Hepatitis C Viruses: Genomes and Molecular Biology Chapter Abstracts

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Chapter 1
HCV Genome and Life Cycle
Stéphane Chevaliez and Jean-Michel Pawlotsky

There is a silent pandemic going on - hepatitis C virus (HCV) infection afflicts more than 170 million people worldwide, with the great majority (~85%) of patients developing chronic HCV infection. It can ultimately result in liver cirrhosis, hepatic failure or hepatocellular carcinoma, which is 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 this devious virus, 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 infection systems should help fill in some of the gaps in understanding the HCV lifecycle in the next few years.

Chapter 2
HCV 5' and 3'UTR: When Translation Meets Replication
Stephanie T. Shi and Michael M. C. Lai

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 two processes.

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

The hepatitis C virus (HCV) nucleocapsid or core protein has been reported to have many functions. During the life cycle of HCV, the main function of the core protein is to form the capsid shell that houses and protects 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. This review highlights important new developments in HCV capsid assembly and HCV core-host interactions.

Chapter 4
HCV Glycoproteins: Assembly of a Functional E1-E2 Heterodimer
Muriel Lavie, Anne Goffard and Jean Dubuisson

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 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.

Chapter 5
HCV NS2/3 Protease
Sarah Welbourn and Arnim Pause

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.

Chapter 6
HCV NS3-4A Serine Protease
Chao Lin

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 non-covalent 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.

Chapter 7
HCV Helicase: Structure, Function, and Inhibition
David N. Frick

The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled by ATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.

Chapter 8
HCV NS4B: from Obscurity to Central Stage
Ella H. Sklan and Jeffrey S. Glenn

The hepatitis C virus (HCV) non-structural 4B (NS4B) protein is a 27kDa hydrophobic protein which for many years was characterized mainly as a protein of unknown function. Recently, however, information about the protein and its involvement in mediating various viral activities and effects on host cells is beginning to accumulate. NS4B has been implicated in modulation of NS5B's RNA dependent RNA polymerase activity and various host signal transduction pathways, a possible role in HCV carcinogenesis, impairment of ER function, and regulation of both viral and host translation. Perhaps most significant, NS4B has recently been found to be responsible for the formation of a novel intracellular membrane structure, termed the membranous web, which appears to be the platform upon which viral replication occurs. Specific domains within NS4B have been identified which likely underlie the mechanisms employed by NS4B to mediate many of the preceding functions. As such, these domains which include an amphipathic helix and nucleotide-binding motif represent attractive targets for new antiviral strategies.

Chapter 9
HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication
Yupeng He, Kirk A. Staschke and Seng-Lai Tan

The hepatitis C virus (HCV) non-structural 5A (NS5A) protein has generated wide interest in HCV research because of its ability to modulate the host cell interferon (IFN) response. The protein is phosphorylated on multiple sites by host cell kinases and interacts with host cell membranes. While no known enzymatic function has been ascribed to NS5A, it is an essential component of the HCV replicase and exerts a wide range of effects on cellular pathways and processes, including innate immunity and host cell growth and proliferation. In this chapter, we review the many studies describing the interaction of NS5A with viral and host cell proteins, its ability to modulate multiple cellular pathways, and its recently described structural attributes, subcellular localization, and function during HCV replication.

Chapter 10
Biochemical Activities of the HCV NS5B RNA-Dependent RNA Polymerase
C. T. Ranjith-Kumar and C. Cheng Kao

Structural and functional studies of the hepatitis C virus (HCV) RNA-dependent RNA polymerase have contributed to our understanding of polymerase mechanism, viral RNA replication, and have generated targets for antiviral development. This review summarizes recent studies on the properties of the HCV polymerase.

Chapter 11
HCV Replicon Systems
Keril J. Blight and Elizabeth A. Norgard

With the remarkable ability of hepatitis C virus (HCV) to establish persistent infections that can lead to progressive liver pathology and the poor response of prevalent HCV genotypes to the current treatment, HCV represents a significant global health problem. Studies of HCV replication in cell culture were virtually impossible until the development of subgenomic replicons that replicate autonomously in the human hepatoma cell line Huh-7. Many improvements to the replicon system have been made allowing the establishment of transient replication assays for HCV genotypes 1a, 1b, and 2a. Specifically, the identification of adaptive mutations that drastically enhance HCV genotype 1 replication and the isolation of highly permissive Huh-7 sublines led to the development of replication-competent full-length genomes in addition to a collection of robustly replicating subgenomes derived from genotype 1 sequences. More recently, the cell tropism of HCV subgenomic replicons has been expanded to non-hepatoma cell lines and mouse hepatocytes. The HCV replicon system has opened new avenues for detailed molecular studies of RNA replication and HCV-host interactions as well as the development of active inhibitors of HCV replication. Finally, the identification of genotype 2a-derived replicons that efficiently replicate in cell culture without adaptive mutations has facilitated the development of systems supporting the complete virus life cycle.

