18. THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS

18. THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS

18. THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS Timothy L. Tellinghuisen and Charles M. Rice INTRODUCTION Hepatitis C virus (HCV) infection is a sig...

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18.

THE MOLECULAR VIROLOGY OF HEPATITIS C VIRUS

Timothy L. Tellinghuisen and Charles M. Rice INTRODUCTION Hepatitis C virus (HCV) infection is a significant public health problem of international scope. Estimates from an epidemiologic study by the World Health Organization (WHO) in 1997 place the number of HCV infected individuals at approximately 170 million, representing nearly 3% of the world’s population (Anonymous, 1997). It is important to note that the HCV infection is five times more prevalent than that of the human immunodeficiency virus (HIV), underscoring the pandemic nature of HCV infection. More recent data from the National Health and Nutrition Examination Survey (NHANES) on HCV infection in the United States indicate 3.9 million Americans have been exposed to HCV (for a summary of the NHANES report see Kim, 2002). The natural course of HCV infection has two distinct virological outcomes, acute infection with subsequent viral clearance, and viral persistence leading to chronic infection. Acute HCV infection is largely asymptomatic and rarely diagnosed. Unfortunately, only 30% of patients are capable of naturally clearing and acute HCV infection, with the vast majority remaining persistently infected (Alter et al., 1992; Alter & Seeff, 2000). The NHANES data places the number of persistently infected individuals in the United States at approximately 2.7 million. HCV replication can occur for decades in these patients, often leading to serious liver disease and a variety of extra hepatic disorders, including autoimmune disorders, cryoglobulinemia, and non-Hodgkin’s lymphoma. The most common hepatic manifestations of a persistent infection are chronic hepatitis and a progressive cirrhosis. Persistent HCV infection has also The Liver in Biology and Disease Principles of Medical Biology, Volume 15, 455–495 © 2004 Published by Elsevier Ltd. ISSN: 1569-2582/doi:10.1016/S1569-2582(04)15018-6

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been linked to an increased risk of hepatocellular carcinoma (for review see Block et al., 2003). Currently, HCV associated liver disease is the leading indicator of liver transplantation in the United States (Fishman et al., 1996). The NHANES data estimated the direct healthcare cost of hepatitis C infection in the United States at more than 1 billion dollars per year in 1998, with predictions of dramatic increases in future years (Kim, 2002). The approved treatment for HCV infection, a combination therapy of pegylated interferon-␣ and ribavirin, is of limited efficacy and is often poorly tolerated by patients (Heathcote et al., 2000; Zeuzem et al., 2000). The efficacy of drug therapy correlates with the genotype of HCV present in the infected individual. There are currently six recognized HCV genotypes, and a number of more closely related subtypes (Bukh et al., 1995). Sequence variability between genotypes is considerable, with the most distantly related genotypes differing by up to 30%. Rates of sustained virologic response of therapy are as high as 80% for genotypes 2 and 3, and as low as 40% with genotype 1 (Chander et al., 2002). The molecular mechanism of variations in drug efficacy for the different HCV genotypes is not clear. In addition to genotypic variations, HCV is present as numerous closely related quasi-species in the infected individual. The diversity of these quasi-species, combined with the high mutation rate of RNA virus replication, greatly complicates the specific targeting of HCV RNA and proteins (Pawlotsky, 2003). Vaccine development has been equally problematic, and despite significant effort, no effective HCV vaccine exists (Lechmann & Liang, 2000). Clearly, much work is needed in the development of effective anti-HCV therapeutics, and understanding of the molecular virology of HCV is of paramount importance to this process.

OVERVIEW OF HCV BIOLOGY The beginning of hepatitis C virus molecular virology heralds to the late 1980s with the identification of HCV as the causative agent for what was termed non-A non-B hepatitis (Choo et al., 1989). The cDNA clone generated in this landmark work has provided the basis for the classification and molecular dissection of HCV (Choo et al., 1991). Infectious consensus clones of a variety of HCV genotypes have been generated (Beard et al., 1999; Bukh et al., 1998; Kolykhalov et al., 1997; Yanagi et al., 1997, 1998, 1999a). Initial examination of these HCV sequences led to the classification of this virus as a member of the diverse Flaviviridae family of enveloped, positive strand RNA viruses. HCV represents the sole member of the Hepacivirus genus within this family (Lindenbach & Rice, 2001). It is worth noting that the Flaviviridae family also contains the genera Flavivirus and Pestivirus,

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which contain numerous important human and animal pathogens, respectively. The HCV genome consists of an RNA molecule of approximately 9.6 kb containing a single open reading frame (ORF) flanked by large, highly structured 5 and 3 non-translated regions (NTRs). The viral RNA lacks both a 5 cap structure and a 3 poly(A) tail. Viral proteins are translated as a polyprotein via an internal ribosome entry site (IRES) located within the 5 NTR. The organization of the polyprotein is similar to that of the other members of the Flaviviridae family, with structural proteins located at the 5 end of the genome, and non-structural proteins downstream. The ten HCV proteins are organized in the polyprotein in the order: NH2 - C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (see Fig. 1) (Grakoui et al., 1993b). The polyprotein processing is complex and involves both host and viral proteinase activities to carry out the numerous co- and posttranslational cleavage events in the maturation of the viral proteins (Grakoui et al., 1993a). An additional HCV protein generated from an overlapping reading frame in the core (C) protein coding sequence, designated ARFP (alternate reading

Fig. 1. Organization of the HCV Genome and Polyprotein Processing. Note: The HCV genome consists of a single, positive sense RNA molecule flanked by structured 5 and 3 non-translated regions (NTRs). The overall organization of the HCV polyprotein is similar to other Flaviviridae, with a single large open reading frame (ORF) with structural proteins (shaded in grey) at the amino terminal end, and non-structural proteins (shaded in white) located downstream. The proteins are organized in the polyprotein in the order; NH2-C-E1E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. The coding sequence of the putative alternate reading frame protein (ARFP), or frame shift protein (F), is indicated with a dark grey box containing the letter F. The locations of known enzymatic activities of the HCV nonstructural proteins have been indicated, including the NS2-NS3 autocatalytic proteinase, the NS3 serine proteinase, the NS3 helicase, and the NS5B RNA dependent RNA polymerase. The cleavage sites utilized in the complex polyprotein processing mechanism have been indicated. Black circles represent the cleavages mediated by the host cell signal peptidase within the structural proteins. The open circle indicates the putative cleavage by signal peptide peptidase involved in generating the mature C protein. The open arrow indicates the site of autocatalytic cleavage by the NS2-NS3 proteinase. The proteolytic cleavage sites processed by the NS3 serine proteinase activity are indicated by black arrows.

