capsids that resemble HCV capsids from infected individuals (Kunkel et al., 2001). Similar results were obtained by expressing truncated core constructs in E. coli (Lorenzo et al., 2001). In contrast, full-length (wild-type) recombinant core assembles into particles with irregular shapes (Kunkel et al., 2001), raising the possibility that host factors or co-ordination of assembly with core synthesis may be required to assemble proper capsids from full-length HCV core. These studies also demonstrated that domain I is sufficient for core assembly. Furthermore, removal of domain II appeared to facilitate capsid assembly, allowing the purified core protein to assemble into more regular-shaped capsids. Together, these systems show that HCV core contains all of the information to assemble into capsid-like structures (in the presence of RNA) (Kunkel and Watowich, 2002). However, because of the minimalist nature of these systems, other systems will be required to determine the mechanism by which wild-type core assembles into capsids within cells, where assembly is likely to be influenced by other events including de novo core translation, host factors, and targeting of core to specific organelles. A cell-free system, a virtual hybrid between in vitro systems and cellular systems, has recently been developed to study HCV assembly. In these systems, cellular

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extracts are used to reconstitute and link translation to post-translational events, such as capsid assembly. Thus, these systems combine the benefits of being able to manipulate the assembly reaction in a test tube while maintaining a cellular context. Cell-free systems faithfully reconstituted HCV capsid assembly when full-length core, either the immature or mature form, was expressed de novo in either wheat germ extracts or rabbit reticulocyte lysate (Klein et al., 2004). Moreover, TEM analysis revealed that capsids formed from full-length core in the cell-free system were morphologically very similar to capsids produced in infected patient serum, both in size and structure (Klein et al., 2004), thereby validating the cell-free system for mechanistic and mutational studies. In addition, cell-free HCV capsid assembly is very efficient, with over 60% of newly-synthesized core polypeptides assembling into immature capsids (Klein et al., 2004). Some cellular systems have also been used to study capsid assembly. When overexpressed in insect cells, core assembles into 30 – 60 nm particles at the ER (Baumert et al., 1998; Baumert et al., 1999; Maillard et al., 2001) that closely resemble capsids produced in vivo. When the envelope proteins E1 and E2 are also expressed, capsids can be seen budding into the ER and cytoplasmic vesicles (Baumert et al., 1998); however, unfortunately no virus-like particles are released (Baumert et al., 1998; Baumert et al., 1999; Maillard et al., 2001). Therefore, this system recapitulates much of what is seen in hepatocytes and supports the notion that capsids assemble at the ER, although virion production is still blocked at a later step in the viral life cycle. Nucleocapsid-like particles have also been observed upon expression of HCV core in yeast (Majeau, 2004). In contrast to these model systems, in general, mammalian cell lines do not support HCV capsid assembly. There have been isolated reports of capsids being produced in cultured mammalian cells (Blanchard, 2002; Ezelle, 20026; Mizuno, 1995); however, the extent of HCV assembly in these cells is unclear. As noted above, even in replicon cells with high levels of HCV core synthesis, HCV assembly is not supported (Pietschmann et al., 2002; Bukh et al., 2002), similar to most cultured mammalian cells (Hope and McLauchlan, 2000). These findings suggest that mammalian cell lines either lack a necessary cellular factor(s) or contain inhibitory factor(s) that cause the majority of core to be targeted away from the ER, as discussed below. This alternate localization of core (Pietschmann et al., 2002), possibly in conjunction with other negative regulatory influences, correlates with failure to assemble HCV capsids or virions in cultured cell lines. Consistent with this, when crude hepatocyte extracts containing membrane-bound organelles are added to the highly permissive cell-free capsid assembly system, efficiency of assembly is reduced (Klein et al., 2004).

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The HCV Core Protein HCV ASSEMBLY: REQUIREMENTS AND MECHANISTIC ANALYSIS

Although an ideal model system for HCV capsid assembly does not exist, much has been elucidated about the requirements and process of capsid assembly from the various systems mentioned above. In vitro studies have been useful for structural analyses, having revealed that HCV core undergoes a conformational change upon assembling into capsid like structures (Kunkel and Watowich, 2002). Meanwhile, the cell-free system for HCV capsid assembly has allowed the process of core assembly to be analyzed mechanistically (Klein et al., 2005; Klein et al., 2004). Pulse chase analyses in the cell-free system have revealed that assembly occurs very quickly, with very little delay between completion of translation and completion of assembly (Klein et al., 2004). Additionally, capsid assembly was not highly dependent on protein concentration or membranes, unlike many other viruses. When HCV core expression was decreased 200 fold, only a 2.3 fold decrease in amount of assembly was observed (Klein et al., 2004). Both the speed of HCV capsid assembly and its relative concentration independence differ from what has been seen with assembly of other types of viral capsids (i.e. lentiviruses and hepadnaviruses) in analogous cell-free systems (Lingappa et al., 1997; Lingappa et al., 1994; Lingappa et al., 2005). Thus, the basic assembly mechanism of HCV capsids may differ from that of many other viral capsids that assemble at the cytoplasmic face of membranes. Assembly may occur in microenvironments, for example on polysomes that contain a high concentration of core protein translating off a single mRNA. The presence of high local concentrations of newly-synthesized HCV core polypeptides, possibly in conjunction with cellular factors, could promote rapid and efficient HCV assembly in permissive cellular extracts, although future studies will be required to test this hypothesis. Model systems for HCV assembly have also been used to define regions of HCV core that are important for HCV capsid assembly. Studies using recombinant HCV core truncation mutants have revealed that domains II and III are dispensable for assembly (Kunkel et al., 2001; Lorenzo et al., 2001). In fact, truncation mutants lacking these domains assemble better than full-length constructs in vitro (Kunkel et al., 2001). Systematic analysis of HCV capsid truncation, deletion, and point mutants in the cell-free HCV capsid assembly system have confirmed that the Cterminus is dispensable for assembly, and also demonstrated that the N-terminal 68 aa are required for capsid assembly (Klein et al., 2005; Klein et al., 2004). This region of HCV core contains numerous basic residues organized into two clusters. Removing either cluster of basic residues, or mutating as few as 4 basic residues to alanines in either cluster, significantly reduces assembly of capsids in wheat germ extracts (Klein et al., 2005). Conversely, when neutral aa were deleted from the same region, no effect on cell-free HCV capsid assembly was observed, suggesting that the critical determinant for assembly is the overall basic charge of the N-terminus. Likewise, deletions or mutations in other regions of HCV core

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did not affect assembly (Klein et al., 2005). While these studies indicate that basic residues in the N-terminus are critical for assembly, it remains unclear whether the N-terminal 68 residues are sufficient for assembly. It should be noted that other domains of core are clearly important for interaction of core with cellular factors and for trafficking of HCV core to distinct cellular locations, as discussed below. Domains involved in core trafficking and cellular protein interactions are likely to influence or even regulate HCV capsid assembly in intact cells, but these events have not been studied together due to lack of cell lines that recapitulate HCV capsid assembly in a robust manner. RNA BINDING AND ENCAPSIDATION BY CORE

Besides multimerization to form the capsid, the other major function performed by core during assembly is RNA encapsidation. Many viruses will encapsidate non-specific cellular RNA if viral genomic RNA is not present. Moreover, many viruses use RNA as a scaffold for assembly, and/or to nucleate the assembly process. HCV core appears to act similarly. Domain I of HCV core is extremely hydrophilic, largely due to the many basic residues clustered in this region. Basic residues are frequently involved in nucleic acid binding because the positive charge can interact with the negative phosphate backbone of nucleic acids. Indeed, HCV core binds RNA (Fan et al., 1999; Santolini et al., 1994; Shimoike et al., 1999) and this association is dependent on the basic N-terminus (Santolini et al., 1994). Consistent with this, and supporting the notion that RNA acts as a scaffold for assembly, RNA was required for in vitro assembly (Kunkel et al., 2001). Additionally HCV core has RNA chaperone capabilities, suggesting that core may also help restructure RNA, which may have implications for specific genomic encapsidation (Cristofari et al., 2004). While the notion that HCV core binds to RNA is well established, it is unclear whether HCV core preferentially binds HCV genomic RNA over cellular RNAs. Core has been shown to bind ribosomal RNA (Santolini et al., 1994), tRNA (Kunkel et al., 2001), and HCV genomic RNA (Cristofari et al., 2004; Fan et al., 1999; Kunkel et al., 2001; Shimoike et al., 1999). It appears that the only requirement is that the RNA should contain significant amounts of secondary structure. When recombinant core was incubated with denatured, or unstructured, RNA, it failed to assemble into capsids suggesting that it could not interact with unstructured RNA. Conversely, when highly structured tRNA or the HCV UTR was used, core assembly was promoted (Kunkel et al., 2001). If core binds to any structured RNA, how does genomic RNA get specifically packaged? Many viral capsid proteins have a higher affinity for specific structures in their cognate genomic RNA, allowing them to preferentially bind the proper RNA. It is unclear whether HCV core has higher affinity for HCV genomic RNA. One

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study demonstrated that the HCV core protein binds specifically to a radiolabeled probe containing the 5' UTR of the genomic RNA. This interaction was abolished by excess unlabeled probe, but not by unlabeled, non-specific RNA, suggesting that core preferentially binds genomic RNA (Fan et al., 1999). This could explain how genomic RNA gets selectively packaged into virions over other cellular RNAs. Conversely, Santolini et al. reported that core fusion proteins bind equally well to HCV genomic RNA and heterologous RNA, suggesting that HCV core does not have enough specificity in its binding to promote genomic RNA encapsidation (Santolini et al., 1994). If HCV core does not specifically bind genomic RNA, then some other mechanism must exist to promote encapsidation of the genome. One possibility is that assembly occurs in microenvironments that contain only a single species of mRNA (i.e. HCV genomic RNA), as discussed above. Unfortunately, RNA encapsidation has not yet been analyzed in conjunction with capsid assembly in any system, so it remains unclear exactly what RNAs are encapsidated and how HCV core selects RNA for encapsidation during synthesis and assembly. CAPSID ASSEMBLY: LIGHT AT THE END OF THE TUNNEL?

As mentioned, for the most part cell culture systems do not support virion production, or even capsid assembly. However, isolated reports have identified infectious virus propagated in special cell culture systems and at low levels. One group infected hepatocytes that were cultured in a radial-flow bioreactor and found that HCV is able to replicate to very low titers (Aizaki et al., 2003). Additionally, at the 11th International Meeting on Hepatitis C and Related Viruses in Heidelberg in October 2004, there were three reports of very low titer infectious virus particle formation in cells transfected with HCV genomic RNA (Murakami et al., 2004b; Pietschmann et al., 2004; Wakita et al., 2004). These initial studies have been confirmed by independent groups (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005) (See Chapter 16). Use of 3-dimensional cultures (Murakami et al., 2004b) or transfection with the JFH strain (Pietschmann et al., 2004; Wakita et al., 2004) resulted in production of infectious particles. In one case infection was receptor mediated, as antibodies to the putative HCV receptor, CD81, blocked infection (Pietschmann et al., 2004). Unfortunately, in all cases, levels of virus production were too low to result in measurable titers or any ultrastructural evidence of virus formation (Aizaki et al., 2003; Murakami et al., 2004b; Pietschmann et al., 2004; Wakita et al., 2004). Most recently, Heller et al. also isolated virus like particles from cell culture after transfecting RNA corresponding to the exact genomic sequence (Heller et al., 2005). This study also showed morphologic data, which suggests that the particles produced are, indeed, virions. Thus, a new wave of data shows evidence that HCV can assemble into capsids and, subsequently, into virions in mammalian cell culture. However, it is unclear whether assembly in these systems is just a stochastic event, what amount of virus or virus like particles are produced, how assembly is regulated in these systems, or what cellular subpopulation, if

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any, is producing the limited number of viruses. By using a combination of all of the current model systems, as well as, newly described cellular systems, new insights into the mechanism by which HCV assembly is regulated in cells should be elucidated. This could allow for enhancements of current cell culture systems that could in turn facilitate the study of late events of the HCV life cycle in cells.

SUB-CELLULAR TARGETING OF HCV CORE IF CORE DOES NOT ASSEMBLE, WHERE DOES IT GO?

While much information has been elucidated from the various model systems outlined above, surprisingly, mammalian cell lines including human liver-derived cell lines fail to produce quantifiable levels of HCV capsids, or virions. For reasons that remain unclear, in these cell lines HCV core polypeptides can be directed to alternate cellular locations upon release from the nascent HCV polyprotein. The fact that core assembles efficiently in infected humans and chimpanzees, but not in intact cultured cell-lines, suggests that HCV assembly can be negatively regulated. Trafficking of core to alternate sites is one possible mechanism for negative regulation of capsid assembly. One approach to studying the subcellular localization of core involves immunostaining liver biopsy specimens from infected patients. This has revealed that the core protein predominantly localizes within the cytoplasm of infected hepatocytes, and often shows a punctate granular distribution within cells (Gonzalez-Peralta et al., 1994; Gowans, 2000; Sansonno et al., 2004; Yap et al., 1994). However, when the core protein alone or the entire viral polyprotein are expressed in mammalian cells, the majority of core has been observed at the ER membrane (Lo et al., 1995), on the surface of lipid droplets (Barba et al., 1997; Hope et al., 2002; McLauchlan et al., 2002; Pietschmann et al., 2002; Shi et al., 2002), and on mitochondrial and mitochondrial-associated membranes (Schwer et al., 2004; Suzuki et al., 2005). In addition, core is also known to target to the nucleus (Lo et al., 1995; Matsuura et al., 1994; Moriishi et al., 2003; Moriya et al., 1998; Yasui et al., 1998), where it can be a substrate for proteasomal degradation. What governs whether core stays at the ER to assemble or traffics to other areas of the cell is not completely understood. Nevertheless, it is clear that such regulation exists and is quite complex. The finding that core targets to lipid droplets and mitochondria, but E1 and E2 do not (Pietschmann et al., 2002; Schwer et al., 2004), raises the possibility that targeting of core away from the ER occurs at a very early time after core synthesis, before core has had time to interact with the envelope glycoproteins. Furthermore, a number of studies suggest that aa in domains II and III direct the post-translational trafficking of core, although agreement is lacking as to which residues are critical. Okamoto et al. has shown that not only the C-

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terminal signal sequence but also aa 128-151 are required for ER retention of the core protein by using a series of N-terminally truncated core protein constructs (Okamoto et al., 2004). Suzuki et al. has reported that a region of aa 112-152 mediates association of the core protein with the ER in the absence of the C-terminal signal sequence (Suzuki et al., 2005). McLauchlan et al. have proposed that a large part of the core protein remains within the cytoplasmic leaflet of the ER membrane after SPP cleavage (McLauchlan et al., 2002). Upon intramembrane cleavage of the transmembrane signal peptide, the processed core protein may traffic along the lipid bilayer from the site of biosynthesis to zones at the ER, where lipid droplets are produced (McLauchlan et al., 2002). Deletion analyses have revealed that domain II (in particular residues between aa 125 - 144) plays a critical role in targeting core to lipid droplets (Hope and McLauchlan, 2000; Hope et al., 2002). Notably, no domain homologous to domain II is present in the core proteins of related pesti- and flavi-viruses. In contrast, the core protein of GB Virus B, from the GB virus group within the Flaviviridae, does contain a homologous domain that also appears to mediate targeting to lipid droplets (Hope et al., 2002). Domain II contains two closely spaced prolines that form a proline knot and appear to be required for targeting core to lipid droplets. The region containing this proline knot can be replaced with a proline knot domain from lipid-associated plant proteins called oleosins (Hope et al., 2002), with preservation of lipid targeting. Lipid targeting of HCV core can also be altered by mutations that affect SPP cleavage. Helix-breaking point mutations within the signal sequence (domain III) eliminate SPP cleavage, but also eliminate trafficking to lipid droplets, leaving core protein on the cytoplasmic face of the ER (McLauchlan et al., 2002; Okamoto et al., 2004). While these alternate pathways for core trafficking are beginning to be defined, the downstream consequences of different post-translational trafficking pathways on core function have not yet been explored. This is in part because using core mutants to study these cellular fates has proven to be relatively tricky. Studies have shown that C-terminally truncated versions of the core protein are localized exclusively to the nucleus (Suzuki et al., 1995). A fraction of the core protein was detected in the nucleus even when full-length HCV core gene was expressed, suggesting that the mature core protein also localizes to the nucleus (Moriya et al., 1997a; Yasui et al., 1998). The N-terminal domain of the core protein contains three stretches of arginine- and lysine- rich sequences. These basic-residue stretches function as nuclear localization signals (NLSs) for translocation of the core protein to the nucleus (Chang et al., 1994; Suzuki et al., 1995). Each of the NLS motifs of the core protein is able to bind importin-α. At least two of them are required for efficient nuclear distribution of the core protein in cells, suggesting that they constitute a bipartite NLS (Suzuki et al., 2005).