Chapter 12
Animal Models for HCV Study
Linda B. Couto and Alexander A. Kolykhalov

The study of HCV biology is complicated by the paucity of relevant animal models. The ideal model for studying HCV would be one that adequately represents most aspects of human HCV infection and disease, is affordable, easily available, and reproducible. Currently, the only widely recognized animal model of HCV infection is the chimpanzee, which does not meet all of these desirable attributes. Recently, other models have been used to dissect various aspects of HCV biology and to evaluate novel therapeutics. Each has a unique set of advantages and limitations. Transgenic mouse models have elucidated the pathophysiology of specific viral proteins, but they are limited by their inability to support HCV replication. Xenograft models provide an environment for human hepatocyte engraftment in mice and subsequent infection with HCV. These models are technically challenging, but once optimized they promise to be extremely useful both for the study of HCV biology and for drug development. Alternatively, the GBV-B virus, which efficiently replicates in tamarins and marmosets, represents a surrogate model for the study of HCV. Chimeras between GBV-B and HCV have been created and will be useful in the development of HCV-targeting drugs.

Chapter 13
HCV Regulation of Host Defense
Spencer Carney and Michael Gale Jr.

Mammalian cells respond to virus challenge by initiating a "host response" characterized by interferon a/b (IFN) production and a cellular antiviral state. The host response is our first line of immune defense against viral pathogens and it imposes several barriers that hepatitis C virus (HCV) must overcome to replicate and persist. HCV evades the host response through a complex combination of virus-host interactions that disrupt intracellular signaling pathways and attenuate the antiviral actions of IFN. Regulation of the host response breaks a link between innate and adaptive immunity and provides a foundation for HCV replication and spread.

Chapter 14
Regulation of Adaptive Immunity by HCV
Xiao-Song He

HCV causes chronic infection in the majority of infected patients, which is associated with attenuated adaptive immunity against the virus. Accumulating data suggest that HCV may modulate the adaptive anti-HCV immunity of the host to facilitate the establishment of viral persistence. Potential mechanisms of this modulation include infection of dendritic cells by HCV, as well as binding of HCV envelope or core proteins to cell surface receptors, resulting in perturbation of the functions of different immune cell subsets. These mechanisms may operate predominantly in the liver, the primary site of infection by HCV, where the unique hepatic environment favors tolerance rather than immunity to foreign antigens. Elucidation of these mechanisms may lead to development of novel therapeutic strategies combining both antiviral drugs and immunotherapy agents.

Chapter 15
Recombinant Vesicular Stomatitis Virus (VSV) and Other Strategies in HCV Vaccine Designs and Immunotherapy
Ayaz M. Majid and Glen N. Barber

Several vaccine strategies have been attempted in chimpanzee and smaller animal models to generate immune responses to hepatitis C virus (HCV). While neutralizing antibody may play a role in preventing HCV infection, studies in chimpanzees and humans during rare cases of acute resolving HCV infection indicate that, HCV immunity appears to be associated with vigorous, sustained and multi-specific Th1 intra hepatic CD8⁺ and CD4⁺ T cell responses. Several new promising technologies utilizing viral based vaccine approaches that appear to generate both antibody and cell mediated immune responses have recently been reported. These include viral vectors that express HCV products and non-replicating viral like particles (VLPs) that appear to induce T-helper type 1 (Th1) immune responses considered important in resolving HCV infection. In addition, viral vectors based on recombinant vesicular stomatitis virus (rVSV) may offer safe yet potent stimulation of both innate and adaptive immune responses. Here, we review the successful application of viral based vaccines, including VSV in generating viral immunity in animal models and describe the potential usefulness of this technology as a strategy for HCV vaccine design and immunotherapy.

Chapter 16
Development of an Infectious HCV Cell Culture System
Takaji Wakita and Takanobu Kato

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a health problem worldwide. Despite the increasing demand for knowledge on viral replication and pathogenesis, detailed examinations of the viral life cycle have been hampered by the lack of efficient viral culture systems, owing in part to its narrow host range. We isolated full-length HCV clone, JFH-1strain, from a fulminant hepatitis C patient. The JFH-1strain fit into the cluster of genotype 2a with notable deviations in the 5'UTR, core, NS3 and NS5A regions, and monoclonality of the hyper-variable region sequence. The JFH-1 subgenomic replicon replicated efficiently in a variety of cell lines without acquiring adaptive mutations in its genome. Transfection of in vitro transcribed full-length RNA into Huh7 cells, efficient replication of JFH-1 RNA and secretion of recombinant viral particles into culture medium. Importantly, secreted viral particles were infectious for both cultured cells and a chimpanzee. Furthermore, infectivity for cultured cells was improved by using permissive cell lines. This infectious HCV system provides for the first time a powerful tool to study the full viral life cycle, to construct anti-viral strategies and to develop effective vaccines.

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