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Fig. 2. Membrane Topology of the HCV Proteins. Note: The membrane topology and membrane association of the processed forms of the HCV proteins is shown in relation to the lumen of the endoplasmic reticulum (ER) and the cytoplasm. The location of amino (N) and carboxy (C) termini of the proteins are indicated. The immature and mature forms of the C protein are shown, with the signal peptide peptides maturation cleavage site shown (SPP). The trans membrane spanning anchor of E1 is colored in black to indicate the reorganization of this sequence required for the correct topological insertion of E2. NS3 is shown associated with NS4A, which is believed to localize this protein to membranes, The unusual horizontal membrane topology of the amino terminal helix of NS5A is shown.

frame protein) or F (frame shift protein), has been proposed. The membrane topology of the mature HCV proteins is shown in Fig. 2. The structural proteins, C, E1, and E2 are cleaved from the polyprotein by the endoplasmic reticulum (ER) signal peptidases, and following maturation, most likely serve as components for the assembly of progeny virions. By analogy to other members of the Flaviviridae, assembly of HCV most likely occurs on ER derived vesicles with budding of virions into internal membrane compartments and subsequent cellular exit via the ER trafficking system. The function of the small hydrophobic p7 protein, located at the polyprotein junction between the structural and non-structural proteins, is only beginning to be unraveled. The HCV non-structural proteins, NS2 through NS5B, are thought to comprise the viral replicase complex. The proteolytic processing of these proteins requires two distinct viral proteases. The NS2 protein, together with the amino terminal region of the NS3 protein, constitutes the NS2–3 proteinase that catalyzes the autocatalytic removal of NS2 from the polyprotein. Following this cleavage, NS2 has no known additional function, and is dispensable for subsequent steps in RNA replication. Once released from NS2, the amino terminal domain of the NS3

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proteins serves as a distinct proteinase for the cleavage of all downstream sites in the polyprotein. The carboxyl-terminal region of NS3 is an NTPase/RNA helicase. The NS4A protein serves as a cofactor/enhancer for the proteolytic activities of NS3. The function of the hydrophobic integral membrane protein NS4B is unknown, but this protein appears to interact with the viral replicase and play a role in the reorganization of cellular membranes, presumably to a conformation is amenable to HCV replication. NS5A is a hydrophilic membrane associated phosphoprotein of unknown function. The NS5B protein comprises the RNA dependent RNA polymerase activity. Viral RNA replication is believed to occur in association with peri-nuclear membranes of ER origin, as has been observed for

Fig. 3. Steps in the HCV Lifecycle. Note: A general overview of the steps of the HCV lifecycle. Following binding of the extracellular virion to the host cell receptors(s) and endocytosis of the virion-receptor complex, the virus penetrates the host cell membrane vesicle via the pH dependent glycoprotein fusion activity. Once release into the cytoplasm, the nucleocapsid disassembles (uncoating) and releases the HCV genomic RNA. The input RNA serves as a template for translation of the polyprotein. Once translation and processing of the polyprotein has occurred, the HCV replicase complex assembles in association with ER derived membranes and generates progeny RNA via a minus strand replicative intermediate. These progeny RNA are then packaged into nucleocapsid structures. Nucleocapsids associate with the mature glycoprotein heterodimers and budding into internal membrane vesicles occurs. Following budding, the virions mature and exit the cell via the host vesicle trafficking system. The figure presented is a general schematic, and it should be noted that these processes are dynamic with numerous overlaps and interactions likely between the steps shown.

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other members of the Flaviviridae. An overview of the HCV lifecycle is presented in Fig. 3. Considerable progress has been made in determining the properties of the HCV proteins and RNA. The complex interactions between these macromolecules required for the processes of RNA replication and virion biogenesis is an area of active research and is not fully understood. The aim of this chapter is to present a brief overview of the systems used for the study of HCV and examine the properties of the HCV RNA and proteins as they relate to the numerous processes of virus replication. It is nearly impossible to provide a detailed discussion the vast body of HCV research articles, so a sample of current and classic literature has been selected for review with the intent of providing the reader a general understanding of HCV molecular virology. This chapter is by no means complete, and wherever possible references for more comprehensive review articles covering specific aspects of HCV virology have been provided.

SYSTEMS FOR THE STUDY OF HEPATITIS C VIRUS Perhaps the most significant difficulty in HCV research has been establishing robust systems for the study of HCV replication. This topic has been recently reviewed (Grakoui et al., 2001; Lanford & Bigger, 2002; Pietschmann et al., 2003). Humans and chimpanzees represent the only known animals capable of being infected with HCV. The use of human samples is complicated by the quantity of research material obtainable and the variability in these samples arising from natural infections outside of a controlled laboratory environment. The use of chimpanzees has alleviated some of the problems associated with human samples by allowing inoculation with defined molecular clones of HCV under controlled laboratory conditions, however both cost and ethical issues limit the number and type of experiments that can be performed using this system. Reviews on the use of the chimpanzee in HCV research are available (Lanford et al., 2001a). Although they have been instrumental in defining the natural course of HCV infection as well as addressing the complex interactions of HCV with the immune response, both the chimpanzee model and available human samples lack the tractability and availability needed for understanding the complete molecular details of HCV replication. The development of a small animal laboratory model for HCV has been difficult. Aside from an isolated report of HCV replication in tree shrews (Xie et al., 1998), attempts at obtaining HCV replication in small laboratory animals have been unsuccessful. A clever small animal model system using immunodeficient

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mouse hybrids (SCID/Alb-uPA) has been recently described (Mercer et al., 2001). Following the elimination of the native murine hepatocytes and the delivery of human hepatocytes up to half of the liver mass can be repopulated with human cells. These animals can be inoculated with infected human sera and develop persistent HCV viremia. Although an important breakthrough, the complexities of this system and the requirement of immunodeficient mice make it far from an ideal small animal model for studying HCV. The ability of the closely related flavivirus, GBV-B to replicate in tamarins and tamarin hepatocytes in culture has led to recent interest in this virus as a model for HCV (Bukh et al., 1999; Lanford et al., 2001b). The study of the properties of HCV ex vivo has been limited to the use of a variety of surrogate expression systems, which although capable of producing HCV proteins, fail to allow for RNA replication. Efficient cell culture HCV RNA replication systems based on replicon technology have recently become available, allowing the molecular dissection of RNA replication (Blight et al., 2000, 2003; Guo et al., 2001; Ikeda et al., 2002; Lohmann et al., 1999, 2001; Yi et al., 2003). In the replicon system, bicistronic RNAs containing the HCV non-structural proteins under translational control of an IRES and a selectable marker under the control of a second IRES are generated with HCV 5 and 3 NTR sequences. In the human hepatoma cell line Huh7, these RNA molecules express HCV non-structural proteins, which then replicate the viral RNA. RNA replication is monitored by either real-time quantitative PCR analysis of HCV RNA, or by monitoring the expression of a reporter gene. Although the initial replicon system was extremely inefficient, a large number of cell culture adaptive mutations have been described that greatly enhance RNA replication (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001). Interestingly, the combination of these adaptive mutations in the same RNA is often deleterious, suggesting multiple mechanisms of adaptation exist (Lohmann et al., 2003). It is important to note that adaptive mutations appear to be a cell culture specific phenomenon, and these changes are debilitating to RNAs in chimpanzee infections (Bukh et al., 2002). The generation of an adapted cell line for replicons has been described, suggesting the importance of host factors (Blight et al., 2002). The generation of genome length HCV replicons has been described, but despite the presence of the HCV structural proteins, these systems fail to generate infectious virus particles (Blight et al., 2002, 2003; Ikeda et al., 2002; Pietschmann et al., 2002). Recently, HCV replicons have been adapted to non-hepatic human epithelial cells and a murine hepatoma cell line, thereby eliminating the previous limitation of replicons to a single cell type (Zhu et al., 2003). A number of reviews detailing the development and use of the HCV replicon system have been published (Pietschmann & Bartenschlager, 2001; Randall & Rice, 2002).

