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Salient molecular features of hepatitis C virus revealed
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Vol.13 No.11 November 2005
Salient molecular features of hepatitis C virus revealed Christoph Seeger Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
Hepatitis C virus (HCV) is a positive strand RNA virus with a narrow host and tissue tropism. It ranks among the most significant of human pathogens, causing inflammation, scarring and cancer of the liver. Recent investigations have shed light on some of the salient molecular features of this virus. These include a requirement for CD81 (a tetraspanin transmembrane protein for viral entry), a novel mechanism for the initiation of RNA synthesis, phosphorylation of a viral protein in the regulation of RNA amplification and virus assembly and, finally, a viral protease suppressing activation of the innate immune response in infected cells.
A major medical challenge Human hepatitis C virus (HCV) is a major blood-borne hepatitis virus that causes chronic infections in an estimated 170 million people, second only to human hepatitis B virus (HBV) [1]. Hepatitis, fibrosis, cirrhosis of the liver and hepatocellular carcinoma are common sequelae of HCV infections. As a consequence, HCV infections are the major cause for orthotopic liver transplantation [2]. Unfortunately, liver grafts are rapidly infected with residual virus and a second, usually more aggressive cycle of the disease takes its course [3]. Vaccines to prevent initial infections are available against HBV but not HCV. HCV stands for a heterogeneous group of positive strand RNA viruses that form the genus Hepacivirus of the Flaviviridae family of viruses. Hepaciviruses are divided into six principal genotypes and their subtypes, which can exhibit as much as 30% sequence diversity [4]. During the course of an infection, subtypes can exist as a quasispecies where the dominant population constantly changes in the face of an immune response directed against B- and T-cell epitopes in the viral polyprotein [5,6]. However, in contrast to HBV, HCV infections can now be cured with a interferon (IFN-a)-based therapy at a remarkable rate – between 50% and 90% depending on the genotype [7]. Unfortunately, the most common genotype – genotype 1 – is also the most refractory to therapy, resulting in a vast cohort of infected patients that are currently without hope of a cure. However, the cloning of HCV in the late 1980s spurred an enormous research effort aimed at understanding the intricacies of HCV and to develop better antiviral therapies [8]. Corresponding author: Seeger, C. ([email protected]). Available online 8 September 2005
This review highlights recent studies on the host and tissue tropism of HCV, the mechanism and regulation of HCV replication, and the regulation of the IFN response. For more detailed descriptions of clinical aspects of HCV infections, and many other investigations on the molecular biology of this virus, the reader is referred to previously published reviews [9,10]. Host and tissue tropism Broad species and tissue specificity is a hallmark of many viruses belonging to the Flaviviridae family. By contrast, HCV infections are restricted to humans and chimpanzees and target primarily hepatocytes. The molecular basis for this narrow tropism is not yet understood because information about the nature of the viral receptor is still incomplete. Several years ago, the tetraspanine transmembrane protein CD81 was identified as a candidate receptor because its large external loop (LEL) could bind to the envelope protein E2 of HCV1a in solution or when expressed on the surface of cells [11,12]. CD81 is expressed in many different cell types where it has a complex role in the reorganization of membranes in response to external stimulation [13]. Subsequent studies demonstrated that HIV particles carrying the HCV E1 and E2 envelope glycoproteins could infect certain human liver or hepatoma-derived cells expressing CD81 [14–18]. Infectivity of these pseudotypes was pH-dependent and could be neutralized with antibodies against E2 and CD81, as well as with a soluble form of CD81. Similarly, the infectivity of an HCV isolate produced in tissue culture cells (described below) could be inhibited with recombinant LEL or anti-CD81 antibodies [19–21]. Thus, these observations are consistent with the original observation predicting a direct interaction between the tetraspanin transmembrane protein and E2. The results also indicated that additional factors required for infectivity are expressed on the surface of hepatocyte-derived cells, in support of the hypothesis that HCV, like HBV, is a hepatotropic virus [17]. Besides CD81, several other candidate receptors have been identified. Among them is a second E2 binding partner, human scavenger receptor class B type I (SR-BI) [22]. SR-BI is expressed primarily in the liver, therefore, the E2–SR-BI interaction is consistent with the apparent tissue specificity of HCV. However, the significance of this interaction for viral infection has been questioned by the demonstration that antibodies against SR-BI could diminish binding of E2 or HCV particles to susceptible
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Upon entry and uncoating, the genome acts as mRNA for the translation of the viral polyprotein. Like with other plus strand RNA viruses, this step is not yet understood. However, it represents a remarkable event in cell biology because it requires that, during an infection, a single RNA molecule is transported to ribosomes with high efficiency. Translation of the polyprotein is directed by an internal ribosomal entry site (IRES, see text). Translation begins with the synthesis of the core protein followed by the two envelope glycoproteins E1 and E2 and the small p7 protein. They undergo cotranslational processing by cellular signal peptidases. In contrast to the structural genes, the remaining six non-structural proteins are processed by the viral NS3 protease with the exception of the NS2-NS3 site, which is cleaved by the NS2-NS3 cysteine protease. Some of the major biochemical and structural properties of the HCV polypeptides have been summarized in Figure 1 and were described in detail elsewhere (e.g. [10]). Translation is interrupted by an unknown signal that activates a reaction leading to the formation of a replication complex on membranes of the endoplasmic reticulum. As a consequence, the mRNA is transformed back to a genome that now acts as a template for the synthesis of minus strand RNA. Viral protein synthesis arrests until plus strands are synthesized and delivered to the ribosome for a new round of RNA amplification. Alternatively, the plus strands are assembled with the structural proteins into virus particles. As observed with other positive strand RNA viruses, plus strands are wtenfold more abundant than minus strands.