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The major fate of core that is targeted to the nucleus is degradation by the nuclear proteasome (Hope et al., 2002; McLauchlan et al., 2002; Moriishi et al., 2003). Whether this is a cellular protein "quality control" mechanism, a normal pathway for core, or a pathway with other functional consequences is unclear. Nevertheless, it appears that constructs encoding mutations in the C terminus of core are less stable in cells than is wild-type core (Moriishi et al., 2003). McLauchlan and colleagues have proposed that the ability of domain II to mediate attachment of core to lipid droplets also protects core from degradation. Furthermore, they demonstrated that core constructs encoding a deletion in domain II are protected from degradation when they also encode a mutation that blocks cleavage of domain III by SPP (McLauchlan et al., 2002). Related to this observation, the mature form of core is much less stable when expressed as such than when expressed as the immature form of core which transiently contains domain III (E1 SS) before undergoing processing (Suzuki et al., 1995; Suzuki et al., 1999; Suzuki et al., 2001). Therefore, while the final product is the same, the presence of domain III during core biogenesis greatly influences core stability. Domain III, while not present in the mature wild-type core protein, plays a complex and important role in core stability. Like domain II, domain III and its cleavage may be involved in linking HCV core to cellular pathways that target it to other regions of the cell and protect it from degradation. Interestingly, although truncations and deletions in domain II lead to rapid degradation in mammalian cells, this phenomenon is not seen in cellfree capsid assembly systems, even when mammalian cell extracts are used (Klein et al., 2005; Klein et al., 2004). This is likely due to the absence of the nucleus in these systems, which prevents targeting to the nuclear proteasome, and allows such mutants to be expressed and analyzed. Core appears to be peripherally associated with mitochondria, since it is accessible to protease digestion and carbonate extraction (Schwer et al., 2004) as is the case at the ER (McLauchlan et al., 2002). Most likely, core traffics from the ER to both the mitochondria and lipid droplets via membrane bridges, since both of these compartments are likely derived from the ER. Mitochondrial targeting appears to be governed by an aa sequence in core. Schwer et al. demonstrated that a short stretch extending from aa 149-158 located in domain II governs mitochondrial targeting (Schwer et al., 2004). Suzuki et al. reported that a region of 41 residues from aa 112-152 is responsible for association between the core protein and mitochondria (Suzuki et al., 2005). This discrepancy may be due to the differences of HCV clones and experimental settings.

POST-TRANSLATIONAL MODIFICATIONS OF HCV CORE Post-translational modification plays crucial roles for regulating the function of the proteins. Several studies have shown post-translational modification of HCV core protein. Phosphorylation of the core protein in insects cells (Lanford et al., 1993), 100


reticulocyte lysates (Shih et al., 1995), and mammalian cells (Lu and Ou, 2002) have been reported. Cellular protein kinase A (PKA) and protein kinase C (PKC) were identified as possible protein kinases responsible for phosphorylation of HCV core protein. Phosphorylation at Ser-116 may regulate nuclear localization of the core protein (Lu and Ou, 2002). Post-translational modification of the core protein by tissue transglutaminase has been reported (Lu et al., 2001). Tissue transglutaminase catalyzes the formation of a γ-carboxyl-ε-lysine isopeptide bond by joining the γ-carboxamide group of glutamine to the amino group of lysine. A small fraction of the core protein has been shown to form a dimer that is highly stable and resistant to denaturation and reduction by SDS and β-mercaptoethanol. A potential role for tissue transglutaminase in core dimer formation has been proposed (Lu et al., 2001). The ubiquitin-proteasome pathway is the major route by which selective protein degradation occurs in eukaryotic cells and is now emerging as an essential mechanism of cellular regulation (Finley et al., 2004; Hershko and Ciechanover, 1998). As mentioned above, the core protein is targeted for ubiquitination and degradation by an unknown ubiquitin ligase. The C-terminus of the core protein is important for regulating stability of the protein (Kato et al., 2003; Suzuki et al., 2001). When the core protein is expressed as the C-terminal truncated forms such as aa 1-173 (21kDa) and 1-152 (17kDa), the core protein is unstable (Kato et al., 2003; Moriishi et al., 2003; Suzuki et al., 2001). Specific proteasome inhibitors stabilize these short-lived forms of the core protein, suggesting that the proteasome machinery is responsible for their degradation (Fig. 2). By contrast, the full-length form of the core protein (aa 1-191) is long-lived. Only the C-terminal truncated form of the core protein can be multi-ubiquitinated, and the predominant stable form of the core protein links to a single or only a few ubiquitin moieties (Suzuki et al., 2001). To understand the mechanism of ubiquitination of the core protein, the specific E3 ubiquitin ligase that acts on HCV core has to be identified. A proteasome activator, PA28γ, has been identified as a core-binding protein by yeast two-hybrid screening (Moriishi et al., 2003). PA28γ can interact with the core protein in cultured cells, as well as in the liver of transgenic mice and chronic hepatitis C patients. PA28γ predominates in the nucleus and forms a homopolymer, which associates with the 20S proteasome (Tanahashi et al., 1997), thereby enhancing proteasome activity (Realini et al., 1997). Over-expression of PA28γ enhanced proteolysis of the core protein, suggesting that PA28γ affects proteasomal activity and regulates stability of the core protein (Moriishi et al., 2003) (Fig. 2). Evidence has been accumulating that ubiquitin-proteasome pathway plays a crucial role in the viral life cycle and in pathogenesis (Banks et al., 2003; Scheffner et al., 1993). However, the biological significance of ubiquitin-dependent degradation of the core protein remains to be elucidated.

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Fig. 2. A model for the processing of HCV precursor and degradation of the core protein by the Ubiquitin-proteasome pathway. The junction between core and E1 is cleaved by the signal peptidase, resulting in production of p23 form of the core protein. Additional cleavage of the core protein by signal peptide peptidase produces p21 form of the core protein. Further processed forms of the core protein, such as p17, are produced by unknown mechanisms. The C-terminal truncated form of the core protein is poly- ubiquitinated by an unidentified E3 ubiquitin ligase and targeted for proteasomal degradation. The immature core protein links to a single or a few ubiquitin moieties and is long-lived. A proteasome activator, PA28γ, enhances proteasomal degradation of the core protein.

HCV CORE-HOST INTERACTIONS Core-host interactions will be discussed in terms of their affects on host antiviral and immune responses, and HCV pathogenesis. The recent finding of core protein in the serum on infected patients has forced one to think that HCV host interactions not only occur within infected cells, but they can also occur extracellularly. EFFECTS ON T CELL FUNCTION

HCV infection in humans is almost invariably associated with viral persistence leading to chronic hepatitis – predisposing the host to development of cirrhosis 102


and hepatocellular carcinoma. CD8+ T cells play a pivotal role in controlling HCV infection; but, in chronic HCV patients, severe CD4+ and CD8+ T cell dysfunction has been observed (Shoukry et al., 2004). This suggests that HCV may employ mechanisms to evade or possibly suppress the host T cell response. In exploring the possible evasion mechanism(s) in order to design strategies for therapeutics and improved immunization, the HCV core protein was identified as an immunomodulatory molecule suppressing T lymphocyte responsiveness through its interaction with complement receptor (gC1qR) (Kittlesen et al., 2000). It was demonstrated that the HCV core protein suppresses an in vivo anti-viral CD8+ T cell response to vaccinia virus, and inhibits the production of IFN-γ and IL-2 in an experimental murine model. A host target protein (gC1qR) on T cells was shown to bind HCV core. Like the natural ligand, C1q, the binding of extracellular core to gC1qR displayed on T cell surface lead to CD4+ T cell deregulation and suppression of CD8+ T cell function. Importantly, HCV core-gC1qR ligation induced the expression of negative signaling molecules (e.g. SHP-1 and SOCS1) in CD4+ T cells. The data suggest that core has potent immunomodulatory functions. EFFECTS ON TOLL-LIKE RECEPTORS

Cells sense the presence of extracellular pathogens via cell surface toll-like receptors (TLRs). There are approximately 10-15 TLRs in mammals, which are responsible for sensing microbial infection, via recognition of pathogen associated molecular patterns (PAMP), such as lipopolysaccharide (LPS; TLR4), double-stranded RNA (dsRNA; TLR3), CpG DNA of bacteria (TLR9), and single-stranded RNA (ssRNA; TLR7) (Iwasaki and Medzhitov, 2004). After binding pathogens, TLR signaling involves coupling of toll-IL-1 receptor (TIR) containing adapter proteins such as TIRAP, TRIF, TIRAP and MAL, and activation of signaling molecules IL-1 receptor associated kinase (IRAK), MyD88, and TNF receptor-associated factor 6 (TRAF-6). Ultimately, transcription factors such as mitogen activated protein kinases (MAPK), NF-κB, and IRF-3 become activated, leading to production of IFN-α/β (Hertzog et al., 2003). Interestingly, DC maturation in vitro is impaired in chronic HCV infection when compared to those subjects with spontaneously resolved infection and normal controls (Anthony et al., 2004; Dolganiuc et al., 2003; Kanto et al., 2004; Murakami et al., 2004a; Sarobe et al., 2003; Tsubouchi et al., 2004a; Tsubouchi et al., 2004b; Wertheimer et al., 2004). Recent studies have provided mechanistic insights into these events. In a study of the effect on the immunostimulatory effects of lipopeptides, 10 of 14 and 9 of 14 HCV core lipopeptides stimulated a reporter gene in TLR2-expressing and TLR4-expressing cells but not in mock-transfected control cells (Duesberg et al., 2002). However, activation was dependent on the lipid moiety since the same free peptides had no stimulatory effect on the TLR2 or TLR4 transfected

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cells. A study by a different group found that addition of recombinant HCV core protein to human monocytes, and human embryonic kidney cells transfected with TLR2 triggered inflammatory cell activation and failed to activate macrophages from TLR2 or MyD88-deficient mice (Dolganiuc et al., 2004). HCV core induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Cell activation required core aa 2-122. Interestingly, HCV core protein was also taken up by macrophages, but this was independent of TLR2 expression. These data indicate that the HCV core protein can trigger innate immune responses. EFFECTS OF HCV CORE ON THE INTERFERON SYSTEM

Several studies have documented that the HCV core protein can activate the interferon (IFN) system. For example, core activates the IFN stimulated genes (ISG) 2-5 OAS (Naganuma et al., 2000) and PKR (Delhem et al., 2001). PKR and 2-5 OAS are two major ISGs that mediate the IFN antiviral response against many viruses. It was also recently shown that HCV core protein activates the innate antiviral cellular response involving interferon regulatory factors (Miller et al., 2004). Core induced IRF-1 transcription and mRNA expression, and caused dose-dependent induction of the IFN-β promoter and IFN-β mRNA expression. In the presence of IFN-α, core expression caused increased IFN-stimulated gene factor 3 (ISGF3) binding to the IFN-stimulated response element (ISRE) and tyrosine phosphorylation of Stat1. Core expression also activated IFN-γ signaling (Miller et al., 2004). The effects of core on innate cellular antiviral responses including TLR and IFN pathways may be critically important during acute infection. Following binding, internalization, and uncoating of HCV virions, core, in the form of nucleocapsid, is the first viral protein to interact with the intercellular milieu of cellular proteins and signaling pathways. Because core mutates during virus replication, HCV core is present as a quasispecies in infected patients (Pawlotsky, 2003). What is not clear at present is whether HCV core's inherent variability influences innate antiviral responses such as TLR signaling and IRF-Jak-Stat activation. Fig. 3 suggests that there is indeed heterogeneity in innate antiviral responses to genetically different HCV core isolates. Fig. 3A depicts the sequence of 2 core proteins (named Core 1 and Core 2) derived from 2 different genotype 1b infected patients. As shown in the figure, the two isolates differed by 7 aa. The 2 core genes were engineered into a tetracycline regulated expression vector, such that in the absence of tetracycline in the medium, both Core 1 and Core 2 proteins were expressed in HeLa cells. Addition of tetracycline to the medium blocked core expression. Fig. 3C presents the effects of Core 1 and Core 2 expression on transcription of an IFN responsive promoter, the ISRE. In the absence of IFN, expression of Core 1 was associated with a 3-fold increase in activation of the ISRE, compared to when

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Fig. 3. Effects of HCV Core Protein Expression on Type I IFN Signal Transduction. A, the sequence of the Core 1 and Core 2 genes are aligned. B, Tetracycline regulated expression of the Core 1 and Core 2 proteins in HeLa cells. Plasmids were transfected into HeLa tet-off cells, grown in the absence and presence of tetracycline to induce and repress core protein expression, respectively, and protein lysates were subjected to Western blot analysis at 48 hours post-transfection. C, Differential effects of Core 1 and Core 2 proteins on ISRE activation. pTRE-Core 1 and pTRE-Core 2 plasmids were cotransfected with an ISRE-luciferase reporter plasmid into HeLa tet-off cells, incubated in the presence or absence of tetracycline for 40 hours, and treated with or without 500 U/ml of IFN-α for 6 hours. Luciferase activity was determined on equal amounts of protein lysates.