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HCV RNA, IRES AND NTRS As mentioned in the introduction, the HCV genome consists of a single positive sense, non-capped and non-polyadenylated, RNA molecule of approximately 9.6 kb flanked by large, highly structured 5 and 3 NTRs. These regions represent areas of considerable sequence conservation among all HCV isolates, suggesting an important role in virus biology. The 5 HCV NTR is a 341 nucleotide element consisting of 4 highly structured domains, designated I though IV. Domains II, III, IV, and a portion of the coding sequence of the C protein comprise the viral IRES, a structure required for the cap-independent translation of the HCV polyprotein. The IRES contains two large stem loop structures and an RNA pseudoknot. A number of the smaller elements of domain III have been visualized in NMR and x-ray structures (Collier et al., 2002; Kieft et al., 2002; Lukavsky et al., 2000), and the entire 5 NTR has been extensively mapped by structure probing. A cryo-electron microscopy image reconstruction (cryo-EM) of the HCV IRES complexed with the 40S ribosomal subunit has been determined, indicating that the IRES is capable of altering the conformation of the ribosomal subunit through a mechanism requiring domain II of the IRES (Spahn et al., 2001). It is believed that this conformational change in the IRES allows HCV to bypass the necessity for the typical canonical translation factors. For a more detailed review on the function of the HCV IRES in translation, the reader is directed to (Hellen et al., 1999; Rijnbrand & Lemon, 2000). The HCV IRES has garnered significant interest as a target for anti-viral therapeutics (Jubin, 2003). Domain I, the most 5 element of the 5 NTR, forms a stable stem loop structure. Deletion of this stem loop positively affects translation, although this region is not required for IRES activity (Honda et al., 1996; Rijnbrand et al., 1995; Yoo et al., 1992). More recently, this region has been shown to be important for RNA replication. Deletion of the 5 terminal 40 nucleotides of the HCV RNA, thereby disrupting this element, abolished RNA replication in the replicon system, while only moderately affecting translation (Friebe et al., 2001). Generation of artificial RNAs containing the first 125 nucleotides of the HCV 5’ NTR has shown this region is sufficient for HCV specific RNA replication, suggesting that domain I and a portion of the HCV IRES, are essential for replication (Friebe et al., 2001). The 3 NTR consists of a short variable region, a polyuridine/polypyrimide tract of approximately 40 nucleotides, and a conserved 98 nucleotide region (designated the 3 X region) containing a stable stem loop structure at the extreme 3 end of the genomic RNA (Kolykhalov et al., 1996). Structural probing of the 3 end of the RNA has partially confirmed the predicted secondary structure (Blight & Rice, 1997). The 3 NTR is essential for HCV RNA replication in cell

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culture and required for infection of chimpanzees (Friebe & Bartenschlager, 2002; Kolykhalov et al., 2000; Yanagi et al., 1999b). Recent experiments have shown the 98 nucleotide 3 X region is essential for RNA replication but plays little role in translation or RNA stability (Friebe & Bartenschlager, 2002). Deletion of the hypervariable region is debilitating to HCV replicons, but is not absolutely required for RNA replication. The polyuridine/polypyrimidine tract is essential for replication, but portions of this region can be replaced with polyuridine homopolymers. The 3 NTR is believed to be the site of initiation of viral RNA synthesis.

HCV Structural Proteins Core The HCV core (C) protein lies at the amino terminus of the viral polyprotein and is the site of initiation of viral translation. The early translation of C is thought to be cytoplasmic, with a redistribution of the nascent polypeptide to the ER following the translation of the C/E1 junction, which functions as an internal signal sequence for ER insertion. Once this sequence has been inserted, the remainder of the HCV polyprotein can be translated and processed in association with the ER. Cleavage of the C/E1 junction by the ER resident signal peptidase generates a 191 amino acid form of C which is inserted in the ER membrane based on the retention of the C/E1 junction signal sequence (Santolini et al., 1994). A second processing event within the ER membrane, presumably by signal peptide peptidase, removes the C/E1 signal sequence peptide and generates what is believed to be the mature 179 amino acid form of the C protein (McLauchlan et al., 2002). The majority of this form of the C protein remains associated with the ER, despite the removal of the carboxy terminal membrane anchor peptide, although other sub cellular localizations of the C have been observed (discussed below). The mature C protein is a small, hydrophilic protein that is believed to be the sole protein component of the HCV nucleocapsid. The binding of C to the HCV 5 NTR has been observed, and this has been proposed to be a potential RNA packaging signal for nucleocapsid assembly (Hwang et al., 1995; Tanaka et al., 2000). This 5 NTR interaction has also been shown to alter translation of the HCV polyprotein, possibly serving as a mechanism to regulate the switch between translation, replication, and virion assembly (Shimoike et al., 1999; Zhang et al., 2002). The non-specific nucleic acid binding activity of C has also been reported, possibly representing the non-specific charge neutralization of RNA required for nucleocapsid assembly (Hwang et al., 1995; Santolini et al., 1994). C has been shown to make homotypic interactions via a tryptophan rich sequence in the amino terminal portion of the protein,

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and these interactions have been proposed to be steps in the assembly of the HCV nucleocapsid (Matsumoto et al., 1996; Nolandt et al., 1997). C protein has been shown to be modified by tissue transglutaminase, resulting in the cross-linking of C proteins into a stable dimeric form that may play a role in the assembly process (Lu et al., 2001). Additionally, interactions of C with the 60S ribosomal subunit have been described, possibly mediating the disassembly of the nucleocapsid during virus entry (Santolini, Migliaccio & La Monica, 1994). The amino terminal region of C has also been shown to contain a number of cryptic nuclear localization signals, although these findings are controversial (Chang et al., 1994). The sub cellular localization of C is complex, with protein found mainly associated with the ER and lipid droplets, although nuclear localization of C, presumably via one or more of the putative nuclear localization signals, has been described (reviewed in McLauchlan, 2000). The localization of C to ER associated complexes containing the HCV structural proteins, non-structural proteins, and presumably RNA is believed to be the most relevant localization observed for HCV replication and virion production (Egger et al., 2002). The association of C with cytoplasmic lipid droplets has been observed, and this interaction may play a role in HCV pathogenesis. The regions of the C protein that are responsible for this association have been mapped to the carboxy terminal hydrophobic region of the protein, and these sequences bear a resemblance to plant olesin, a lipid binding protein (Hope et al., 2002). Additionally, C has been shown to bind to apolipoprotein II, possibly mediating lipid interactions (Perlemuter et al., 2002; Sabile et al., 1999; Shi et al., 2002). The interaction of C with lipid vesicles has been implicated in HCV related steatosis (Moriya et al., 1997). Transgenic mice expressing C develop steatosis (Moriya et al., 1997; Perlemuter et al., 2002) and liver cancer (Moriya et al., 1998), although the later observation appears to be mouse strain specific. The C protein has been shown to lead to a reduction in microsomal triglyceride transfer protein activity, leading to defects in the assembly and secretion of very low-density lipoproteins (Perlemuter et al., 2002). The C protein may play a role in lipid metabolism, lipid reorganization and trafficking, but the functional significance of these observations to HCV replication remain unclear. As mentioned previously, the C protein has been proposed to localize to the nucleus via several cryptic nuclear localization signals, although efficient localization requires artificial constructs lacking the hydrophobic carboxy terminal region of C (Chang et al., 1994; Liu et al., 1997; Lo et al., 1995; Ravaggi et al., 1994; Suzuki et al., 1995; Yasui et al., 1998). The nuclear localization of C is a significant area of debate. A large number of transcriptional regulatory activities have been proposed for the nuclear form of C. In addition to the role

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of C in nuclear transcriptional regulation, the cytoplasmic form of C has been proposed to interact with numerous cellular signaling cascades, suggesting links to transcription, apoptosis, carcinogenesis, and evasion of the host immune system. Much of the data regarding both the nuclear and cytoplasmic roles of C in gene expression are controversial and contradictory, and in most cases, it is unclear if these interactions occur in the course of a normal infection. The reader is directed to (McLauchlan, 2000) for an extensive review on these activities of C.