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Replication of the viral genome Following infection and uncoating, the HCV RNA transits from genome to mRNA (Box 1). A salient feature of HCV lies in the properties of the internal ribosomal entry site (IRES), which controls translation of the viral polyprotein (Figure 1). The IRES can engage 40S ribosomes in the absence of canonical translation factors, similar to a prokaryotic mRNA [29]. Binding of the IRES induces conformational changes in the 40S subunit that are believed to correctly position the AUG initiation codon at the P site of the ribosome [30,31]. The binary IRES–40S structure binds to eIF3 and the ternary eIF2–Met-tRNA– GTP complex to enable formation of the IRES–80S translation machinery [32]. Thus, in contrast to the
primary hepatocytes but could not inhibit infection of these cells [23]. Obviously, the HCV receptor story is still incomplete leading to the debate about the tropism of this virus. Many studies have provided evidence for the presence of HCV RNA in lymphocytes and other tissues. The question remains whether the virus replicates in these cells and whether they release infectious HCV into the bloodstream. Viral RNA levels are low, on average only a few copies per hepatocyte, thus, the demonstration of viral replication
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requires the selective detection of minus strands (intermediates of viral replication) that are present at tenfold lower levels compared with plus strands. Under conditions where such a distinction was possible with strand-specific PCR, minus strands were only detected in infected liver tissue but not in lymphocytes or other tissues of infected chimpanzees [24]. Moreover, under the same conditions, minus strands could not be detected in peripheral blood mononuclear cells (PBMC) from infected patients [24,25]. Nevertheless, studies providing support for HCV replication in extrahepatic tissues and immortalized lymphocytes continue to flare up and fuel the debate about tissue tropism of HCV (see references [26,27]). HCV RNA replication can occur in cells of non-hepatic origin and even in mouse cells, therefore, it is likely that host and tissue tropism is determined during an early step of the viral life cycle [28]. Thus, clarification of this important problem will require more detailed information about the viral receptor and the mechanism of entry.
Box 1. Model for the replication of HCV
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Figure 1. Physical and functional map of Hepatitis C virus (HCV). The open reading frame encodes four structural (core, envelope 1 and 2 and p7) and six non-structural proteins (NS2–5B). It is flanked by the 5 0 and 3 0 untranslated regions (UTRs). A more detailed description of the 3 0 UTR is provided in Figure 2. Asterisks denote proposed functions of p7 and NS4B. The detailed map of NS5A depicts the three major structural domains (D 1–3) with the helical anchor domain (H), the cysteine residues (C) forming the Zn2C binding domain, the interferon sensitivity determining region (ISDR) and two regions that are not essential for the replication of viral subgenomes in tissue culture cells (D). The amino acid sequence of a segment containing the most frequently observed mutations in adapted replicons (black spheres) is shown at the bottom of the figure. The numbering corresponds to the HCV1b isolate Con1 (AJ238799). Abbreviations: HVR, hypervariable region; Rep. Complex, replication complex; SL, stem-loop; VR, variable region. www.sciencedirect.com
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were defects in the assembly and secretion of virus and, most probably as a consequence of this, lack of infectivity and spread after intrahepatic inoculation into chimpanzees [37]. Recently, the generality of these findings has been challenged with an HCV clone belonging to genotype 2 that replicated with high efficiency in different cell lines without the requirement for adaptive mutations and permitted the production of infectious virus in tissue culture cells [19–21,38] (reviewed in Ref. [39]). However, unlike the other clones used to date, this isolate was obtained from a patient with fulminant hepatitis, a rare form of HCV-induced disease that results in massive liver damage with often fatal outcome. Although it is conceivable that this clone represents a cytopathic variant of a normally non-cytopathic virus with mutations that interfere with controlled amplification of viral RNA, infection of Huh7 cells and a chimpanzee did so far not support this view [19–21,38]. The value of the replicon system for a better understanding of the HCV life cycle has been underscored recently by the discovery of a novel signal near the 3 0 end of the NS5B coding region, which is required for RNA replication. Initial genetic studies revealed stringent requirements for the nucleotide sequence composition of the 3 0 untranslated regions (UTR) that can fold into three stem-loop (SL) structures [40,41] (Figure 2a). Comparative sequence analyses revealed the presence of several stem-loop structures overlapping the coding region of the polymerase that were conserved among HCV isolates [42–44]. Genetic