gene expression was repressed. In the presence of IFN, Core 1 induced a 2-fold increase in luciferase activity. Expression of Core 2 resulted in only marginal ISRE stimulation. These data demonstrate that 2 genetically different HCV core proteins activate a canonical IFN promoter to varying degrees. The data suggest that HCV quasispecies differentially modulate host cell responses. Indeed, other studies have demonstrated that NS5A mediated transcriptional activation varies among clinical quasispecies isolates (Pellerin et al., 2004). Thus, future studies should take into account genetic and structural heterogeneity of HCV isolates as being important factors in host responsiveness to HCV infection. This concept may have clinical implications. It can be hypothesized that genetic and structural variants of HCV proteins such core could differentially trigger

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innate antiviral responses during acute infection. Thus, some HCV infections may be "silent" because they minimally activate the TLR and/or IFN cellular defense systems. This would have obvious selective advantage for the virus and could contribute to the establishment of chronic infection. Alternatively, when a virus enters cells in a "noisy" fashion, it has a poor chance of establishing chronic infection because the innate antiviral responses would quickly shut down virus replication. Finally, stimulation of the IFN system by the HCV core protein may be required to balance the anti-IFN functions of other HCV proteins such as E2 (Taylor et al., 1999), NS5A (Gale et al., 1997; Polyak et al., 2001), and NS3 (Foy et al., 2003) during certain stages of the HCV replication cycle. EXTRACELLULAR VERSUS INTRACELLULAR EFFECTS OF CORE

HCV core is found within infected cells as well as in patient serum (Kashiwakuma et al., 1996; Widell et al., 2002). Extracellular core protein likely affects the modulation of T cell function, TLR signaling and DC function as described above. Thus, it is important to consider the contribution of extracellular and intracellular core protein to the biological activity in question. Indeed, CD81 engagement by the HCV envelope glycoprotein E2 inhibits NK and T cell cytotoxic function and signal transduction (Crotta et al., 2002; Tseng and Klimpel, 2002; Wack et al., 2001), and induced pro-inflammatory chemokine expression in hepatocytes (Balasubramanian et al., 2003). Thus, immune function may be altered as cells "sample" the microenvironment through HCV-host interactions that are limited to molecules on the cell surface, such as the HCV core-TLR or core-C1qR interaction. Moreover, these extracellular HCV-host interactions may also contribute to HCV pathogenesis. HCV CORE AND PATHOGENESIS

Recent work has demonstrated that the HCV core protein may also participate in the pathogenesis of liver disease. Development of fibrosis is characterized histologically with infiltration of inflammatory lymphocytes, hepatocellular apoptosis, and Kupffer cell activation. HSC proliferate and undergo and become highly activated, which involves secretion of large amounts of extracellular matrix proteins (Bataller and Brenner, 2005). Despite a large body of literature from clinical and animal studies on fibrosis development, very little is known about how HCV causes fibrosis. A recent study found that addition of recombinant core protein to activated human hepatic stellate cells (HSC) stimulated intracellular signaling pathways, while viral transduction of HCV core into HSCs caused increased cell proliferation (Bataller et al., 2004). Interestingly, the HSC response appeared to differ between core and other HCV non-structural proteins. The data suggest that HCV core and non-structural proteins can modulate the activity of HSC, which may contribute to fibrosis. This study also reinforces the notion that HCV proteins can have intracellular as well as extracellular effects on a variety of cells. 106


A second important point from the study of Bataller et al., (Bataller and Brenner, 2005), is that HCV proteins including core induce oxidative stress on HSC which is involved in HSC activation. Indeed, antioxidant therapy reduces the effects of HCV proteins on HSCs. This finding is in line with the current thinking that oxidative stress is central to induction of fibrosis in many model systems. HCV core induced oxidative stress also affects mitochondrial physiology. HCV CORE AND MITOCHONDRIAL DYSFUNCTION

Expression of HCV core protein in transgenic mice and in cell culture induces oxidative stress. It has been shown that core protein localizes to mitochondria, between the mitochondrial outer membrane and ER (Moriya et al., 1998; Moriya et al., 2001a; Okuda et al., 2002; Schwer et al., 2004; Suzuki et al., 2005), as described in the targeting section above. Core protein expression and mitochondrial localization inhibits electron transport at complex I, increases complex I reactive oxygen species (ROS) production, decreases mitochondrial glutathione, and increases mitochondrial permeability transition in response to exogenous oxidants such as alcohol (Korenaga et al., 2005; Okuda et al., 2002; Wen et al., 2004). These effects are associated with increased hepatocyte apoptosis in the presence of HCV core protein, ethanol and cytochrome P4502E1. Like the case with HSC, core and ethanol metabolism effects on apoptosis can be prevented with antioxidants (Otani et al., 2005). HEPATITC STEATOSIS AND HEPATOCARCINOGENESIS

Evidence has been accumulating that HCV core protein is directly involved in pathogenesis (Giannini and Brechot, 2003; McLauchlan, 2000). As shown in Table 1, many cellular proteins, which interact with core protein have been identified. Several studies have suggested that the core protein plays a crucial role for hepatocarcinogenesis (Chang et al., 1998; Moriya et al., 1998; Ray et al., 1996). Recent studies have highlighted steatosis as a basis of HCV-associated HCC (Lerat et al., 2002; Moriya et al., 1998). Steatosis, which is an accumulation of fat deposits in hepatocytes, is one of the histological features of chronic hepatitis C (Bach et al., 1992; Lefkowitch, 2003). In vitro studies have shown that HCV core protein associates with cellular lipid droplets, via direct interaction with apolipoprotein A2 (Barba et al., 1997; Shi et al., 2002). The mice transgenic for HCV core gene have been shown to develop steatosis and hepatocellular carcinoma (HCC) (Moriya et al., 1998; Moriya et al., 1997b). Steatosis in the core-transgenic mice is age-dependent and characterized by the appearance of micro- and macro-vesicular lipid droplets (Moriya et al., 1998). Lerat et al. have confirmed that transgenic mice expressing the whole genome of HCV also develops steatosis and HCC (Lerat et al., 2002).

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Polyak et al. Table 1. Cellular proteins that bind to the HCV core protein. The list contains cellular proteins with various cellular functions that interact with HCV core. The interaction of HCV core with these cellular proteins may have pathogenic implications. Please refer to the text for details. Core-Interacting protein

Function

Reference

Apolipoprotein AII

lipid metabolism

Sabile et al., 1999; Shi et al., 2002

CAP-Rf

RNA helicase

You et al., 1999

complement receptor gC1qR

T-cell response

Kittlesen et al., 2000

cyclin-dependent kinase 7

cell cycle

Ohkawa et al., 2004

DEAD box protein

RNA helicase

Mamiya and Worman, 1999

DEAD box protein 3 heterogeneous nuclear ribonucleoprotein K

RNA helicase

Owsianka and Patel, 1999

transcriptional control

Hsieh et al., 1998

JAK1/2

signal transduction

Hosui et al., 2003

lymphotoxin-β receptor

cytotoxicity

Chen et al., 1997

p53


Otsuka et al., 2000

p73


Alisi et al., 2003

proteasome activator PA28γ

protein stability

Moriishi et al., 2003

retinoid X receptor α


Tsutsumi et al., 2002

Smad3


Cheng et al., 2004

Sp110b


Watashi et al., 2003

STAT3

cell transformation

Yoshida et al., 2002

TAFII28


Otsuka et al., 2000

Tumor necrosis factor receptor 1 apoptosis

Zhu et al., 2001

14-3-3 protein

Aoki et al., 2000

signal transduction

Although the molecular mechanisms of steatosis caused by the core protein is still unclear, the core protein may alter lipid metabolism by interacting with cellular proteins involved in lipid accumulation and storage in hepatocytes (Barba et al., 1997; Sabile et al., 1999; Shi et al., 2002). The concentration of carbon 18 monosaturated fatty acids were increased in the livers of the core-transgenic mice and chronic hepatitis C patients, suggesting that HCV core affects a specific pathway in lipid metabolism (Moriya et al., 2001b). Nonetheless, transgenic mouse lines established by other groups did not show either steatosis nor HCC (Kawamura et al., 1997; Pasquinelli et al., 1997). These discrepancies suggest that not only the viral proteins but also other factors are involved in hepatocarcinogenesis. These discrepancies may be due to differences in genetic backgrounds of the mice and expression levels of the viral proteins.

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ACKNOWLEDGEMENTS SJP is partially supported by NIH grants AA13301 and DK62187, and the University of Washington Royalty Research Fund. JRL received support from Puget Sound Partners, and KCK received support from NIH training grant T32 CA09229. IS and TM are supported in part by grants from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, RandD Promotion and Product Review of Japan (ID:01-3) and the Second Term Comprehensive 10-year Strategy for Cancer Control of the Ministry of Health, Labor, and Welfare of Japan. IS and TM also thank their colleagues, T.Tsutsumi, K.Ishii, H.Aizaki, K. Murakami, R.Suzuki, T, Suzuki, (National Institute of Infectious Diseases), Y.Shintani, H.Fujie, K. Moriya, K.Koike, (Tokyo University) and K.Moriishi, Y. Matsuura (Osaka University) for contribution.

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Chapter 4

HCV Glycoproteins: Assembly of a Functional E1-E2 Heterodimer Muriel Lavie, Anne Goffard and Jean Dubuisson

ABSTRACT The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1-E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1-E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have been limited by the lack of a robust cell culture system for HCV replication and viral particle production. Recently, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins, allowing the characterization of functional E1-E2 envelope glycoproteins. Here, we review our understanding to date on the assembly of the functional HCV glycoprotein heterodimer.

INTRODUCTION As obligate intracellular parasites, all viruses have evolved ways of entering target cells to initiate replication and infection. The first step in virus entry is the recognition of host cells through cell surface receptor(s). This initial engagement can mediate attachment as well as act as a primer for subsequent conformational alteration, leading to virus entry into host cell. In many cases, interaction with a receptor is important for defining the tropism of a virus for a particular organism, tissue or cell type. Enveloped viruses possess a lipid bilayer that surrounds their nucleocapsid. The glycoproteins present in their envelope are involved in the receptor-binding step. After attachment, the entry of these viruses into cells requires the fusion of the viral and a cellular membrane by a process that is also driven by the viral envelope glycoproteins. To fulfill these functions, viral envelope glycoproteins have to adopt dramatically different conformations during the virus lifecycle. In addition, these conformational changes have to occur at a precise time of the virus lifecycle, and thus, have to be tightly modulated.

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HCV encodes two envelope glycoproteins, named E1 and E2. For a long time, the lack of a cell culture system supporting efficient HCV replication and particle assembly has hampered the characterization of the envelope proteins present on the virion. Cell culture transient expression systems have allowed investigators to characterize the first steps in the biogenesis of HCV envelope glycoproteins (reviewed in: Op De Beeck et al., 2001). In addition, surrogate models have also been developed to study the entry steps of HCV lifecycle (reviewed in: Op De Beeck and Dubuisson, 2003). However, it is only recently that a model mimicking the entry process of HCV lifecycle has been developed. This has been achieved by pseudotyping retroviral particles with native HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). This new tool allows, for the first time, the characterization of the assembly of functional HCV envelope glycoproteins.

BIOGENESIS OF HCV ENVELOPE GLYCOPROTEINS CLEAVAGE OF HCV GLYCOPROTEINS FROM THE VIRAL POLYPROTEIN

As for the other members of the Flaviviridae family, the genome of HCV encodes a single polyprotein. This ~3010 amino acid polyprotein is processed by cellular (signal peptidase and signal peptide peptidase) and viral proteases (NS2-3 and NS3-4A) to generate at least 10 polypeptides (reviewed in: Penin et al., 2004). The nonstructural proteins are released from the polyprotein after cleavage by HCV proteases NS2-3 and NS3-4A, whereas the structural proteins are released by host endoplasmic reticulum (ER) signal peptidase(s) (Fig. 1)(reviewed in Reed and Rice, 2000). Further processing mediated by a signal peptide peptidase also occurs at the C-terminus of the capsid protein (McLauchlan et al., 2002). Most cleavages in the polyprotein precursor proceed to completion during or immediately after translation (Grakoui et al., 1993; Dubuisson et al., 1994; Lin et al., 1994; Mizushima et al., 1994; Dubuisson and Rice, 1996). Partial cleavages occur at the E2/p7 and p7/NS2 sites, leading to the production of an uncleaved E2p7NS2 molecule. While most of NS2 is progressively cleaved from the E2p7NS2 precursor, the cleavage between E2 and p7 does not change over time, at least for most HCV strains analyzed (Dubuisson, 2000). Thus, this results in cleavage products consisting of E2, E2p7, p7, and NS2. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can efficiently function as signal peptides. Indeed, when fused to a reporter protein, the signal peptides of p7 and NS2 are efficiently cleaved (Carrère-Kremer et al., 2004). These data indicate that inefficiency of cleavage at E2/p7 and p7/NS2 sites is not due to the presence of suboptimal signal peptides. The p7 polypeptide is a polytopic membrane protein containing two transmembrane domains with both its N- and C-termini oriented toward the ER lumen (Fig. 1)(Carrère-Kremer et al.,

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Fig. 1. Processing of the N-terminal one-third of HCV polyprotein. The arrows show host signal peptidase cleavages. Partial cleavages at E2/p7 and p7/NS2 sites are indicated by dotted arrows. Cleavage by the host cell signal peptide peptidase (SPP) is indicated by scissors. The signal peptide and signals of reinitiation of translocation are shown as a black cylinder and light grey cylinders, respectively. The transmembrane domains of HCV envelope glycoproteins are represented in their pre-cleavage topology. Post-cleavage reorientation of the glycoprotein signals of reinitiation of translocation is indicated by curved arrows.