F Protein(s) or ARFP(s) One of the most surprising observations in recent HCV research is the presence of multiple overlapping reading frames in the core protein coding sequence that give rise to what has been called the frame shift (F) or alternate reading frame protein (ARFP) (Varaklioti et al., 2002; Xu et al., 2001). The majority of HCV isolates have been shown to contain an open reading frame in the −2/+1 frame that overlap the core protein coding sequence. Analysis of non-synonymous codon usage in the core protein coding sequence has indicated an unusual conservation of codons, presumably to maintain the integrity of the ARFP ORF. The translation of the ARFP ORF via a ribosomal frame-shifting event can generate a protein of up to 180 amino acids, although the exact size and composition of the ARFP is not clear. The generation of the ARFP requires only codons 8−14 of the core protein-coding sequence, a region that has been designated the HCV type I frame shift sequence (Xu et al., 2001). The frame shift junction that generates ARFP is believed to be located at codon 11 within this sequence. A double stem-loop structure located downstream of the frame shift signal has been shown to enhance frame shifting in the presence of the puromycin (Choi et al., 2003). More recent data suggests the generation of an additional 1.5 kDa ARFP using the −1/+2 frame of the core protein (Choi et al., 2003). This smaller ARFP is largely uncharacterized. Immunoflourescence studies suggest the larger ARFP, like many of the other HCV proteins, is localized to ER or ER derived membranes (Xu et al., 2003). Pulse chase experiments reveal a surprisingly short, 10-minute half-life of ARFP in Huh7 cells (Xu et al., 2003). The ARFP is most likely degraded by the proteosome complex, the final resting place of many misfolded proteins, as proteosome inhibitors seem to stabilize the ARFP (Xu, 2003). It is easy to dismiss the generation of the ARFP as an artifact of the in vitro translation and over expression systems used in the description of this phenomenon, save the presence of antibodies directed against ARFP sequences observed in infected HCV patient sera. The presence of these anti-ARFP antibodies in patient sera suggests this protein is generated in the natural

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course of an HCV infection (Varaklioti et al., 2002; Xu et al., 2001). The function of the ARFP(s) remain to be elucidated.

E1 and E2 E1 and E2 represent the HCV viral glycoproteins and are presumably the virion components required for receptor binding and fusion with the cellular membranes. E1 and E2 are type I trans membrane proteins with large amino terminal ectodomains facing the lumen of the ER. The ectodomain of E1 consists of 160 amino acids, and this region of E2 is considerable larger, consisting of 334 residues. Both proteins contain small (approximately 30 residue) trans membrane spanning anchors (TM) located at their carboxy termini. The requirement of both ectodomains to be within the ER lumen, combined with the location of the TMs, necessitates a complex interaction of the proteins with the ER translocation machinery in which the TM of E1 must be repositioned to allow for the correct topology of E2 (Cocquerel et al., 2002). In addition to their role in anchoring the glycoproteins, the TMs of E1 and E2 are involved in the formation of noncovalent E1-E2 heterodimers (Cocquerel et al., 1998; Michalak et al., 1997; Selby et al., 1994). A number of reports have also demonstrated the formation of large disulfide linked aggregates of E1 and E2 (Dubuisson et al., 1994; Grakoui et al., 1993b). The relevant disulfide bond formations are believed to be solely intramolecular. Interactions between the ectodomains of E1 and E2 are important in stability and processing of the proteins (Cocquerel et al., 2001; Patel et al., 2001), and a recent publication has demonstrated the importance of C in this process (Merola et al., 2001). Both E1 and E2 have been shown to interact with the ER chaperones BiP, calnexin, calreticulin, and the enzyme protein disulfide isomerase at various stages in the maturation process (Choukhi et al., 1998). E1 and E2 are heavily modified with complex N-linked glycosylation, containing 5 and 11 such modifications, respectively. The membrane insertion, folding, disulfide bond formation, glycosylation, and oligomerization of the envelope proteins are complex events that have been reviewed in detail elsewhere (Op De Beeck et al., 2001). A structural model of E2 has recently been proposed based on the solved structure of the related flavivirus tick borne encephalitis virus E protein, and the organization of the proteins is believed to be similar (Yagnik et al., 2000). Little is known about the structure of E1, or the structure of the mature dimeric glycoproteins. The search for the cellular receptor for HCV binding and entry has a long history, beginning with demonstration of the binding of a soluble form of E2

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to cells and potential receptor molecules, and eventually leading to the use of artificial HCV like particles, including artificial membrane vesicles containing the HCV glycoproteins, virus like particles, and virus pseudotypes (reviewed in Flint et al., 2001). Current experimental data has implicated tetraspanin CD81, low density lipoprotein receptor, scavenger receptor class B type I, dendritic cellspecific intracellular adhesion molecule 3 grabbing nonintergrin (DC-SIGN), the related molecule DC-SIGNR, liver/lymph node specific intracellular adhesion molecule 3 grabbing integrin (L-SIGN), and heparan sulfate as potential HCV receptor molecules (Barth et al., 2003; Bartosch et al., 2003; Flint et al., 1999a; Gardner et al., 2003; Pohlmann et al., 2003; Scarselli et al., 2002). Many of these interactions can be blocked by anti-E2 neutralizing antibodies, suggesting the specific nature of the observed interactions. An emerging body of evidence suggests that a number of these molecules are required in a “receptor complex” for productive binding and entry of HCV pseudotypes, and that no one molecule is the HCV receptor. Little is known about the interactions of the glycoproteins involved in membrane fusion, although pseudotype virus infection studies indicate this is a pH dependent mechanism (Hsu et al., 2003; Meyer et al., 2000). A 26 amino acid region of E1 is similar to other viral fusion peptides, but the demonstration of this sequence as a functional fusion peptide has not been performed (Flint et al., 1999b). It is important to note that psuedotyped viruses may not completely represent the properties of the HCV glycoproteins as they exist in a native virus particle. Nonetheless, the ability to generate psuedotypes virions bearing the HCV glycoproteins is an exciting and powerful tool, and when used with appropriate experimental controls, is quite useful for characterizing the early steps in HCV infection. The reader is directed to a recent review on pseudotypes and the study of HCV entry (Castet, 2003). Undoubtedly, the generation of a system capable of producing infectious HCV virions will greatly aid in the understanding of the mechanisms of receptor binding and fusion. An unusual and surprising finding for the E2 protein is the reported interaction and inhibition the activity of the double stranded RNA dependent protein kinase (PKR) (Taylor et al., 1999). Interactions between E2 and PKR have been observed in cells over expressing E2, and similar interactions have been observed using an in vitro binding system (Taylor et al., 2001). It is unclear how the ectodomain of E2, located in the ER lumen, would interact with the cytoplasmic PKR protein. A newer report indicates that the glycosylated form of E2 in the ER does not interact with PKR, but rather a novel, non-glycosylated cytoplasmic E2 mediates this interaction (Pavio et al., 2002). How exactly this cytoplasmic form of E2 is generated in the normal course of polyprotein translation and processing is difficult to envision. A recent publication has demonstrated that no correlation

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exists between the presence of the E2-PKR interaction sequence of E2 and response to interferon therapy (Abid et al., 2000). In addition to the proposed role in PKR signaling, E2 has been shown to induce the unfolded protein response signal cascade of the ER (Liberman et al., 1999).

p7 Protein The p7 protein is a small, 63 amino acid protein found at the junction of the structural and non-structural proteins in the HCV polyprotein. p7 is an integral membrane protein that crosses the ER membrane twice, leaving the amino and carboxy termini of the protein on the same membrane surface, most likely the ER luminal facet (Carrere-Kremer et al., 2002). The transmembrane spanning regions of p7 have been modeled as ␣-helices, and spatial conservation of residues suggests these transmembrane regions are involved in specific helix-helix interactions (Carrere-Kremer et al., 2002). The orientation of p7 places the E2/p7 and p7/NS2 cleavage sites within the ER lumen, where they are likely cleaved by signal peptidase. Unlike the other HCV polyprotein processing events, the cleavage of both termini of the p7 protein is inefficient, with intermediates of E2-p7 and E2p7-NS2 readily observable. This delayed cleavage of p7 has led to speculation of a regulatory role of p7 processing in virion assembly and downstream protein processing. The p7 protein is dispensable for RNA replication in HCV replicon systems (Blight et al., 2000; Lohmann et al., 1999). Evidence of a possible role for p7 in virion morphogenesis can be found in studies on the analogous p7 protein of the pestiviruses. Although not associated with mature virions, the pestivirus p7 protein is required for the production of infectious progeny (Harada et al., 2000). The p7 protein can be supplied in trans in these systems, suggesting more than a simple protein topology and processing role for p7. Immunoflouresence studies on HCV p7 sub-cellular localization indicate that p7, in addition to the aforementioned ER localization, is also found on internal vesicles and the cell surface, suggesting a role in modulation of cellular vesicular trafficking for progeny virion maturation and release (Carrere-Kremer et al., 2002). Recent data has proposed another role for p7, that of a viroporin ion channel (Griffin et al., 2003). Cross-linking studies suggest p7 forms discrete hexameric structures with a ring-like appearance (Griffin et al., 2003). In artificial membrane/p7 peptide systems, this protein allows ion flux across membranes that can be blocked with a variety of ion channel blockers (Griffin et al., 2003; Pavlovic et al., 2003). The function of p7 as a viroporin in the course of a natural infection has been postulated to be involved in virion morphogenesis. As with the other HCV proteins that have been proposed to be involved in morphogenesis, p7 will likely remain a mystery until a system for

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generating infectious HCV virions that is amenable to molecular techniques is developed.

HCV VIRIONS AND HCV ASSEMBLY The determination of the structure and composition of the HCV virion is one of the most daunting tasks in HCV research. Presumably, the virion is composed of the C protein complexed with RNA in a nucleocapsid structure, enveloped by a host derived lipid bilayer containing the E1 and E2 glycoproteins. Experiments demonstrating the composition of the virion are greatly complicated by the inability to culture HCV in a tractable laboratory system, and little is therefore known about virus particles. Filtration experiments of infected materials suggest the virus particle is between 30 and 80 nm (Bradley et al., 1985; He et al., 1987; Yuasa et al., 1991). Density gradient centrifugation of infected chimpanzee serum suggests a buoyant density of 1.03−1.1 g/ml, consistent with an enveloped virus (Carrick et al., 1992; Hijikata et al., 1993b). It should be noted that considerable variation in density of HCV particles have been observed, presumably through interaction with immunoglobulins and lowdensity lipoproteins (Andre et al., 2002; Carrick et al., 1992; Hijikata et al., 1993b; Thomssen et al., 1992, 1993; Watson et al., 1996). Stripping the membranes off of HCV particles with chloroform or detergents releases what is believed to be the nucleocapsid (buoyant density of 1.17−1.25 g/ml) (Hijikata et al., 1993b; Kanto et al., 1994; Miyamoto et al., 1992). Nucelocapsids have been directly observed in the cytoplasm of infected human hepatocytes in at least one report (Falcon et al., 2003). A system for the in vitro assembly of nucleocapsids has been developed using purified C protein (Kunkel et al., 2001). Preliminary in vitro assembly data suggests that C protein undergoes conformation changes during oligomerization and nucleocapsid assembly. Additionally, in vitro assembled nucleocapsids are RNAse sensitive, suggesting a structural role for nucleic acid in maintenance of the nucleocapsid structure. The mechanism of nucleocapsid and virion formation in infected cells is unknown. A single report has demonstrated a weak interaction between C and E1 during immunoprecipitation, hinting at an interaction involved in budding (Lo et al., 1996). Interactions of the C protein with the HCV 5 NTR may represent an early step in nucleocapsid assembly (Hwang et al., 1995; Tanaka et al., 2000). The aforementioned heterodimerization of the glycoproteins is believed to be a requirement for virion assembly. Complete HCV virions have been directly visualized in pooled human plasma and both chimpanzee and human liver samples by electron microscopy (De Vos et al., 2002; Falcon et al., 2003; Prince et al., 1996; Takahashi et al., 1992). These particles appear to be enveloped

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structures with a diameter of approximately 50−60 nm. A recent publication has observed these particles in both cytoplasmic membrane vesicles and the ER of infected human hepatocytes (Falcon et al., 2003). This correlates well with the hypothesis that HCV assembly is similar to that observed for other members of the Flavivirdae, with budding into internal membranes and release via host vesicle trafficking system. A number of reports using surrogate viruses to express the HCV structural proteins, thereby generating what are referred to as virus like particles (VLPs) have been published (Baumert et al., 1998; Ezelle et al., 2002). The VLPs generated by these systems appear to have a similar size and morphology to the native HCV virions. These VLPs, in addition to their use in potential vaccine development and receptor binding studies, may provide important insights into the composition of the native HCV virion (Baumert et al., 1999). Additional data about the structure of the HCV virion may be gleaned from the recent cryo-EM image reconstruction of the flavivirus Dengue, although it is not clear how similar this particle is to the HCV virion (Kuhn et al., 2002). The major impasse in understanding the HCV virion is the lack of a robust system for the generation of particles.

HCV Non-Structural Proteins NS2 The NS2 protein is an integral membrane protein of approximately 23 kD. The membrane topology of NS2, although not completely understood, is an area of active research with current models suggesting four transmembrane spanning ␣-helices with the amino and carboxy termini of the protein located in the ER lumen. Although the topology of the p7 protein places the amino terminus of NS2 in the ER lumen, the protein appears to associate with membranes when expressed alone, due to the presence of at least 2 internal signal sequences (Yamaga et al., 2002). The localization of the carboxy terminus of NS2 in the ER lumen by glycosylation site mapping is somewhat controversial, as the NS2/NS3 cleavage site must be located on the cytoplasmic face of the ER membrane to generate a cytoplasmically localized NS3 protein (Yamaga et al., 2002). The carboxy terminus of NS2 has been proposed to insert into the ER membrane after the cleavage of the NS2/NS3 junction. The cleavage of the NS2/NS3 junction is performed in a co-translational manner by the NS2–3 autoproteinase activity composed of approximately half of the NS2 protein and the amino terminal 180 amino acids of NS3 (Grakoui et al., 1993a; Hijikata et al., 1993a). This cleavage event marks the only known function of the NS2 protein, which is dispensable for RNA replication in cell culture (Blight et al., 2000;

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Lohmann et al., 1999). It is important to note that the NS2–3 autoproteinase activity is distinct from the serine proteinase activity of the amino-terminal region of NS3, despite the physical overlap of these activities in the NS3 protein sequence. The NS2–3 protease activity is stimulated by addition of microsomal membranes, detergent and zinc in cell free translation extracts (Grakoui et al., 1993a; Hijikata et al., 1993a; Pieroni et al., 1997; Santolini et al., 1995). Although the stimulatory nature of zinc, and the conversely inhibitory activity of EDTA on the NS2–3 proteinase suggest a metalloproteinase activity, the presence of a structural zinc atom in the amino terminal region of NS3 required for the NS2–3 and NS3 proteinase activities complicates this interpretation. Site-directed mutagenesis implicates amino acids His 143 and Cys 184 of NS2 in the NS2–3 proteinase activity, with current models placing these two residues as the catalytic diad of a thiol protease (Neddermann et al., 1997). The NS2–3 proteinase has been shown to require, presumably through a direct protein-protein interaction, the host cell chaperone protein hsp90 for proper cis-cleavage activity (Waxman et al., 2001). A cellular J-domain protein has been shown to be involved in the NS2–3 protein cleavage in a pestivirus, and similar interactions have been proposed for HCV (Rinck et al., 2001). In pestiviruses the processing of the NS2/NS3 junction is often incomplete, and the production of cleaved NS3 has been correlated with pathogenesis (discussed in Lindenbach & Rice, 2001). Additionally, processing in the NS2/NS3 region has been shown to be involved in virion morphogenesis (Kummerer & Rice, 2002). The cleavage of the NS2/NS3 junction appears to be complete in HCV. The possible link between NS2/NS3 processing and HCV pathogenesis or virion morphogenesis have not yet been addressed. NS3 and NS4A The NS3 protein is a large (approximately 70 kD) multifunctional enzyme comprised of two domains; a serine protease domain at the amino terminus (independent of the NS2–3 proteinase activity), and an NTPase/helicase domain at the carboxy terminus. The NS3 serine protease activity is modulated by the small (54 amino acid) NS4A protein (Failla et al., 1994; Pang et al., 2002), and NS4A plays an important structural role in the serine protease (reviewed in (De Francesco & Steinkuhler, 2000)). In addition NS4A is also responsible for localizing the cytoplasmic NS3 to perinuclear ER membranes via an amino terminal hydrophobic region (W¨olk et al., 2000). For these reasons NS3 and NS4A are usually considered as a complex. NS3–4A represents the best characterized of the HCV proteins, with numerous crystal structures of the NS3 protease domain alone and complexed with NS4A, the NS3 NTPase/helicase domain with and without nucleic acid, and the full length NS3 protein complexed with the NS4A protein are available (Cho et al., 1998;