2002). Interestingly, the presence of the first transmembrane domain of p7 reduces the efficiency of p7/NS2 cleavage (Carrère-Kremer et al., 2004). Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both E2/p7 and p7/NS2 sites (Carrère-Kremer et al., 2004). In addition, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane α-helix located downstream of the cleavage sites might impose additional structural constraints to these cleavage sites (Fig. 1). Such constraints in the processing of a polyprotein precursor are likely essential for HCV to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins. The processing at the E2p7 site has been further explored. It has been reported to be more efficient in genotype 1b (strain BK) than in the genotype 1a (strain H77c) (Dubuisson et al., 1994; Lin et al., 1994). A sequence comparison of p7 signal peptides of these two viral strains has identified a difference of 3 amino acids and mutational analysis has shown that the V720L change in the H77c sequence substantially increases the efficiency of processing at the E2/p7 site (Isherwood and Patel, 2005). Although, when expressed alone, p7 protein has been shown to adopt a double membrane spanning topology with both extremities orientated luminally in the ER (Carrère-Kremer et al., 2002), the C-terminal part of E2p7 proteins has been found to be located in the cytosol (Isherwood and Patel, 2005). These data suggest 123

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that p7 can potentially adopt a dual transmembrane topology. It remains, however, to be shown whether an E2p7 with a cytosolic orientation of the C-terminus of p7 exists when this protein is expressed in the context of the polyprotein. Since p7 and NS2 are not essential for HCV genomic replication (Lohmann et al., 1999; Blight et al., 2000), they will likely play their role in virion assembly, a process that is supposed to be tightly regulated. It has recently been shown that p7 reconstituted into artificial lipid membranes homo-oligomerizes and behaves as an ion channel protein (Griffin et al., 2003; Pavlovic et al., 2003; Premkumar et al., 2004). It is likely that, when bound to E2, p7 cannot oligomerize and function as an ion channel, and the existence of E2p7 would therefore reduce the amount of functional p7 molecules available. Production of precursors like E2p7NS2 and E2p7 might be a means to maintain p7 inactive during the phase of the accumulation of E2 molecules required for HCV envelope formation. Alternatively, such precursors might also control the temporal release of E2 and NS2. GLYCOSYLATION OF HCV ENVELOPE GLYCOPROTEINS

N-linked glycosylation is one of the most common types of protein modification, and it occurs by the transfer of an oligosaccharide from a lipid intermediate to an Asn residue in the consensus sequence Asn-X-Thr/Ser of a nascent protein, where X is any amino acid except Pro (Kornfeld and Kornfeld, 1985; Gavel and von Heijne, 1990). The addition of this glycan is catalyzed by the oligosaccharyltransferase, which is closely associated with the translocon through which the nascent peptidic chains emerge in the ER lumen (Silberstein and Gilmore, 1996). However, not every tripeptide sequence in a protein sequence is used for carbohydrate addition (Gavel and von Heijne, 1990). In the early secretory pathway, the glycans play a role in protein folding, quality control and certain sorting events. Viral envelope proteins usually contain N-linked glycans that can play a major role in their folding, in their entry functions or in modulating the immune response (Hebert et al., 1997; Ohuchi et al., 1997a; Ohuchi et al., 1997b; van Kooyk and Geijtenbeek, 2003; von Messling and Cattaneo, 2003; Wei et al., 2003). The ectodomains of HCV envelope glycoproteins E1 and E2 are highly modified by N-linked glycans. E1 and E2 possess up to 6 and 11 potential glycosylation sites, respectively (Fig. 2). Sequence analyses of E1 indicate that 5 potential Nglycosylation sites are strongly conserved among HCV genotypes (Goffard and Dubuisson, 2003; Zhang et al., 2004b). However, in one case the presence of a proline residue immediately downstream the glycosylation site is unfavorable for glycosylation, and it has been confirmed experimentally that this site is not glycosylated (Meunier et al., 1999). Interestingly, the glycosylation site of E1 at position 250 is poorly conserved; this site is indeed only observed in genotypes 1b and 6 (Fig. 2)(Goffard and Dubuisson, 2003). Most E2 glycosylation sites are

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Fig. 2. Schematic representation of E1 and E2 features. Positions of N-linked glycans are indicated as an N followed by a number related to the relative position of the potential glycosylation site in each glycoprotein. The numbers correspond to the positions in the polyprotein of reference strain H (acc. Number AF009606). Glycans involved in HCVpp entry are indicated with a black square (Goffard et al., 2005). Glycosylation sites for which the mutation alters E1E2 folding are indicated with a grey circle (Goffard et al., 2005). The hypervariable region 1 (HVR1) of E2 is shown as a grey box. The black boxes correspond to E2 epitopes recognized by neutralizing antibodies (Hsu et al., 2003). The sequences of the transmembrane domains of HCV envelope glycoproteins are indicated above their corresponding region in E1 and E2. The two hydrophobic segments in these regions are underlined. The charged residues present between the two hydrophobic stretches are in white lettering. Arrows indicate the positions of inserted alanine residues that disrupt HCV E1E2 heterodimerization (Op De Beeck et al., 2000).

also well conserved. Indeed, global sequence analyses of potential glycosylation sites in E2 indicate that nine of the eleven sites are strongly conserved (Goffard and Dubuisson, 2003; Zhang et al., 2004b). The two remaining sites, N5 and N7, show conservation levels of 75% and 89%, respectively (Goffard and Dubuisson, 2003). Mutants of E1 and E2 have been produced to characterize the glycosylation of these proteins (Meunier et al., 1999; Nakano et al., 1999; Slater-Handshy et al., 2004; Goffard et al., 2005). In the context of the H strain, the 4 potential glycosylation sites of E1 were shown to be occupied by glycans (Meunier et al., 1999; Goffard et al., 2005). In the case of E2, a first study has shown that mutation of some glycosylation sites in the context of a truncated form of E2 alters the recognition by sera from HCV patients (Nakano et al., 1999); however, these mutants were not characterized in terms of glycosylation and no clear conclusion can be drawn from this study. More recently, glycosylation mutants have been produced in the context of a truncated form of E2 ending at position 660 (Slater-Handshy et al., 2004). The E2 sequence of HCV isolate used in this study contains 10 instead of 11 potential 125

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glycosylation sites, the site N5 at position 476 being missing (Fig. 2). Interestingly, the last two glycosylation sites, N10 and N11, were not occupied in E2660 (SlaterHandshy et al., 2004). However, at least one of these sites was occupied in the context of full-length E2. A more recent mutagenesis study, in the context of an E2 glycoprotein containing 11 potential glycosylation sites, has shown that all the sites are occupied by glycans (Goffard et al., 2005). In this case, E2 was expressed as a polyprotein containing full-length E1 and E2. Altogether, these data indicate that full-length and truncated forms of E2 can have different properties. The addition of the glycan precursor is catalyzed by the oligosaccharyltransferase, and this enzyme is thought to have access only to nascent chains as they emerge from the ribosome at the luminal face of the rough ER (Silberstein and Gilmore, 1996). The glycosylation process of HCV envelope glycoprotein E1 has been analyzed in the context of a Man-P-Dol-deficient cell line (B3F7) and it has been shown to occur post-translationally (Duvet et al., 2002), indicating that the oligosaccharyltransferase has also access to the E1 glycoprotein for more than an hour after its translation. A characterization of HCV glycoprotein E1 has also shown that, in the absence of E2, different glycoforms of E1 are produced and the glycosylation of E1 is improved by co-expression of E2 in cis (Dubuisson et al., 2000). FOLDING OF HCV ENVELOPE GLYCOPROTEINS

HCV envelope glycoproteins have been shown to assemble as a noncovalent E1E2 heterodimer (Deleersnyder et al., 1997). However, at least in heterologous expression systems, HCV envelope glycoproteins have a tendency to also form misfolded aggregates stabilized by disulfide bonds (reviewed in (Dubuisson, 2000)). Analyses of HCV envelope glycoproteins with conformation-sensitive antibodies are therefore necessary to discriminate noncovalent heterodimers from misfolded complexes (Deleersnyder et al., 1997; Cocquerel et al., 2003b). Alternatively, such discrimination can also be made by analyzing disulfide-bond formation by migrating HCV envelope glycoproteins on SDS-PAGE under nonreducing conditions (Dubuisson and Rice, 1996; Brazzoli et al., 2005). Analyses of the formation of conformation-dependent epitopes and disulfide-bond formation indicate that folding of HCV envelope glycoproteins is a slow process (Deleersnyder et al., 1997; Dubuisson and Rice, 1996; Duvet et al., 1998; Brazzoli et al., 2005). Interestingly, the folding of E1 has been shown to be dependent on the co-expression of E2 (Michalak et al., 1997; Patel et al., 2001). In addition, it has also been shown that the folding of E2 is also dependent on the co-expression of E1 (Cocquerel et al., 2003a; Brazzoli et al., 2005). Altogether, these observations indicate that HCV envelope glycoproteins cooperate for the formation of a functional complex. These observations also indicate that, although some degree of folding can be observed in E2 expressed alone (Michalak et al., 1997; Cocquerel et al., 2003a), both glycoproteins need to be co-expressed to analyze their functional properties.

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During their folding, HCV envelope glycoproteins have been shown to interact with calnexin (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001; Brazzoli et al., 2005), a lectin-like ER chaperone, which shows an affinity for monoglucosylated N-linked oligosaccharides (Trombetta and Helenius, 1998). Both E1 and E2 have been found to associate rapidly with calnexin and dissociate slowly, suggesting a role of this chaperone in the folding of HCV envelope glycoproteins (Dubuisson and Rice, 1996; Choukhi et al., 1998; Merola et al., 2001). However, more recent data suggest that only E1 interacts with calnexin (Brazzoli et al., 2005). Differences in the cell lines used and/or in the levels of expression of the envelope glycoproteins might potentially explain these discrepancies. Further experiments in cell cultures infected with native HCV particles will be needed to confirm the involvement of calnexin in the folding E2. The presence of glycans on HCV envelope glycoproteins can potentially affect their folding either directly or through interaction with calnexin. Site-directed mutagenesis studies have indeed shown that the absence of some glycans in E1 (N1 and N4) and E2 (N8 and N10) leads to misfolding of HCV envelope glycoproteins (Fig. 2)(Meunier et al., 1999; Goffard et al., 2005). This alteration in folding was not due to the lack of interaction of HCV envelope glycoproteins with calnexin, suggesting that the mutations would rather have a direct effect on protein folding. The presence of a large polar saccharide is indeed known to affect the folding at least locally by orienting polypeptide segments toward the surface of protein domains (Imperiali and O'Connor, 1999; Wormald and Dwek, 1999). INVOLVEMENT OF THE TRANSMEMBRANE DOMAINS IN THE BIOGENESIS OF E1E2 HETERODIMER MEMBRANE ANCHOR AND SIGNAL SEQUENCE

Due to their resistance to alkaline or salt extraction, HCV envelope glycoproteins have been confirmed to be membrane associated proteins (Ralston et al., 1993; Cocquerel et al., 2001). In addition, deletion of the C-terminal hydrophobic regions of these proteins leads to their secretion, indicating that these regions are involved in membrane anchoring (Michalak et al., 1997). Sequence analysis of a large number of HCV isolates has shown that the C-termini of E1 and E2 contain hydrophobic sequences that are less than 30 amino acid residues long (Fig. 2)(Cocquerel et al., 2000). As in other viruses of the Flaviviridae family, these regions are composed of two stretches of hydrophobic residues separated by a short segment containing at least one fully conserved positively charged residue (Cocquerel et al., 2000). Interestingly, when fused to a reporter protein the second hydrophobic stretch functions as a signal sequence (Cocquerel et al., 2002), which is in agreement with the observation that HCV envelope glycoproteins are released from the polyprotein precursor after cleavage by host signal peptidase(s) (Dubuisson et al., 2002). It is worth noting that in the context of HCV polyprotein, only the sequence located at 127

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the C-terminus of the immature form of the capsid protein is a true signal peptide that will interact with the signal recognition particle (Santolini et al., 1994). The sequence present at the C-terminus of E1 and E2 do not interact with the signal recognition particle, and they should be called signals of reinitiation of translocation (Fig. 1). Deletion of these signals leads to the secretion of E1 and E2, indicating that these signals are involved in their membrane anchoring (Cocquerel et al., 2000). ER RETENTION FUNCTION

HCV envelope glycoproteins are retained in the ER (Dubuisson et al., 1994; Deleersnyder et al., 1997; Duvet et al., 1998), and ER retention signals are present in the transmembrane domains of E1 and E2 (Cocquerel et al., 1998; Cocquerel et al., 1999). In addition, the charged residues of the transmembrane domains of E1 (Lys) and E2 (Asp and Arg) play a key role in the ER retention of these glycoproteins (Cocquerel et al., 2000). It has been proposed that an additional ER retention signal might also be present in the ectodomain of E1 (Mottola et al., 2000). Interestingly, in some conditions of overexpression a small fraction of HCV envelope glycoproteins has been shown to accumulate at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). Cell surface expression of E1 and E2 is likely due to the accumulation of small amounts of glycoproteins escaping the ER-retention machinery, due to saturation of this mechanism. ROLE IN HETERODIMERIZATION

In addition to their anchoring, signal sequence and ER retention functions, the transmembrane domains of HCV envelope glycoproteins have also been shown to play a major role in the assembly of E1E2 heterodimer. Indeed, deletion of the transmembrane domain of E2 or its replacement by the anchor signal of another protein abolishes the formation of E1E2 heterodimer (Selby et al., 1994; Michalak et al., 1997; Cocquerel et al., 1998; Patel et al., 2001). Other studies by site-directed mutagenesis or alanine scanning insertion mutagenesis (Cocquerel et al., 2000; Op De Beeck et al., 2000) have confirmed that the transmembrane domains of E1 and E2 play a direct role in E1E2 assembly. In addition, alanine scanning insertion mutagenesis allowed to identify two distinct segments in the transmembrane domain of E1 and one in the transmembrane domain of E2 that were specifically involved in E1E2 assembly (Fig. 2). Interestingly, at least one region located outside of the transmembrane domains has also been shown to be involved in heterodimerization (Drummer and Poumbourios, 2004). TOPOLOGICAL CHANGE IN THE TRANSMEMBRANE DOMAIN OF HCV GLYCOPROTEINS

The topology of the transmembrane domain of HCV envelope glycoproteins has given rise to some controversy. Indeed the presence of a first hydrophobic stretch and a signal sequence function separated by charged residues in the transmembrane domains of E1 and E2 has suggested that they might be composed of two membrane 128


spanning segments with the charged residues facing the cytosol (Charloteaux et al., 2002). This type of organization has been observed in the C-terminal region of the envelope glycoprotein E2 of the alphaviruses as well as for the envelope proteins of the flaviviruses (Strauss and Strauss, 1994; Op De Beeck et al., 2003; Zhang et al., 2003). However sequence analysis and data of alanine scanning insertion mutagenesis were in favor of a single spanning topology of E1 and E2 transmembrane domain (Cocquerel et al., 2000; Op De Beeck et al., 2000). A study of the topology of the transmembrane domains of HCV envelope proteins has been performed by determining the accessibility of their N- and C-termini in selectively permeabilized cells (Cocquerel et al., 2002). This work has shown that before signal sequence cleavage at their C-terminus, the transmembrane domains form a hairpin structure (Fig. 1). However, after cleavage between E1 and E2 or between E2 and p7, the second C-terminal hydrophobic stretch is reoriented towards the cytosol, leading to the formation of a single membrane-spanning domain. Here again, the charged residues located in the middle of the transmembrane domains were shown to play a crucial role in their structural dynamics (Cocquerel et al., 2002).