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Kim et al., 1996, 1998; Love et al., 1996a; Yan et al., 1998; Yao et al., 1997, 1999). The intricacies of these crystal structures are beyond the scope of this review, and the reader is thereby directed to (De Francesco et al., 2003; De Francesco & Steinkuhler, 2000; Kwong et al., 1999) for a review of these structures. The amino terminal serine proteinase activity of NS3–4A catalyzes the cis cleavage of the NS3/NS4A junction, as well as the downstream trans cleavages of the NS4A/4B, NS4B/NS5A, and NS5A/NS5B junctions. The NS3 serine protease has a overall fold reminiscent of chymotrypsin, with two six stranded squashed ␤-barrel sub-domains forming an active site cleft/substrate binding pocket between the sub-domain interface. The function of NS4A in the NS3–4A protease activity becomes clear when the structures of the protease domain with and without NS4A are compared (Kim et al., 1996; Love et al., 1996b; Yan et al., 1998). NS4A forms a ␤-strand that interacts with the amino terminal residues of NS3 to generate a two strand antiparallel ␤-sheet that is important in orienting the active site catalytic triad. A structural zinc atom is also important for the proper folding and activity of the protease domain of NS3 (reviewed in De Francesco et al., 1998). Some of the residues responsible for coordinating this zinc atom lie within the loop connecting the two ␤ barrels and therefore may affect the geometry of the active site that lies between the barrels. These features are indicated in the crystal structure of the protease domain complexed with an NS4A peptide presented in Fig. 4A (Yan et al., 1998). The association of NS3–4A protease with trans substrates can be inferred by the crystal structures of this complex with peptide mimetic drugs (Di Marco et al., 2000). Insights into the cis cleavage of the NS3/4A site can be gleaned from the crystal structure of a recombinant single chain NS3NS4A protein construct that generates the 14 residues of NS4A responsible for interaction with the NS3 protease domain as an amino terminal extension of the complete NS3 protein (Yao et al., 1999). In this structure the carboxy terminus of NS3 lies adjacent to the NS3 active site, presumably in a conformation similar to that seen in cis cleavage of NS3-NS4A (Fig. 4C) (Yao et al., 1999). What rearrangements of the carboxy terminus of NS3 render the active site accessible for subsequent trans cleavages remains to be demonstrated. The crystal structure of this NS3-NS4A construct indicates the true multi-domain nature of NS3, with the protease and NTPase/helicase domains clearly separated by a flexible loop region. Nevertheless, some interdomain contacts exist, most notably the interaction of the protease domain in generating a portion of the nucleic acid binding site on the helicase domain and the more compact subdomain contacts within the helicase induced by the protease domain. The NTPase/helicase region of NS3 is similar to members of helicase superfamily 2. This domain of NS3 is comprised of three subdomains, two ␤-␣-␤ subdomains and a third helical subdomain (Cho et al., 1998;

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Fig. 4. Overview of HCV NS3 and NS5B Structures. Note: Panel A. The crystal structure of the NS3 protease domain complexed with a peptide corresponding to a portion of NS4A (Love et al., 1996b). The active site cleft between the two ␤-barrel subdomains is designated. The structural zinc atom required for protease activity is shown (Zn) coordinated by the loop structure connecting the two subdomains. The amino (N) and carboxy (C) termini of the NS4A peptide are shown. Panel B. Crystal structure of the helicase domain of NS3 complexed with a short synthetic nucleic acid (Yao et al., 1997). The locations of the 3 subdomains of NS3 are indicated, as is the location of the bound nucleic acid. Panel C. The crystal structure the entire NS3 protein and a portion of NS4A (Yan et al., 1998). The well separated protease and helicase domains are shown. The position of the carboxy terminus of NS3, adjacent to the protease domain active site, is labeled (C). Panel D. The crystal structure of the NS5B RNA dependent RNA polymerase (Lesburg et al., 1999). The classic palm, fingers, thumb domain organization of the polymerase is shown. The active site (AS) region of the polymerase is designated. Note that the active site is completely enclosed by interactions between the finger and thumb domains.

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Kim et al., 1998; Yao et al., 1999). The crystal structure of the helicase domain of NS3 complexed with nucleic acid is shown in Fig. 4B (Kim et al., 1998). The ␤-␣-␤ subdomains are arranged with a central hydrophobic core of ␤-sheets and flanking ␣-helices. The two ␤-␣-␤ subdomains are structurally similar, each containing a 6 stranded parallel ␤ sheet, with the exceptions of the presence of a single antiparallel ␤-strand in subdomain I and two anti-parallel ␤-strands in subdomain II and the arrangement of the flanking helices of each subdomain. Subdomain I contains the NTPase activity, with subdomain II involved in RNA binding. The subdomains are arranged in configuration similar to the shape of the letter Y, with clefts between subdomains that contain the conserved helicase motifs (between subdomains I and II) and RNA binding site (between subdomain III and I and II). Both the helicase domain alone and full length NS3 have in vitro helicase activity, although the activity of the full length protein is enhanced compared to the helicase domain alone, likely due to the aforementioned role of the protease domain in the generation of the RNA binding site (Gallinari et al., 1998; Howe et al., 1999; Kumar et al., 1997; Urvil et al., 1997). The NS3 helicase activity unwinds double stranded RNA and DNA, as well as RNA/DNA heteropolymers in a 3 –5 orientation in the presence of ATP and the appropriate divalent cations, Mn++ and Mg++ (Tai et al., 1996). The NTPase/helicase activities of NS3 show what appear to be a complex regulation, presumably via cross-talk between subdomains (Levin et al., 2003). The regulation of this enzyme in the complex process of genome replication is undoubtedly regulated in an intricate manner, but the exact nature of this regulation remains unclear. The NS3 protein has recently been shown to bind to the poly(U/C) tract located in the 3 NTR of the HCV genome, and it is an attractive hypothesis that this protein is involved in unwinding structured RNAs during replication or unpairing of plus and minus strand replicative intermediates (Banerjee & Dasgupta, 2001). The binding of NS3 to the HCV RNA polymerase further suggests a direct role in the manipulation of RNA during replication (Piccininni et al., 2002). The NS3 and NS3–4A proteins have further been demonstrated to interact with and modulate the phosphorylation of the NS5A protein, a putative component of the replicase (Koch & Bartenschlager, 1999; Neddermann et al., 1999). The NS3 NTPase and helicase activities are absolutely required for RNA replication in HCV and the related flavivurses and pestiviruses, yet the actual role of these activities in RNA replication is undefined (Gu et al., 2000; Matusan et al., 2001). Nevertheless, the absolute requirement for both the NS3 protease and NTPase/helicase activities for replication has led to the development of exciting new anti-viral drugs targeting NS3 (De Francesco et al., 2003). NS3 has been proposed to interact with a large number of cellular proteins with a myriad of effects, but the relevance of these interactions to HCV replication

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remain to be demonstrated. The majority of the proposed interactions for NS3 involve alteration of normal cellular signaling pathways, a feature that has been proposed for many of the HCV proteins. Sequences in the NS3 protein resemble the consensus target sequences and autophosphorylation sequences of both PKA and PKC (Borowski et al., 1996, 1997, 1999a, 1999c, 2000). NS3 has been shown to interact with the catalytic subunit of PKA and with PKC, thereby inhibiting the activity of these kinases by both blocking their interaction with normal cellular targets and preventing the relocalization of these proteins upon activation (Borowski et al., 1996, 1997, 1999a, 1999c, 2000). The function of these interactions in an infected cell is not clear. The NS3 protease domain appears to be weakly oncogenic in cell culture, possibly through an observed interaction with the p53 tumor suppressor (Ishido et al., 1997; Ishido & Hotta, 1998; Sakamuro et al., 1995). NS3 can interact with several histone proteins in vitro via an internal histone binding sequence, possibly allowing the modification of host cell transcription, although it is unclear how a cytoplasmic protein can interact with nuclear histone proteins (Borowski et al., 1999b). Recent publications demonstrate the covalent modification of NS3 by arginine methyltransferase 1, possibly modifying the interaction of NS3 with other proteins (Rho et al., 2001). NS4B NS4B is probably the least well characterized of the HCV proteins. NS4B is small hydrophobic integral membrane protein that co-translationaly associates with ER membranes via an internal signal sequence. The protein has been predicted to cross the ER membrane between four and six times, resulting in the orientation of the amino and carboxy termini in the cytosol (H¨ugle et al., 2001). Despite the large number of predicted membrane spanning regions and relative hydrophobicity of the protein, the bulk of NS4B appears to be on the cytoplasmic face of the ER membrane (H¨ugle et al., 2001). Immunoflouresence studies indicate NS4B is associated with ER or ER-like membranes, and when expressed alone this protein alters the ER into convoluted vesicular structures referred to as membranous webs (Egger et al., 2002; H¨ugle et al., 2001). Further characterization of these membranous webs has shown the presence of all of the HCV non-structural and structural proteins as well as replicating HCV RNA within these structures (Gosert et al., 2003). The membranous web has therefore been proposed to be the site of HCV RNA replication and possibly the site of the early stages of virion assembly. An additional role of NS4B has been proposed. NS4B protein may have oncogenic properties, based on transformation of cells expressing NS4B in conjunction with Ha-ras, but this observation has yet to be linked to HCV replication or pathogenesis (Park et al., 2000).

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NS5A NS5A is a large (56–58 kDa), hydrophilic protein of unknown function. The ability of adaptive mutations in NS5A to greatly stimulate HCV replication in cell culture, and the association of this protein with other members of the putative replicase suggests NS5A plays an important role in RNA replication (Blight et al., 2000; Lohmann et al., 2001). NS5A is associated with ER derived membranes via an amino-terminal amphipathic ␣-helix that has been proposed to be partially buried in one leaflet of the cellular membrane (Brass et al., 2002). Deletion of this helix leads to a diffuse cytoplasmic localization of NS5A and is lethal for RNA replication. Although NS5A has been clearly shown to be an ER associated protein in cells with actively replicating HCV replicons, numerous publications suggest an alternate nuclear localization. The presence of a cryptic nuclear localization signal in the interior of NS5A has been proposed (Ide et al., 1996). The exposure of this nuclear localization signal by a caspase mediated cleavage in apoptotic cells has been observed to allow the nuclear localization of NS5A, where is has been proposed to function as a PKA-regulated transcription factor (Goh et al., 2001; Satoh et al., 2000). The nuclear localization of NS5A and its function as a transcription factor are areas of significant controversy in the HCV community. The NS5A protein exists in multiple phosphorylation states, designated p56 (basal) and p58 (hyper) based on their migration on SDS-PAGE gels. The majority of phosphorylation occurs on serine residues, although some threonine phosphorylation has been observed (Kaneko et al., 1994; Reed et al., 1997). A number of phosphorylation sites have been mapped for 1a and 1b genotype NS5A sequences (Katze et al., 2000; Reed & Rice, 1999). The hyper phosphorylation of NS5A appears to require the presence of other HCV non-structural proteins in cis (Asabe et al., 1997). NS5A appears to be directly associated with the cellular kinase(s) responsible for its phosphorylation ( Reed et al., 1997; Tanji et al., 1995). A number of kinases have been proposed to be responsible for NS5A phosphorylation (Arima et al., 2001; Kim et al., 1999), but inhibitor studies suggest that a yet to be identified enzyme of the CMGC group (an abbreviation reflecting the best characterized members; CDK, MAPK, GSK3, CKII) of kinases is responsible (Reed et al., 1997). The same kinase activity is believed to phosphorylate the NS5A and NS5 proteins of pestiviruses and flaviviruses, respectively (Reed et al., 1998). Much of the work to date regarding the characterization and identification of the NS5A associated kinase activity has been performed in surrogate expression systems, and although the kinase activity appears to be evolutionarily conserved in yeast, insect and mammalian cells, the consequences of phosphorylation have not been examined in the context of HCV RNA replication. The NS5A phosphorylation state varies in a number of adaptive NS5A mutations in HCV replicons (Blight et al., 2000), but the link between NS5A phosphorylation and replication is unknown.

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The phosphorylation of NS5A, and its direct association with a cellular kinase or kinases, has led to the investigation of interactions between NS5A and cellular signal transduction pathways. The interactions observed are too numerous and convoluted to discuss in detail herein, and the reader is directed to (Reyes, 2002) for a review. The vast majority of these publications rely on the over expression of NS5A in the absence of a functional replicase, thereby complicating the interpretation of the data in the context of HCV replication. In addition, many of these studies are contradictory, with NS5A activating and inhibiting some of the same cellular pathways in different experimental systems. Another area of active research, and active debate, in the HCV community is the interaction of NS5A protein with PKR and the IFN/chemokine systems. The NS5A protein contains a short sequence in the central region of the protein referred to as the interferon sensitivity determining region (ISDR), named for the weak association of hypermutation in this region with response to IFN therapy for patients infected with HCV genotype 1b (Enomoto et al., 1996). Surprisingly, deletion of the ISDR does not affect the IFN sensitivity of HCV replicons (Lohmann et al., 2001). NS5A has been shown to bind PKR, via the ISDR, and inhibit the IFN induced activity of PKR on downstream targets, most notably eIF2␣, thereby preventing the antiviral effects of IFN (Gale et al., 1997, 1998, 1999; Gale & Katze, 1998; Gale, Korth & Katze, 1998; Pawlotsky et al., 1998; Pawlotsky, 1999). Another interaction of NS5A with the IFN response has been observed with the ability of NS5A to induce interleukin 8, leading to the inhibition of the antiviral effects of IFN (Pflugheber et al., 2002). The intricacies of the interaction of NS5A with PKR and IFN have been reviewed elsewhere (Reyes, 2002; Tan & Katze, 2001). NS5B The NS5B protein comprises the viral RNA dependent RNA polymerase activity (RdRp) required for the generation of a minus strand complimentary genome templates, and the subsequent synthesis of progeny plus strand genomic RNAs from this replicative intermediate. NS5B was initially predicted to function as an RNA polymerase based on the presence of the conserved GDD motif common to the active site of other polymerases (Choo et al., 1989). Mutation of this GDD motif abolishes infectivity of HCV transcripts in chimpanzees and blocks RNA replication in the replicon system (Blight et al., 2000; Kolykhalov et al., 2000; Lohmann et al., 1999). NS5B is a large (68 kD) hydrophilic protein that is found associated with ER derived membranes (Ivashkina et al., 2002; Schmidt-Mende et al., 2001). The association of NS5B with membranes has been determined require a hydrophobic 21 amino acid sequence at the carboxy terminus of the protein that has been proposed to form an ␣-helix (Ivashkina