ROLE OF HCV ENVELOPE GLYCOPROTEINS IN VIRUS ENTRY For most viruses, entry into the cytosol is a multistep process, during which the host cell assists the incoming virus. Viruses first attach themselves to components of the plasma membrane, which they use as non-specific attachment factors or as specific cell surface receptors. Viral attachment is mediated by the binding of a protein present at the surface of the virion to a molecule on the cell surface acting as a virus receptor. The envelope glycoprotein complex E1E2 is the viral component thought to be present at the surface of HCV particles and it is therefore the obvious candidate ligand for cellular receptors. Receptor binding can activate cellular endocytic pathways through which viruses are internalized in endosomes. When they reach the appropriate intracellular location, viruses are activated for penetration by cellular signals and make their way through the membrane of the endosome, or through the plasma membrane for those that do not enter by endocytosis. Enveloped viruses fuse their lipid envelope with the plasma membrane or the membrane of an endosome, resulting in the release of the nucleocapsid into the cytosol. MODELS TO STUDY HCV ENTRY

In the absence of a robust cell culture system to amplify HCV, several models have been developed to study HCV entry. In a first approach, a soluble form of HCV glycoprotein E2 has been used to identify cell surface proteins potentially involved in HCV entry (Rosa et al., 1996; Pileri et al., 1998). Although this approach is potentially interesting in protein-protein interactions studies, it cannot be used to study the entire entry process. In addition, as discussed above, due to their cooperative role in folding, both glycoproteins need to be co-expressed to analyze their functional properties. To study the role of E1E2 envelope glycoproteins in HCV

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entry, several surrogate models of HCV particles have therefore been developed. As a first approach, virus-like particles have been produced in insect cells infected by a recombinant baculovirus containing the cDNA of HCV structural proteins (Baumert et al., 1998). However these particles are not infectious and they are retained in an intracellular compartment. It is therefore difficult to evaluate how close these virus-like particles are to native virion. In addition, due to the absence of infectivity, these particles cannot be used to study the fusion process. Another approach to study HCV entry has been to produce virosomes by incorporating E1E2 heterodimers into liposomes (Lambot et al., 2002). These virosomes can be used to study the interactions between E1E2 heterodimers and cell surface receptors. However, it has not been shown whether the envelope glycoproteins incorporated into these liposomes can induce fusion. Other models have been based on pseudotyping of viral vectors. The first model that has been developed was based on vesicular stomatitis virus (VSV) pseudotyped with modified E1 and/or E2 glycoproteins (Lagging et al., 1998; Matsuura et al., 2001). In these particles, the transmembrane domains of HCV envelope glycoproteins have been replaced by the transmembrane domain and cytoplasmic tail of the VSV envelope glycoprotein G. This allows the export of HCV envelope glycoproteins to the cell surface (Takikawa et al., 2000). However, some doubts have been raised on the infectivity of such VSV pseudotyped particles (Buonocore et al., 2002). In addition, replacement of HCV envelope glycoproteins has been shown to alter their entry function (Hsu et al., 2003). More recently, retroviruses have also been used to produce pseudotyped particles containing HCV envelope glycoproteins (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Murine leukemia virus (MLV) or human immunodeficiency virus (HIV) vectors were used. Retroviruses are indeed well known to be able to incorporate in their envelope a variety of cellular and viral glycoproteins (Ott, 1997; Sandrin et al., 2002). In addition, they can easily package and integrate genetic markers into host cell DNA (Negre et al., 2002). All these properties were exploited to produce viral pseudoparticles expressing E1E2 at their surface and packaging a reporter gene that allows to monitor viral infection of the target cell. HCV pseudoparticles (HCVpp) are produced by transfecting 293T cells with three expression vectors encoding the E1E2 polyprotein, the retroviral core proteins and a packaging-competent retrovirus-derived genome containing a marker gene (Fig. 3). Because MLV and HIV are supposed to assemble at the plasma membrane and HCV glycoproteins are retained in the ER, a first approach has been to modify the transmembrane domains of E1 and E2 to re-address them at the plasma membrane (Hsu et al., 2003; Pohlmann et al., 2003). However pseudoparticles bearing such modified HCV envelope glycoproteins were not infectious. Surprisingly, in the absence of any modification of HCV envelope glycoproteins, infectious

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Fig. 3. Production of HCV pseudoparticules (HCVpp). For the production of HCVpp, human embryo kidney cells 293T are transfected with three expression vectors. The first vector encodes retroviral Gag and Pol proteins. Gag proteins are responsible for particle budding at the plasma membrane and RNA encapsidation via recognition of the specific retroviral encapsidation sequence (ψ). The second vector harbors a ψ sequence for encapsidation and encodes a reporter protein (Luciferase). This vector also contains retroviral sequences that are necessary for the reverse transcription of genomic RNA into proviral DNA and for integration of the proviral DNA in the host genomic DNA by the retroviral protein Pol encoded by the first vector. The third vector encodes HCV envelope glycoproteins, which are responsible for the cell tropism and fusion of HCVpp with the target cell membrane. HCVpp contain Gag, Pol, E1 and E2 proteins as well as the RNA encoding the luciferase protein. Infectivity of HCVpp is evaluated by measuring the amount of luciferase expressed in target cells.

pseudoparticles were produced (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003). Interestingly, due to saturation of the ER retention machinery, the cells used to produce HCVpp were shown to express a small fraction of HCV 131

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envelope glycoproteins at the plasma membrane (Bartosch et al., 2003b; Drummer et al., 2003; Hsu et al., 2003; Op De Beeck et al., 2004). This accumulation at the plasma membrane might therefore be sufficient to incorporate native HCV envelope glycoproteins into retroviral pseudotyped particles. The data that have been accumulated on these pseudoparticles strongly suggest that they mimic the early steps of HCV infection. Indeed, they exhibit a preferential tropism for hepatic cells and they are specifically neutralized by anti-E2 monoclonal antibodies as well as sera from HCV-infected patients (Bartosch et al., 2003b; Hsu et al., 2003; Op De Beeck et al., 2004). These HCVpp therefore represent the best tool available to study functional HCV envelope glycoproteins. An analysis of the glycoproteins associated with HCVpp has shown the heterogeneous nature of E1 and E2 incorporated into HCVpp (Flint et al., 2004). This highlights the difficulty in identifying forms of the HCV glycoproteins that initiate infection. However, characterization of HCVpp envelope glycoproteins with conformationsensitive neutralizing monoclonal antibodies has shown that the functional unit is a noncovalent E1E2 heterodimer (Op De Beeck et al., 2004). In addition, coexpression of both envelope glycoproteins has been shown to be necessary to produce infectious pseudoparticles (Bartosch et al., 2003b), confirming that only the E1E2 heterodimer is functional. HCV RECEPTORS

As a first approach to identify potential HCV receptor(s), a soluble form of HCV glycoprotein E2 has been used. This allowed to identify the CD81 tetraspanin (Levy and Shoham, 2005) as a putative receptor for HCV (Pileri et al., 1998). A very similar approach identified the scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002), a high-density lipoprotein (HDL)-binding molecule (Connelly and Williams, 2004), and the mannose binding lectins DC-SIGN and L-SIGN (van Kooyk and Geijtenbeek, 2003) as additional candidate receptors for HCV (Gardner et al., 2003; Lozach et al., 2003; Pohlmann et al., 2003; Ludwig et al., 2004). Heparan sulfate has also been shown to interact with HCV glycoprotein E2, suggesting that this type of molecule can play a role in HCV entry (Barth et al., 2003). An approach using virus-like particles produced in insect cells has led to the identification of the asialoglycoprotein receptor as another candidate receptor for HCV (Saunier et al., 2003). Finally, because of the physical association of HCV with low- or very-low-density lipoproteins (LDL or VLDL) in serum, the LDL receptor has also been proposed as another candidate receptor for HCV. (Agnello et al., 1999; Monazahian et al., 1999). A number of cell-surface molecules bind viral envelope glycoproteins without mediating entry, and validation of a viral receptor or co-receptor requires proof that the putative receptor is necessary for infection. This is not easy for HCV due

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to the absence of a robust cell culture system to amplify this virus. The recent development of HCVpp has allowed to further investigate the role of candidate receptors in virus entry. Among all the candidate receptors, only CD81 and SR-BI have been shown to play a direct role in HCVpp entry. Indeed, antibodies directed against CD81 or SR-BI as well as siRNA targeting these receptors reduce HCVpp infectivity (Bartosch et al., 2003b; Bartosch et al., 2003c; Hsu et al., 2003; Cormier et al., 2004b; Zhang et al., 2004a; Lavillette et al., 2005b). A soluble domain of CD81 is also able to compete with HCVpp infectivity (Bartosch et al., 2003b; Hsu et al., 2003). In addition, HDL, the natural ligands of SR-BI, are able to markedly enhance HCVpp entry (Meunier et al., 2005; Voisset et al., 2005). This HDLmediated enhancement of HCVpp entry involves a complex interplay between SR-BI, HDL and HCV envelope glycoproteins (Voisset et al., 2005). Interestingly, the involvement of CD81 and SR-BI in HCVpp entry seems to be conserved among all the HCV genotypes (McKeating et al., 2004; Lavillette et al., 2005b). Interactions between viral envelope glycoproteins and potential receptors can have other consequences than virus entry. It has been shown that intracellular interaction between HCV envelope glycoproteins and CD81 can lead to secretion of exosomes containing E1 and E2 glycoproteins (Masciopinto et al., 2004). Interestingly, a soluble form of E2 is also able to bind CD81 at the surface of natural killer cells, and this interaction inhibits cytotoxicity and cytokine production by these cells (Crotta et al., 2002; Tseng and Klimpel, 2002). Binding of a soluble form of E2 can also provide a co-stimulatory signal for T cells (Wack et al., 2001; Soldaini et al., 2003;) and up-regulate matrix metalloproteinase-2 in human hepatic stellate cells (Mazzocca et al., 2005). It remains however to be determined whether HCV glycoprotein expressed in the context of native particles will have the same effects on cell functions. HCVpp have also been used to investigate the role of other candidate receptors in HCV entry. HCVpp as well as native HCV particles have been shown to bind to cells expressing L-SIGN and DC-SIGN (Gardner et al., 2003; Pohlmann et al., 2003; Lozach et al., 2004). Although these molecules are not expressed on hepatocytes, HCV interactions with L-SIGN and DC-SIGN may contribute to establishment or persistence of infection both by the capture and delivery of virus to the liver and by modulating dendritic cell functions as recently suggested (Cormier et al., 2004a; Lozach et al., 2004). Finally, there is no clear evidence that the LDL receptor is a major receptor for HCVpp (Bartosch et al., 2003b). Interestingly, all the cells permissive to HCVpp co-express CD81 and SR-BI and are of liver origin (Bartosch et al., 2003c; Hsu et al., 2003; Zhang et al., 2004a). However, there are some other cell lines coexpressing CD81 and SR-BI that are non-permissive to infection and which are of non-hepatic origin. These results

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suggest that additional molecule(s), expressed in hepatic cells only, are necessary for HCV entry. Further investigations with HCVpp should allow to identify such molecule(s). FUNCTIONAL REGIONS OF HCV ENVELOPE GLYCOPROTEINS

HCVpp have been used to investigate the functional role of some regions of HCV envelope glycoproteins in virus entry. Mutagenesis studies of the transmembrane domains of HCV envelope glycoproteins have shown that some mutations can affect the entry function of HCVpp without alteration in the biogenesis of E1E2 heterodimer and their incorporation into HCVpp (Ciczora Y, Callens N, Montpellier C, Bartosch B, Cosset FL, Op De Beeck A, Dubuisson J, unpublished data). This suggests that in addition to their role in E1E2 heterodimerization, the transmembrane domains of HCV glycoproteins might play a role in coordinating protein reorganization for the fusion process to occur. Studies of E2-CD81 interactions and identification of epitopes recognized by antibodies that inhibit these interactions suggest that the CD81-binding region consists of discrete segments of E2 that are rearranged within the same domain during E2 folding (Flint et al., 1999a; Forns et al., 2000a; Yagnik et al., 2000; Owsianka et al., 2001; Clayton et al., 2002; Hsu et al., 2003). Besides this putative binding region, the hypervariable region 1 (HVR1)(Weiner et al., 1991), a 27-amino acid long segment found at the N-terminus of E2 (Fig. 2), has also been suggested to play a role in cell attachment (Penin et al., 2001; Scarselli et al., 2002). This region evolves rapidly in infected individuals, suggesting that it is under strong immune pressure (reviewed in Mondelli et al., 2003). Although an HCV clone lacking HVR1 was shown to be infectious in chimpanzee, this mutant virus was attenuated, suggesting that HVR1 plays a facilitating role in HCV infectivity (Forns et al., 2000b). In addition, deletion of HVR1 reduces HCVpp infectivity (Bartosch et al., 2003c) and abolishes HDL-mediated enhancement of HCVpp infectivity (Voisset et al., 2005). Despite strong amino acid sequence variability related to strong pressure towards change, the chemicophysical properties and conformation of HVR1 are highly conserved, and HVR1 is a globally basic stretch, with basic residues located at specific sequence positions. Functional studies of HCVpp containing mutations in HVR1 indicate that infectivity increases with the number of basic residues in HVR1 (Callens N, Ciczora Y, Bartosch B, Vu-Dac N, Cosset FL, Pawlotsky JM, Penin F, Dubuisson J, unpublished data). In addition, a shift in position of some charged residues modulates infectivity. These data suggest that HVR1 is a region involved in interaction with a host molecule involved in HCV entry. However, it remains to be determined whether SR-BI or another putative receptor is involved in this interaction.

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HCV envelope glycoproteins are highly glycosylated and some maturation of these glycans has been observed on HCV envelope glycoproteins associated with HCVpp (Flint et al., 2004; Lozach et al., 2004; Op De Beeck et al., 2004). Mutation of some glycosylation sites in HCV envelope glycoproteins can reduce or abolish HCVpp infectivity without apparently affecting folding and incorporation of the glycoproteins into the particles (Goffard et al., 2005). N-linked glycans at position N2 and N4 of E2 have indeed been shown to be essential for the entry functions of HCV envelope glycoproteins (Fig. 2). In addition, some other glycans (N2 of E1 and N5, N6 and N11 of E2) can also modulate HCVpp entry. Further studies will be necessary to determine whether these mutations affect receptor binding or the fusion properties of HCV envelope glycoproteins. MECHANISMS OF HCV ENTRY

Virus attachment to receptors initiates a series of events that lead to virus entry. For enveloped viruses, the entry process is controlled by viral surface glycoproteins that undergo triggered conformational changes from a metastable state to a lower energy state. This structural change leads to the exposure of a buried functional element, named the fusion peptide and is believed to provide the energy required for the merging of the lipid bilayers (reviewed in Colman and Lawrence, 2003). So far, viral fusion proteins have been shown to fall into two different structural classes designated as class I and II (reviewed in Earp et al., 2005). Class I fusion proteins possess N-terminal or N-proximal fusion peptides, and they are synthesized as a precursor that is cleaved into two subunits by host cell proteases. In some cases (e.g., influenza HA), the two subunits remain associated through a disulfide bond, whereas in others (e.g. HIV Env) the two subunits remain associated through noncovalent interactions. The proteolytic processing event creates the metastable state of the fusion protein (Colman and Lawrence, 2003). In their native metastable conformation, class I fusion proteins form trimeric spikes at the surface of the virions with the fusion subunit being highly helical. Upon a fusion trigger event (receptor binding at the cell surface or low pH in endosomes), the trimeric proteins transiently form an extended conformation allowing the hydrophobic fusion peptide to insert into the target membrane. Protein refolding leads then to the formation of very stable trimeric structures in which both the N-proximal fusion peptide and the C-proximal membrane anchor are juxtaposed at the same end to allow virus and cell membrane connection and hemifusion (reviewed in Colman and Lawrence, 2003). Class II viral fusion proteins have a completely different structure. They are predominantly non-helical, instead having a beta-sheet type structure; they are not cleaved during biosynthesis; and they possess an internal fusion peptide with a loop conformation (reviewed in Earp et al., 2005). The proteins are oriented parallel to the membrane, and they have a three-domain architecture with domain I beginning at the N-terminus, domain II containing the internal fusion loop, and domain III

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being at the C-terminus. In addition, class II fusion proteins are synthesized as a complex with a second membrane glycoprotein (prM for flaviviruses; pE2 for alphaviruses). Newly synthesized E and prM proteins of the tick borne encephalitis virus associate to form noncovalent heterodimers (Fig. 4) that are incorporated into immature virions by budding into the ER lumen (Allison et al., 1995; Mackenzie and Westaway, 2001). These particles are then transported through the secretory pathway and shortly before release from the cell, the activation of the fusogenic potential occurs by the cleavage of the accessory protein prM by a cellular furin protease in the trans-Golgi network (Stadler et al., 1997). After prM cleavage, the E protein exists as a metastable homodimers at the virion surface. The ectodomains of the dimers are orientated antiparallel to one another (Rey et al., 1995; Lescar et al., 2001; Modis et al., 2003). The architecture of the alphavirus Semliki Forest virus spike is similar to that of tick borne encephalitis virus E, but in this case, the metastable oligomer is a heterodimer of the fusion protein E1 and the companion protein E2 with an associated small protein E3 (Lescar et al., 2001). In addition,

Fig. 4. Comparison of flavivirus and hepacivirus envelope proteins. In the Flaviviridae family, class II fusion proteins (depicted in light grey) have been described in the flaviviruses (E protein of tick born encephalitis and dengue viruses). They are synthesized as a complex with a second membrane glycoprotein (depicted in dark grey). Shortly before release from the cell, activation of the fusogenic potential occurs by cleavage of the accessory protein (arrow). HCV envelope glycoproteins are supposed to belong to the class II fusion proteins, but contrary to flaviviruses, HCV envelope proteins are highly glycosylated and are not matured by a cellular endoprotease during their transport through the secretory pathway.

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contrary to flaviviruses, alphaviruses have been shown to bud from the plasma membrane. Both alphaviruses and flaviviruses enter target cells by receptor-mediated endocytosis. The receptor recognition function is carried by the fusion protein itself for the flaviviruses (E) and by the companion protein (E2) for the alphaviruses. Exposure to the acidic pH of the endosomes triggers a major conformational change of the envelope involving dissociation of the native homodimer (for flaviviruses) or heterodimer (for alphaviruses) and the irreversible formation of homotrimers of the fusion proteins (Earp et al., 2005; Mukhopadhyay et al., 2005). Based on its classification in the Flaviviridae family, HCV envelope has been proposed to contain a class II fusion protein (Yagnik et al., 2000). As found in the case of alphaviruses and flaviruses, HCVpp entry is pH dependent (Bartosch et al., 2003c; Hsu et al., 2003). These observations indicate that HCV may enter the cells through endocytosis. The cell surface receptor(s) recognized by HCV should therefore traffic cell-bound virions to endosomal compartments. However, characterization of the route of HCV entry needs further investigations. Contrary to what is observed for other class II envelope proteins, there is no evidence that HCV envelope glycoproteins are matured by a cellular endoprotease during their transport through the secretory pathway (Op De Beeck et al., 2004). In addition, HCV envelope glycoproteins are highly glycosylated, whereas other described class II envelope proteins contain a very low number of glycans (Fig. 4). Interestingly, some of the glycans present on HCV envelope glycoproteins seem to be involved in controlling HCV entry (Goffard et al., 2005). There remains some controversy on the identity of HCV fusion protein. It has been proposed that E1 might be a good candidate because sequence analyses suggest that it might contain a putative fusion peptide in its ectodomain (Flint et al., 1999b; Garry and Dash, 2003). On the other hand, potential structural homology with other class II fusion proteins suggests that E2 could be the fusion protein (Yagnik et al., 2000). Mutagenesis studies in the putative fusion peptides of the envelope glycoproteins associated with HCVpp as described for the flavivirus envelope protein E (Allison et al., 2001), should be helpful for further characterization of HCV fusion protein. In addition, a high-resolution structure of HCV envelope glycoproteins would also help understanding the fusion mechanism of the virus. INHIBITION OF HCV ENVELOPE GLYCOPROTEIN FUNCTIONS BY NEUTRALIZING ANTIBODIES

Because they are exposed at the surface of the virion, the envelope proteins are targets of neutralizing antibodies. These antibodies block a viral infection by inhibiting virion binding or membrane fusion. Understanding the mechanisms of neutralization needs therefore a good knowledge of the mechanism of entry. The 137

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role of neutralizing antibodies in HCV infection and disease progression remained unclear for a long time, largely because of the lack of assays to measure and quantify their activity. Previous experiments showed that serum from a chronically infected patient could neutralize HCV infectivity in a chimpanzee model, giving evidence for antibody-mediated neutralization of HCV (Farci et al., 1994). Neutralizing antibodies could also be identified by their ability to prevent HCV replication in a lymphoid cell line (Shimizu et al., 1994; Shimizu et al., 1996). The recent development of HCVpp offers the possibility to study HCV neutralization with defined sequences of HCV envelope glycoproteins, and the use of HCVpp in neutralization studies has been validated (Bartosch et al., 2003a). As determined with HCVpp, it seems that the majority of chronically infected patients have cross-reactive neutralizing antibodies (Logvinoff et al., 2004; Meunier et al., 2005). In contrast, neutralizing antibodies have not been detected in several cases of acute resolving infection (Logvinoff et al., 2004; Meunier et al., 2005), and the detection of neutralizing antibodies in acutely infected individuals did not seem to be associated with viral clearance (Logvinoff et al., 2004). However, another study has shown in some patients a progressive emergence of a relatively strong neutralizing response in correlation with a decrease in viremia (Lavillette et al., 2005a). Further investigations on a large number of acutely infected patients will be necessary to determine the role of neutralizing antibodies in controlling HCV infections. Interestingly, it has been observed that HCVpp infectivity is enhanced by human sera, and this enhancement of infectivity can partly mask the presence of neutralizing antibodies (Lavillette et al., 2005a; Meunier et al., 2005). In addition, HDL have been identified as the component responsible for serum-mediated enhancement of infectivity (Meunier et al., 2005; Voisset et al., 2005). For a long time, the HVR1 sequence of E2 has been proposed to be a major target for neutralizing antibodies (Kato et al., 1993; Farci et al., 1996). However, data obtained with the HCVpp model indicate that neutralizing epitopes located outside of HVR1 also exist (Bartosch et al., 2003a). Interestingly, characterization of HCVpp with monoclonal antibodies has allowed to identify conformation-dependent and -independent neutralizing epitopes outside of HVR1 (Fig. 2)(Bartosch et al., 2003b; Hsu et al., 2003; Keck et al., 2004; Op De Beeck et al., 2004). Conformationdependent human monoclonal antibodies have also allowed to identify three immunogenic domains in E2 with neutralizing antibodies being restricted to two of these domains (Keck et al., 2004). Whether E2 domains identified with these monoclonal antibodies are similar to the antigenic structural and functional domains of the envelope protein E of the flaviviruses (Rey et al., 1995) remains to be determined.

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CONCLUSION Studies of the biogenesis of HCV envelope glycoproteins have shown the pivotal role of the transmembrane domains in the assembly of a noncovalent E1E2 heterodimer in the ER. More recently, the development of the HCVpp model has allowed to investigate the role of E1E2 heterodimer in virus entry. Functional regions in HCV envelope glycoproteins can now be identified and potential receptors can also be validated. Entry is an essential step in the life cycle of a virus, which can potentially be blocked by neutralizing antibodies or antiviral drugs that target the envelope proteins of the virus. Understanding the viral and cellular components involved in HCV invasion into the host cell, combined with a comprehension of the mechanisms that govern this process, should therefore open the possibility of developing new therapeutic approaches.

FUTURE TRENDS The development of the HCVpp model has allowed to initiate the characterization of the entry function of HCV envelope glycoproteins. The use of HCVpp will continue to provide additional information on the role of HCV envelope glycoproteins in viral entry. However, the recent development of a full-length clone that is infectious in cell culture (see chapter 16) provides new opportunities to study the functions of HCV envelope glycoproteins. A comparison of the properties of HCV envelope glycoproteins produced in HCVpp and in this infectious clone will be very useful to validate the data that have been generated during the past three years. In addition, this infectious clone will allow for the first time to decipher the role of HCV envelope glycoproteins in virion assembly. Finally, obtaining a high-resolution structure of HCV envelope glycoproteins will also be necessary to understand the fusion mechanism of this virus.

ACKNOWLEDGMENTS We thank Sophana Ung for preparing the illustrations. Our research was supported by EU grant QLRT-2000-01120 and QLRT-2001-01329 and grants from the "Agence Nationale de Recherche sur le Sida et les hépatites virales" (ANRS), INSERM "ATCHépatite C" and the "Association pour la Recherche sur le Cancer" (ARC).

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Chapter 5

HCV NS2/3 Protease Sarah Welbourn and Arnim Pause

ABSTRACT The hepatitis C virus NS2/3 protein is a highly hydrophobic protease responsible for the cleavage of the viral polypeptide between non-structural proteins NS2 and NS3. However, many aspects of the NS2/3 protease's role in the viral life cycle and mechanism of action remain unknown or controversial. NS2/3 has been proposed to function as either a cysteine or metalloprotease despite its lack of sequence homology to proteases of known function. In addition, although shown to be required for persistent infection in a chimpanzee, the role of NS2/3 cleavage in the viral life cycle has not yet been fully investigated due to the lack of an in vitro system in which to study all aspects of HCV replication. However, several recent studies are beginning to clarify possible roles of the cleaved NS2 protein in modulation of host cell gene expression and apoptosis.

INTRODUCTION The NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. Extending from amino acids 810-1206, NS2/3 is the first non-structural (NS) protein translated and is responsible for the intramolecular cleavage between NS2 and NS3 (see Fig. 1). The amino terminus of NS2 is cleaved from the adjacent p7 protein by host signal peptidases in a membrane-dependent

C E1

E2

P7 NS2

NS3

NS2/3

810

NS2 810

NS4A NS4B

NS5A

NS5B

1206

NS3 1026 1027

1206

Fig. 1. The HCV NS2/3 protease. The NS2/3 protease is shown in the context of the HCV polyprotein. NS2 and the protease domain of NS3 (from aa 810 to 1206) constitute NS2/3, which undergoes autocatalytic cleavage between aa 1026 and 1027.

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manner, while the chymotrypsin-like serine protease located in NS3 is responsible for the cleavage at the NS3/4A and downstream junctions. The HCV NS2/3 protein is an autoprotease whose activity is separate from NS3 protease functions (see Chapter 6). Although many studies have focused on the residues and sequences required for efficient NS2/3 processing, the exact nature of the protease has still not been firmly established, with it being proposed to function as either a novel cysteine or metalloprotease. Furthermore, although all NS proteins are proposed to play a role in viral replication, the exact functions of HCV NS2/3, as well as cleaved NS2 remain largely unexplored; however, some interesting potential functions have emerged in recent years. This chapter will focus on the known properties of the NS2/3 protease as well as the possible functions of both the NS2/3 protease and the NS2 protein.

NS2/3 CATALYTIC CLEAVAGE GENERAL STRUCTURAL FEATURES OF NS2/3

The NS2/3 protease is responsible for the intramolecular cleavage of NS2 from NS3 between aa 1026 and 1027 (Grakoui et al., 1993; Hijikata et al., 1993a). Fig. 2 shows the main structural and functional domains of the protein. NS2 contains a highly hydrophobic N-terminal region suggested to contain multiple transmembrane segments; however, this region is not required for efficient cleavage at the NS2/3 site (Hijikata et al., 1993a; Pallaoro et al., 2001; Thibeault et al., 2001). The minimal domain for activity of the enzyme has been mapped to aa 907-1206 (Pallaoro et al., 2001). This encompasses the C-terminal portion of NS2, immediately following the hydrophobic region, as well as the N-terminal protease domain of NS3. Although these sequences are required and sufficient for cleavage activity, processing is not dependent the NS3 serine protease activity (Grakoui et al., 1993; Hijikata et al., 1993a). This differs from the NS2B protein of flaviviruses, in which the NS3 NS3 Structural Zinc Binding Sites

Hydrophobic Region

810

H952

*

E972 C993

*

C1123 C1125C1171 H1175

**

*

907

** 1206

1026

Minimal Region for NS2/3 activity

NS2

NS3

Fig. 2. Functional domains of the NS2/3 protease. The NS2/3 protease encompasses an N-terminal hydrophobic region, with a minimal domain required for activity between aa 907 and 1206. Residues in NS2 required for NS2/3 processing (H942, E972, C993), as well as residues in NS3 responsible for the coordination of a zinc atom are shown.