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et al., 2002). The insertion of this sequence into membranes is believed to be posttranslational, making NS5B a member of the tail-anchored class of membrane proteins (Ivashkina et al., 2002). Deletion of this sequence leads to a predominantly nuclear localization of the polymerase (Ivashkina et al., 2002). Removal of this sequence in heterologous expression systems has allowed to generation of a soluble form of NS5B that retains enzymatic activity in vitro (Ferrari et al., 1999; Lohmann et al., 1997). A number of crystal structures of the soluble form of NS5B have been generated (Ago et al., 1999; Bressanelli et al., 1999, 2002; Lesburg et al., 1999; O’Farrell et al., 2003). These structures have been reviewed elsewhere in great detail (De Francesco et al., 2003; Hagedorn et al., 1999). The overall fold of NS5B is similar to that of other single chain polymerases, with a classic right hand topology containing distinct palm, finger and thumb subdomians (see Fig. 4D). Extensive interactions exist between the finger and thumb subdomains, thereby restricting movement of these domains relative to each other, resulting in a fully enclosed active site capable of binding nucleotides without further conformational changes (Lesburg et al., 1999). It is therefore believed that the structures of NS5B represent a polymerase during initiation events, and further conformational changes that have yet to be observed are required for elongation. Another unique feature of the NS5B polymerase is the presence of a ␤-hairpin in the thumb subdomain that is located close to the enzyme active site. This loop has been shown to restrict access to the active site and is believed to play a role in the initiation of RNA synthesis (Bressanelli et al., 2002; Hong et al., 2001). The thumb subdomain also contains an allosteric regulatory site that has been shown to bind GTP (Bressanelli et al., 2002). Recent structures of NS5B complexed with nonnucleoside inhibitors suggest the importance of the region of the thumb subdomain near this allosteric site in conformational changes required for the transition from initiation to elongation (O’Farrell et al., 2003). NS5B has been an important target for the development of antiviral drugs (see De Francesco, 2003 for review). The enzymatic activity of NS5B has been extensively studied (see De Francesco et al., 1996; Hagedorn et al., 1999; Lohmann et al., 2000 for review). NS5B is capable of the extension of both RNA and DNA primers in vitro using a variety of templates (Al et al., 1997, 1998; Behrens et al., 1996; Lohmann et al., 1997; Yamashita et al., 1998). Additionally, NS5B is capable of the synthesis of the entire HCV genome in vitro via a copy back method in which the 3 end of the genome serves as an artificial primer for the extension of the nascent RNA (Behrens et al., 1996; Lohmann et al., 1997). The polymerase is also capable of de novo synthesis of RNA in the absence of any primer, and this mechanism of action is widely accepted as the relevant mechanism of action for NS5B (Luo et al., 2000; Zhong et al., 2000). A recent crystal structure of NS5B with bound nucleotides strongly supports the de novo synthesis of HCV RNA, as this structure is similar to the structure of the de

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novo initiation complex of the bacteriophage phi 6 polymerase (Bressanelli et al., 2002). An oligomerization of the HCV polymerase has been observed that may be important in cooperative activity of the enzyme (Qin et al., 2002; Wang et al., 2002). NS5B has been shown to directly interact with NS3, NS4A, and NS5A, and some of these interactions modify the enzymatic activity of NS5B (Ishido et al., 1998; Shirota et al., 2002). It is exciting to speculate these contacts mimic interactions in the functional HCV replicase.

HCV GENOME REPLICATION AND THE HCV REPLICASE The general mechanism of HCV RNA replication is believed to involve de novo initiation of RNA synthesis at the 3 end of the genome followed by extension in the 5 to 3 direction to generate a minus strand complimentary genome template, and the subsequent synthesis of progeny plus strand genomic RNAs from this minus strand replicative intermediate. Examination of RNA copy number from cells bearing HCV replicons suggest 50–5,000 genomic RNAs are present per cell (Blight et al., 2000; Lohmann et al., 1999). The ratio of plus strand to minus strand RNAs in these systems is approximately 5–10:1. The mechanism for regulating the ratio of positive and negative strand RNA synthesis is unknown. Similar numbers have been determined from the examination of infected hepatocytes (Lanford et al., 1995). Replication is believed to occur in association with ER membranes and require the activity of the viral polymerase, helicase, and a mixture of the other nonstructural and structural proteins. Numerous host factors are likely to be involved in this process as well. The RNA replication complex has recently been observed in Huh7 cells containing HCV replicons, and this structure has been termed the membranous web (Gosert et al., 2003). The membranous web has been shown to contain all of the HCV structural and non-structural proteins as well as actively replicating RNA (Gosert et al., 2003). This structure is similar in appearance to the sponge-like inclusions seen in infected chimpanzee hepatocytes, and is most likely a modification of the host ER membrane (Pfeifer et al., 1980). The interaction of all of the HCV proteins with ER membranes has been described (see Dubuisson et al., 2002, for review). Recent biochemical assays have described several novel homo- and heterotypic interactions among HCV non-structural proteins that may be involved in the replicase (Dimitrova et al., 2003). Cell culture adaptive mutations that increase RNA replication efficiency in replicons have been observed in all of the non-structural proteins, suggesting the entire non-structural region is important for replication (Blight et al., 2000; Lohmann et al., 1999). The incompatibility seen when combining adaptive mutations in the same RNA suggests these mutations

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affect multiple phenomena to generate increased HCV RNA replication (Lohmann et al., 2003), although the mechanism of action of is not clear. Adapted cell lines that allow increased HCV replication suggest the importance of host cell factors in the replicase (Blight et al., 2002). Numerous host factors have been proposed to play a role in replication, but none of these have been validated (Tellinghuisen & Rice, 2002). Despite all of these observations, little is known about what constitutes a functional HCV replicase. Advances in the efficiency of HCV replicon systems in the past few years, yielding a tractable system for reverse genetics, will likely aid the understanding of HCV replication in future years (Blight et al., 2000, 2003). The recent development of a system allowing replication of HCV RNA in cell lysates generated from replicon bearing Huh7 cells may be a valuable tool in defining the relevant components of the HCV replicase, as has been performed for poliovirus (Ali et al., 2002; Hardy et al., 2003). Unfortunately, our understanding HCV replication and the HCV replicase are in their infancy, but this is an active area of research, and significant progress is expected in future years.

CONCLUSIONS In just 14 short years since the initial discovery of HCV as an infectious agent, significant progress has been made in understanding the molecular virology of this important pathogen, despite the numerous experimental difficulties associated with HCV research. A number of powerful experimental systems for the study of HCV have been developed, and through the use of these tools the biology of HCV has slowly started to emerge. The processing, localization, membrane association/topology, and putative functions of the majority of the HCV proteins have been determined. Considerable characterization of the HCV IRES and the viral NTRs has been performed. The site of HCV replication in cells bearing replicons has been observed. Considerable enzymatic characterization of HCV proteins with known activities has been performed. Molecular structures of half of the non-structural proteins have been determined to atomic resolution. Additional structural characterization of portions of other HCV proteins and the viral RNA has been performed. The structural and biochemical data generated to date has served to further the development of anti-HCV therapeutics, with many new potential pharmacological agents in development. Literally hundreds of interactions of the various HCV proteins and RNA with host cell proteins have been described, with effects attributed to many important cellular processes. The structure and properties of the HCV virion and the interactions of the virion components in assembly, receptor binding, penetration, and disassembly have only begun to be characterized, but newly described systems for studying these processes will likely

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lead to a greater understanding of HCV morphogenesis and infection. Despite the amazing progress made in understanding HCV, many questions remain to be answered. It is perhaps the greatest challenge in HCV research to distill the information in hand, as well as future observations, into concise mechanisms to describe the molecular virology of HCV.

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