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protease performs a cis-cleavage at the NS2B site and then uses NS2B as a cofactor for the processing of the downstream NS polypeptide (Chambers et al., 1991; Chambers et al., 1990; Falgout et al., 1991). NS2/3 PROCESSING REQUIREMENTS

The HCV NS2/3 protease shows no sequence motifs typical of known proteases; however, sequence alignments show similarity with the GBV NS2/3 protein as well as the bovine viral diarrhea virus (BVDV) NS2/3 protein (Lackner et al., 2004). Residues H952, E972 and C993 are conserved among all genotypes of HCV and mutation of H952 or C993 to alanine completely inhibits NS2/3 cleavage activity while a glutamic acid 972 to glutamine substitution also significantly affects processing (Grakoui et al., 1993; Hijikata et al., 1993a). Furthermore, although NS3 serine protease activity is not required for NS2/3 processing, the full NS3 protease domain must be present and cannot be substituted for another NS protein (Santolini et al., 1995). In addition, mutation of cysteine residues 1123, 1127 and 1171 in NS3, which together with H1175 participate in the coordination of a zinc molecule (Kim et al., 1996; Love et al., 1996), abolishes both NS3 and NS2/3 activities (Hijikata et al., 1993a), presumably by disrupting folding of the enzymes. This therefore suggests that the NS3 protease domain is required to play a structural role in the folding of the enzyme. Proper folding of the NS2/3 protein and cleavage site plays an important role in the efficiency of NS2/3 processing. Residues surrounding the cleavage site, WRLL↓APIT, are highly conserved between HCV genotypes but are remarkably resistant to mutations (Hirowatari et al., 1993; Reed et al., 1995). Only mutations severely affecting the conformation of the cleavage site (such as deletion or proline substitution of P1 or P1') severely inhibit cleavage. Furthermore, NS4A-derived peptides that upon binding cause a conformational rearrangement of the NS3 Nterminus are potent inhibitors of NS2/3 activity, likely by altering the positioning of the cleavage site (Darke et al., 1999; Thibeault et al., 2001). The presence of microsomal membranes or non-ionic detergents has been found to be required for in vitro processing at the NS2/3 site in certain genotypes (Pieroni et al., 1997; Santolini et al., 1995), while increasing the efficiency of cleavage of others (Grakoui et al., 1993; Santolini et al., 1995), suggesting the hydrophobic environment is necessary for proper folding of the enzyme and positioning of the cleavage site. Similarly, Waxman et al. (2001) have demonstrated the requirement for the ATP hydrolyzing ability of molecular chaperone HSP90 for efficient cleavage in in vitro and cell based assays. A similar phenomenon has been described for the BVDV NS2/3 protein where a cellular DnaJ chaperone protein, Jiv, has been found to associate with and modulate NS2/3 activity, possibly by causing a conformational change in the protein (Rinck et al., 2001). Although the mechanisms are still unclear, this could point to a role of cellular chaperones in inducing/maintaining the proper conformation of NS2/3 required for cleavage. 153

Welbourn and Pause MECHANISM OF ACTION: CYSTEINE OR METALLOPROTEASE?

Initial studies showing NS2/3 activity is inhibited by EDTA and stimulated by zinc led to the early suggestion that NS2/3 functions as a zinc-dependent metalloprotease (Hijikata et al., 1993a). However, with the discovery of the importance of zinc for the structural integrity of the NS3 protease domain, others have proposed NS2/3 may be a novel cysteine protease with a catalytic dyad comprised of H952 and C993 with the possible involvement of E972 as the third residue of a catalytic triad. Inhibition studies both in in vitro translation systems and with purified proteins have failed to yield a definite classification (Pallaoro et al., 2001; Pieroni et al., 1997; Thibeault et al., 2001). Although inhibited by metal chelators such as phenanthroline and EDTA, this inhibition is relieved by the addition of ZnCl2, CdCl2 or MgCl2. This could therefore point to a structural rather than catalytic role for the zinc molecule as Cd has not traditionally been able to functionally replace Zn in other metalloproteases (Angleton and Van Wart, 1988; Cha et al., 1996; Holland et al., 1995). However, although classical cysteine protease inhibitors iodoacetamide and N-ethylmaleimide show strong inhibition of NS2/3 processing, no single cysteine has been found to be more susceptible to these alkylating agents (Pallaoro et al., 2001). Recently, conserved His, Cys and Glu have also been found to be present in BVDV strains and required for NS2/3 cleavage in vitro (Lackner et al., 2004), suggesting a similar mechanism of action of the two proteases. However, several differences exist. In addition to the necessity of the N-terminal hydrophobic region of NS2, BVDV NS2/3 does not require the full NS3 protease domain for activity, but rather possesses a conserved zinc-binding site within NS2 itself (Lackner et al., 2004). Although no traditional metal-binding sequences have been identified in HCV NS2, the presence of an additional catalytic zinc in NS2 or a catalytic role for the NS3 zinc molecule cannot be definitely ruled out. The elucidation of the so far unknown crystal structure of NS2/3 should bring important insights into the mechanism of cleavage of this enzyme. NS2/3 BIMOLECULAR CLEAVAGE

Bimolecular cleavage of NS2/3 has been shown to occur, albeit inefficiently, in cell transfection experiments (Grakoui et al., 1993; Reed et al., 1995). In this system, NS2/3 proteins with mutations/deletions in either the NS2 or NS3 domains could support cleavage provided the missing functional region was co-expressed on a separate polypeptide. In addition, catalytically inactive NS2/3 mutants were also found to inhibit processing of a wild-type protein when expressed in trans. The observation that a recombinant NS2/3 protein forms dimers in vitro is consistent with these findings (Pallaoro et al., 2001). However, no trans cleavage has been observed using purified proteins (Pallaoro et al., 2001; Thibeault et al., 2001). Interestingly, NS2/3 activity in vitro was found to be concentration dependent, supporting the notion that dimer formation is essential for the reaction (Pallaoro et al., 2001). Dimitrova et al. (2003) have also demonstrated the homo-association of 154

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the NS2 protein in various systems and suggest that the cleavage between NS2 and NS3 could potentially be performed by dimers of NS2/3 encoded on neighbouring polyprotein chains. As NS2/3 cleavage is widely believed to be an intramolecular event, the significance of bimolecular cleavage in the polyprotein processing events of HCV infection in vivo remains to be determined.

ROLE OF NS2/3 CLEAVAGE IN VIRAL REPLICATION The role of the NS2/3 protease in HCV replication remains to be fully understood. NS2/3 cleavage is required for viral replication in vivo, as demonstrated by an HCV clone devoid of NS2/3 activity that fails to cause a persistent infection in a chimpanzee (Kolykhalov et al., 2000). However, NS3-3' UTR subgenomic replicons not encoding the NS2 protein replicate efficiently in Huh-7 cells (Lohmann et al., 1999), suggesting NS2/3 is not strictly required for genome replication. If cleavage at the NS2/3 site occurs solely for the release of the NS2 protein, what is the advantage for the virus of encoding two distinct proteases for polyprotein processing? Although several roles have been proposed for the cleaved NS2 protein, the NS2/3 protease itself appears unique in that its activity subsequently causes its inactivation. However, potential regulation of the cleavage reaction could have other implications for the viral life cycle, as is known for BVDV NS2/3 processing. BVDV stains are present in two forms, non-cytopathic (noncp) which expresses primarily uncleaved NS2/3 and has the ability to cause persistent infection and cytopathic (cp) strains expressing cleaved NS3 (Donis and Dubovi, 1987; Pocock et al., 1987). For this pestivirus, RNA replication levels have been shown to correlate with amount of cleaved NS3 protein (Lackner et al., 2004), whereas the uncleaved NS2/3 is required for viral infectivity (Agapov et al., 2004). Evolution of a cp strain from a non-cp strain occurs through the activation of the NS2/3 cleavage by a variety of mutations, deletions, duplications and rearrangements within the NS2 region (Kummerer et al., 1998; Meyers et al., 1992; Tautz et al., 1996; Tautz et al., 1994). However, it has recently been suggested that BVDV NS2/3 is an autoprotease whose temporal regulation is involved in modulating the different stages of RNA replication and viral morphogenesis (Lackner et al., 2004). Whether HCV NS2/3 could perform a similar regulatory role remains to be determined. Although NS2/3 processing appears to be a very efficient event in cell expression systems, the possible role for an uncleaved NS2/3 precursor in the complete viral life cycle has not been ruled out. . NS2 AS PART OF THE REPLICATION COMPLEX?

HCV RNA replication has been proposed to occur via the formation of a membrane bound replication complex that comprises the association of the NS proteins required for genome replication (NS3-5B). However, due to the lack of an efficient cell culture system to study the viral life cycle, studies focusing on the

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replication complex have been so far limited to the subgenomic replicon system (see Chapter 11), where NS2 is not expressed. Several studies have indicated that NS2 is an integral membrane protein that is targeted to the endoplasmic reticulum (ER) (Santolini et al., 1995; Yamaga and Ou, 2002). Interestingly, NS2 has been found by one group to be inserted into the membrane only when expressed in the context of the NS2/3 protein, and only after cleavage from NS3 (Santolini et al., 1995). NS2 has also been found to interact with all other HCV NS proteins in in vitro pull-down, as well as cell-based co-localization and co-immunoprecipitation experiments (Dimitrova et al., 2003; Hijikata et al., 1993b). Therefore, although not required for RNA replication, the possible presence of NS2 in this complex as an accessory protein is plausible and warrants further investigation.

ROLES OF CLEAVED NS2 HCV NS2 IS AN INTEGRAL MEMBRANE PROTEIN

The NS2 protein derived from the cleavage of NS2/3 is inserted into the ER membrane through its N-terminal hydrophobic domain. However, the exact mechanisms of translocation as well as the membrane topology of the protein remain controversial. Membrane association has been found to be dependent on SRP-SRP receptor targeting (Santolini et al., 1995). It was originally proposed that a signal sequence present in upstream p7 was required for membrane association co-translationally, although NS2 translocation has subsequently been demonstrated by several groups to be p7 independent (Santolini et al., 1995; Yamaga and Ou, 2002). Furthermore, although the cleavage at the p7-NS2 junction is performed in a membrane-dependent fashion by signal peptidase (Lin et al., 1994; Mizushima et al., 1994) and the presence of membranes is stimulatory (and for some strains required) for NS2/3 cleavage, one group has shown that the integration of NS2 into the membrane is performed post-translationally, and only after cleavage from NS3 (Santolini et al., 1995). However, Yamaga and Ou (2002) have since proposed that translocation could occur co-translationally and therefore the exact mechanisms of integration remain unclear. The amino terminal region of NS2 is likely to span the membrane several times (Pallaoro et al., 2001; Yamaga and Ou, 2002). However, the exact number of transmembrane domains, as well as the orientation of the protein in the membrane have not been conclusively determined. NS2 AND NS5A HYPERPHOSPHORYLATION

HCV NS5A has many roles in both RNA replication and the modulation of the host cell environment during infection and has been found to be present in two distinct phosphorylated forms: p56 and p58 (see Chapter 9). Liu et al. have reported the importance of NS2 for the generation of hyperphosphorylated NS5A (p58) (Liu et al., 1999). Using plasmids expressing various sections of the HCV polyprotein in transient transfection experiments, they demonstrate the requirement of NS2 generated by the cleavage of NS2/3 for the formation of p58. However, while 156

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performing similar experiments, other groups have demonstrated the appearance of p58 without the presence of NS2 (Koch and Bartenschlager, 1999; Neddermann et al., 1999). Indeed, Neddermann et al. (1999) therefore suggested that NS2 itself is not required for the hyperphosphorylation process, but rather that it could be the authentic N-terminus of NS3, generated by NS2/3 cleavage, that is of importance. NS2 INHIBITION OF GENE EXPRESSION

NS2 may also play a role in modulating cellular gene expression in infected cells. One study by Dumoulin et al. (2003) found that NS2 exerted a general inhibitory effect on the expression of a reporter gene expressed from a variety of different promoters (human ferrochelatase promoter, NFkappaB binding sites, SV40 promoter/enhancer sequences, full length, as well as minimal TNF-alpha promoters and cytomegalovirus immediate-early promoter) in several different hepatic and non-hepatic cell types. The amino-terminal (810-940) region of NS2 was sufficient to cause this effect, suggesting inhibition of gene expression is not dependent on the activity of the NS2/3 protease itself. It was therefore suggested that NS2 could potentially regulate host cell protein levels by interfering with a general aspect of transcription or translation. Indeed, several other HCV-encoded proteins, including core (Chapter 3), NS4B (Chapter 8) and NS5A (Chapter 9), have been demonstrated to alter cellular gene expression though a variety of mechanisms (Kato et al., 1997; Kato et al., 2000; Naganuma et al., 2000; Ray et al., 1995). This aspect of NS2 function will require further confirmation and careful investigation as it indicates a potential role for NS2 in the modulation of the host cell environment which has important implications for both the establishment of persistent infection and the pathogenesis of chronic HCV. NS2 AND APOPTOSIS

In order to establish a persistent infection, many viruses have evolved mechanisms to interfere with cellular apoptosis. In this manner, the virus is then able to replicate to sufficient levels without the elimination of the host cell. Several HCV proteins have been implicated in the modulation of cell signalling and apoptosis, including core, E2, NS5A and NS2 (Gale et al., 1997; Honda et al., 2000; Machida et al., 2001; Ruggieri et al., 1997). Machida et al. (2001) have reported that Fas-mediated apoptosis is inhibited in transgenic mice expressing HCV core, E1, E2 and NS2 proteins. The expression of these proteins in the liver prevented cytochrome c release from the mitochondria as well as preventing the activation of caspase 9 and caspase 3/7, but did not affect caspase 8. Therefore, this implicates these HCV proteins in the mitochondrial intrinsic apoptotic pathway, which involves mitochondrial membrane permeabilization and the release of pro-apoptotic factors, resulting in cell death. Furthermore, Erdtmann et al. (2003) showed that NS2 inhibits CIDE-B-induced apoptosis in co-expression experiments. CIDE-B (cell death-inducing DFF45like effector) is a mitochondrial pro-apoptotic protein whose overexpression has 157

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been shown to induce cell death (Inohara et al., 1998). CIDE-B-induced apoptosis requires mitochondrial localization and dimerization of the protein, both of which are mediated by a region in its C-terminal domain (Chen et al., 2000). NS2 was found to interact specifically with the C-terminal region of CIDE-B and block cytochrome c release from the mitochondria as well as cell death (Erdtmann et al., 2003). NS2 could therefore potentially prevent the dimerization of CIDE-B required for activity. However, the mechanism of inhibition remains unclear as NS2 is thought to be localized at the ER membrane. In this case, NS2 could potentially bind and sequester CIDE-B, preventing its localization at the mitochondria. The roles of mature cleaved NS2 remain largely unexplored. Although some possible functions have been proposed and are described here, the lack of an efficient cell culture system remains a major hurdle in identifying the main tasks of NS2 in the various events of the viral life cycle. Furthermore, it has been observed that NS2 is a short-lived protein in replicon cells (Franck et al., 2005). Franck et al. (2005) showed that NS2 is a target for phosphorylation by CK2 and is subsequently rapidly degraded by the proteasome. This appears to be a ubiquitin-independent process and the exact mechanisms involved have yet to be identified. However, the regulation of this process could have important implications for the understanding of the various functions of NS2 and the sequential events of the viral life cycle.

CONCLUSIONS Much work is still required in the study of the NS2/3 protease. Although several studies over the past decade have focussed on NS2/3 cleavage, the catalytic mechanism of the enzyme remains controversial. Initial attempts at characterizing the enzyme were limited to in vitro and cell expression systems and despite the development in recent years of in vitro systems in which the processing reaction can be studied using purified recombinant proteins, a definitive classification has not yet been determined. A three dimensional structure of NS2/3 is very much needed and will likely yield important insights into the mechanism of action of the enzyme. Similarly, a robust cell culture system for the study of the viral life cycle is of urgent need (see Chapter 16). Such a system will be crucial to precisely define the roles of NS2/3 cleavage and the NS2 protein in the complete viral life cycle. Of particular interest are the observations that NS2 could potentially modulate the host cell environment during HCV infection through interference with gene expression and cellular apoptosis. However, it will be necessary to validate these findings in a more physiologically relevant setting. Although its mode of action is unclear, NS2/3 cleavage is absolutely required for persistent viral infection in a chimpanzee. The HCV NS2/3 protease shares no obvious sequence homology to any known proteases in the animal kingdom and 158

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would therefore make an attractive target for antiviral therapy. The elucidation of the crystal structure of NS2/3, its mechanism of action and precise functions in replication will help to generate important information for the development of strategies for inhibition of NS2/3 processing, which could become the basis for novel HCV therapies in the future.

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Falgout, B., Pethel, M., Zhang, Y. M., and Lai, C. J. (1991). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65, 2467-2475. Franck, N., Le Seyec, J., Guguen-Guillouzo, C., and Erdtmann, L. (2005). Hepatitis C Virus NS2 Protein Is Phosphorylated by the Protein Kinase CK2 and Targeted for Degradation to the Proteasome. J Virol 79, 2700-2708. Gale, M. J., Jr., Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R., and Katze, M. G. (1997). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217-227. Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M., and Rice, C. M. (1993). A second hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A 90, 10583-10587. Hijikata, M., Mizushima, H., Akagi, T., Mori, S., Kakiuchi, N., Kato, N., Tanaka, T., Kimura, K., and Shimotohno, K. (1993a). Two distinct proteinase activities required for the processing of a putative nonstructural precursor protein of hepatitis C virus. J Virol 67, 4665-4675. Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K., and Shimotohno, K. (1993b). Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90, 10773-10777. Hirowatari, Y., Hijikata, M., Tanji, Y., Nyunoya, H., Mizushima, H., Kimura, K., Tanaka, T., Kato, N., and Shimotohno, K. (1993). Two proteinase activities in HCV polypeptide expressed in insect cells using baculovirus vector. Arch Virol 133, 349-356. Holland, D. R., Hausrath, A. C., Juers, D., and Matthews, B. W. (1995). Structural analysis of zinc substitutions in the active site of thermolysin. Protein Sci 4, 1955-1965. Honda, A., Hatano, M., Kohara, M., Arai, Y., Hartatik, T., Moriyama, T., Imawari, M., Koike, K., Yokosuka, O., Shimotohno, K., and Tokuhisa, T. (2000). HCV-core protein accelerates recovery from the insensitivity of liver cells to Fas-mediated apoptosis induced by an injection of anti-Fas antibody in mice. J Hepatol 33, 440-447. Inohara, N., Koseki, T., Chen, S., Wu, X., and Nunez, G. (1998). CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor. Embo J 17, 2526-2533. Kato, N., Lan, K. H., Ono-Nita, S. K., Shiratori, Y., and Omata, M. (1997). Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator. J Virol 71, 8856-8859. Kato, N., Yoshida, H., Kioko Ono-Nita, S., Kato, J., Goto, T., Otsuka, M., Lan, K., Matsushima, K., Shiratori, Y., and Omata, M. (2000). Activation of intracellular signaling by hepatitis B and C viruses: C-viral core is the most potent signal inducer. Hepatology 32, 405-412. 160

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Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., et al. (1996). Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87, 343-355. Koch, J. O., and Bartenschlager, R. (1999). Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol 73, 7138-7146. Kolykhalov, A. A., Mihalik, K., Feinstone, S. M., and Rice, C. M. (2000). Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. J Virol 74, 20462051. Kummerer, B. M., Stoll, D., and Meyers, G. (1998). Bovine Viral Diarrhea Virus Strain Oregon: a Novel Mechanism for Processing of NS2-3 Based on Point Mutations. J Virol 72, 4127-4138. Lackner, T., Muller, A., Pankraz, A., Becher, P., Thiel, H. J., Gorbalenya, A. E., and Tautz, N. (2004). Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J Virol 78, 10765-10775. Lin, C., Lindenbach, B. D., Pragai, B. M., McCourt, D. W., and Rice, C. M. (1994). Processing in the hepatitis C virus E2-NS2 region: identification of p7 and two distinct E2-specific products with different C termini. J Virol 68, 5063-5073. Liu, Q., Bhat, R. A., Prince, A. M., and Zhang, P. (1999). The hepatitis C virus NS2 protein generated by NS2-3 autocleavage is required for NS5A phosphorylation. Biochem Biophys Res Commun 254, 572-577. Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L., and Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110-113. Love, R. A., Parge, H. E., Wickersham, J. A., Hostomsky, Z., Habuka, N., Moomaw, E. W., Adachi, T., and Hostomska, Z. (1996). The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell 87, 331-342. Machida, K., Tsukiyama-Kohara, K., Seike, E., Tone, S., Shibasaki, F., Shimizu, M., Takahashi, H., Hayashi, Y., Funata, N., Taya, C., et al. (2001). Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins. J Biol Chem 276, 12140-12146. Meyers, G., Tautz, N., Stark, R., Brownlie, J., Dubovi, E. J., Collett, M. S., and Thiel, H. J. (1992). Rearrangement of viral sequences in cytopathogenic pestiviruses. Virology 191, 368-386. Mizushima, H., Hijikata, M., Tanji, Y., Kimura, K., and Shimotohno, K. (1994). Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2. J Virol 68, 2731-2734. Naganuma, A., Nozaki, A., Tanaka, T., Sugiyama, K., Takagi, H., Mori, M., Shimotohno, K., and Kato, N. (2000). Activation of the interferon-inducible 2'-

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5'-oligoadenylate synthetase gene by hepatitis C virus core protein. J Virol 74, 8744-8750. Neddermann, P., Clementi, A., and De Francesco, R. (1999). Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol 73, 9984-9991. Pallaoro, M., Lahm, A., Biasiol, G., Brunetti, M., Nardella, C., Orsatti, L., Bonelli, F., Orru, S., Narjes, F., and Steinkuhler, C. (2001). Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein. J Virol 75, 9939-9946. Pieroni, L., Santolini, E., Fipaldini, C., Pacini, L., Migliaccio, G., and La Monica, N. (1997). In vitro study of the NS2-3 protease of hepatitis C virus. J Virol 71, 6373-6380. Pocock, D. H., Howard, C. J., Clarke, M. C., and Brownlie, J. (1987). Variation in the intracellular polypeptide profiles from different isolates of bovine virus diarrhoea virus. Arch Virol 94, 43-53. Ray, R. B., Lagging, L. M., Meyer, K., Steele, R., and Ray, R. (1995). Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Res 37, 209-220. Reed, K. E., Grakoui, A., and Rice, C. M. (1995). Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage. J Virol 69, 4127-4136. Rinck, G., Birghan, C., Harada, T., Meyers, G., Thiel, H. J., and Tautz, N. (2001). A cellular J-domain protein modulates polyprotein processing and cytopathogenicity of a pestivirus. J Virol 75, 9470-9482. Ruggieri, A., Harada, T., Matsuura, Y., and Miyamura, T. (1997). Sensitization to Fas-mediated apoptosis by hepatitis C virus core protein. Virology 229, 68-76. Santolini, E., Pacini, L., Fipaldini, C., Migliaccio, G., and Monica, N. (1995). The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J Virol 69, 7461-7471. Tautz, N., Meyers, G., Stark, R., Dubovi, E. J., and Thiel, H. J. (1996). Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion. J Virol 70, 7851-7858. Tautz, N., Thiel, H. J., Dubovi, E. J., and Meyers, G. (1994). Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion. J Virol 68, 3289-3297. Thibeault, D., Maurice, R., Pilote, L., Lamarre, D., and Pause, A. (2001). In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus. J Biol Chem 276, 46678-46684. Waxman, L., Whitney, M., Pollok, B. A., Kuo, L. C., and Darke, P. L. (2001). Host cell factor requirement for hepatitis C virus enzyme maturation. Proc Natl Acad Sci U S A 98, 13931-13935. Yamaga, A. K., and Ou, J. H. (2002). Membrane topology of the hepatitis C virus NS2 protein. J Biol Chem 277, 33228-33234. 162

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Chapter 6

HCV NS3-4A Serine Protease Chao Lin

ABSTRACT The 9.6 kb plus-strand RNA genome of HCV encodes a long polyprotein precursor of ~3,000 amino acids, which is processed by cellular and viral proteases to 10 individual proteins. One of the HCV proteases, NS3-4A serine protease, is a noncovalent heterodimer consisting of a catalytic subunit (the N-terminal one-third of NS3 protein) and an activating cofactor (NS4A protein), and is responsible for cleavage at four sites of the HCV polyprotein. HCV NS3-4A protease is essential for viral replication in cell culture and in chimpanzees, and has been considered as one of the most attractive targets for developing novel anti-HCV therapies. However, discovery of small-molecule, selective inhibitors against HCV NS3-4A protease as oral drug candidates has been hampered by its shallow substrate-binding groove and the lack of robust, reproducible viral replication models in cell culture or in small animals. Nevertheless, decade-long intense efforts by many groups have largely overcome these two obstacles and provided fruitful understanding of its biological functions, biochemistry, and three-dimensional structures, culminating in recent demonstration of proof-of-concept anti-HCV activities in patients. This chapter will review key findings in these areas, and focus on the discovery and clinical development of HCV NS3-4A protease inhibitors as novel antiviral therapies.

INTRODUCTION The hepatitis C virus (HCV) epidemic, affecting ~170 million people worldwide, has been widely discussed (Memon and Memon, 2002; Wasley and Alter, 2000). The current standard therapy for chronic hepatitis C patients is a combination of weekly injections of pegylated interferon (IFN)-α, and daily oral doses of ribavirin (for a review, see Anonymous, 2002; Strader et al., 2004 and references therein). Both drugs are indirect antivirals because they do not target a specific HCV protein or RNA element. A sustained viral response (SVR), which is defined as treated patients remaining HCV-free (undetectable viral load) for 6 months after the termination of therapy, is achieved in only half of the treated patients and in less than half of patients with genotype 1 HCV or with high viral load (Fried et al., 2002; Hadziyannis et al., 2004; Manns et al., 2001). The standard therapy is associated with considerable adverse effects, including depression, fatigue, and "flu-like" symptoms caused by IFN-α, and hemolytic anemia by ribavirin. There is a huge unmet medical need

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for orally available, small-molecule, direct anti-HCV drugs to provide hepatitis C patients more effective treatments with fewer side effects. HCV, a member of the Flaviviridae family of viruses, has a 9.6 kb plus-strand RNA genome that encodes a long polyprotein precursor of ~3,000 amino acids, which is processed proteolytically upon translation by both cellular and viral proteases to at least 10 individual proteins, including four structural proteins (C, E1, E2 and p7) and six nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Fig. 1) (for a review see Lindenbach and Rice, 2001). The NS3 protein is a multi-functional protein, with a serine protease domain in its N-terminal one-third and a helicase domain in the C-terminal two-third (reviewed in chapter 7). The NS3-4A serine protease is a non-covalent, heterodimer complex formed by two HCV-encoded proteins, the N-terminal serine protease domain of NS3 (catalytic subunit) and the NS4A cofactor (activation subunit). The NS3-4A serine protease is responsible for the proteolytic cleavage at four junctions of the HCV polyprotein precursor: NS3/NS4A (self cleavage), NS4A/NS4B, NS4B/NS5A, and NS5A/ NS5B (Fig. 1) (Bartenschlager et al., 1993; Bartenschlager et al., 1995b; Failla et al., 1995; Grakoui et al., 1993a; Grakoui et al., 1993b; Hijikata et al., 1993b; Kim et al., 1996; Lin and Rice, 1995; Lin et al., 1995; Tanji et al., 1995; Tomei et al., 1993). HCV encodes four viral enzymes in its nonstructural protein region: NS2-3 autoprotease (reviewed in chapter 5) and NS3-4A serine protease (reviewed in this chapter), NS3 helicase (reviewed in chapter 7) and NS5B RNA-depdendent RNA polymerase (reviewed in chapter 10), all of which are essential for HCV replication or infectivity in chimpanzees (Kolykhalov et al., 2000). Among them, NS3-4A serine protease and NS5B RNA-dependent RNA polymerase are generally considered to be the most attractive targets for design of new anti-HCV oral drugs. The success of HIV protease inhibitor drugs demonstrates that viral proteases, such as the HCV NS3-4A protease, could be excellent targets for a structure-based drug design approach. However, the shallow substrate-binding groove of the HCV

Fig. 1. A schematic diagram of the HCV genome. The 5' and 3' untranslated regions (UTR) are shown with putative secondary structures. The polyprotein encoded by the long open reading frame is shown as a long box, in which individual mature protein products are labeled as core (C), envelope proteins 1 and 2 (E1 and E2), p7, followed by six nonstructural proteins (NS) 2, 3, 4A, 4B, 5A, and 5B. The cleavage sites are marked for cellular signal peptidase (filled triangle), HCV NS2-3 auto-protease (filled arrow), and NS3-4A serine protease (open arrow).

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NS3-4A serine protease observed in an X-ray crystal structure (Kim et al., 1996) suggested that discovery of a potent, small-molecule, and orally available drug candidate would be an enormously challenging task. Despite of the lack of a robust and consistent HCV infection cell culture, a subgenomic replicon system developed by Lohmann et al. (1999) became the workhorse as the standard assay of antiviral activity of the HCV NS3-4A protease inhibitors. In addition, the lack of a robust HCV infection model in small animals has generally forced scientists to rely on a combination of anti-HCV activity in cell culture and animal pharmacokinetics as surrogate indicators of efficacy prior to clinical trials in human. Nevertheless, significant progress has been made in recent years to identify potent small-molecule inhibitors against the HCV protease. Clinical proof-of-concept for HCV NS3-4A protease inhibitors has recently been obtained with BILN 2061 (a non-covalent inhibitor) and VX-950 (a covalent but reversible inhibitor). Viral load in chronic hepatitis C patients was reduced by 2-3 log10 after a treatment with BILN 2061 (Lamarre et al., 2003) or VX-950 (Reesink et al., 2005) for 2–3 days. At the end of a 14-day treatment with VX-950, up to a 4-log10 reduction in HCV viral load was observed, while in some patients the virus became undetectable (

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Frontiers in Viral Hepatitis

Compute's Computer Viruses

Viruses, Plagues, and History

Bacteria and Viruses

Hepatitis C Viruses