well-characterized type I and type II IRES element of poliovirus and encephalomyocarditis virus (EMCV), respectively, translation in HCV does not depend on eIF4G and eIF4A (reviewed in Ref. [33]). Although the significance of this unusual mechanism for HCV replication is not known, it might be required for efficient translation in hepatocytes that are normally in a growth arrested state (Go). Like translation, the formation of functional replication complexes also appears to depend on mechanisms that set HCV apart from other plus strand RNA viruses. In tissue culture cells, replication occurs only under selected conditions that are just beginning to be understood. The so-called ‘HCV replicon system’ was developed based on previous studies with West Nile virus (WNV) and bovine viral diarrhoea virus (BVDV), which demonstrated that the structural genes were not required for RNA replication and that subgenomic replicons carrying a neomycin resistance gene could be used to establish cell lines expressing replicons [34–36]. However, the scenario with HCV differed in a major way in that subgenomes prepared from available infectious HCV clones from genotype 1 generally replicated with extremely low efficiency or not at all in most cell lines used for transfection. Efficient replication required either mutations in the non-structural (NS) proteins or, alternatively, depended on cellular factors that are enriched in certain subclones of the hepatoma cell line Huh7 (reviewed in Ref. [10]). Notable features of full length replicons with adaptive mutations (a) SL VI
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Figure 2. Initiation of RNA replication. (a) The proposed secondary and tertiary structures at the 3 0 end of HCV RNA. Stem-loops (SL) VII–IV are located in the coding region of the polymerase (NS5B), whereas SLs III–I are present in the 3 0 UTR region. The two clusters of SLs are separated by the variable region (VR) and the poly(U/C) tract. The positions are numbered according to the sequence of HCV1b Con1 (AJ238799). The figure is not drawn to scale. (b) A model for the initiation of minus strand RNA, which predicts that the 3 0 UTR folds into a kissing-loop structure with the help of Watson–Crick base pairs that can form between the apical loops in SL V and SL II (step 1, [44]). The polymerase then binds to the tertiary structure to initiate minus strand RNA synthesis (steps 2 and 3). www.sciencedirect.com
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experiments revealed that the structure of at least one of these stem-loop structures (SL V, Figure 2) was essential for RNA replication. Importantly, it was demonstrated that the loop region of SL V can base-pair with the loop of SL II present in the 3 0 UTR and form a kissing-loop interaction [44] (Figure 2). Finally, biochemical studies indicated that the NS5B RNA polymerase binds with high affinity to SL V and adjacent sequences in the coding region of NS5B [43,45]. These results helped to refine the model for the initiation of minus strands, which predicts that the 3 0 end of HCV RNA is in a closed loop formation that aligns the binding site for the polymerase with the 3 0 end of the RNA to facilitate the initiation of minus strand RNA synthesis. It is not yet known whether RNA synthesis initiates at the 3 0 of the RNA or whether the polymerase copies a few nucleotides from an internal site and then translocates a short primer to the 3 0 end, essentially as it has been proposed for protein-primed replication in poliovirus and hepatitis B virus [46,47]. In this model, the poly (U/C) tract separating the two stem-loop clusters could act as a linker providing the flexibility necessary for the formation of the tertiary interaction between SL II and SL V. Consistent with such a model, deletion of the tract inhibited RNA replication, probably because it either prevented loop formation or caused premature termination of minus strand synthesis. This last phenomenon might be due to a steric problem created by the proximity of SL V, the binding site for the polymerase and the nascent minus strand (Figure 2b). In support of this possibility, artificially truncated poly(U/C) tracts expanded to their natural size during replication [48]. Thus, if the loop is too small, the polymerase might arrest, slip back and expand the loop until it can continue RNA synthesis. Does NS5A regulate HCV replication? A major question concerning the life cycle of HCV is how the persistence of viral RNA is controlled in infected cells. HCV is a non-cytolytic virus, therefore, RNA accumulation must reach a steady state following an initial exponential amplification phase. Although the mechanism regulating this process is not yet understood, it is tempting to speculate that the NS5A protein might be an important player. NS5A has three major domains that are connected by two protease sensitive hinge regions [49]. The N-terminal domain consists of an amphipathic a-helix proposed to act as a membrane anchor attached to a Zn2C binding site composed of four conserved cysteine residues. Mutations in any of the four residues inhibit HCV replication in tissue culture cells [49]. X-ray crystallographic analysis of the structure of this domain confirmed the presence of the Zn-coordination motif and revealed the presence of a disulfide bond [50]. However, because the cysteine residues required for proposed formation of disulfide bonds were not essential for replication in tissue culture cells, it is uncertain whether they have a role for NS5A function. The first hinge region is the target for many adaptive mutations that are necessary for the persistent replication of HCV replicons in Huh7 cells. The most commonly found adaptive mutations map to conserved serine residues and www.sciencedirect.com
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are associated with a reduction in the levels of p58, the hyperphosphorylated form of NS5A, suggesting that modification of NS5A has an important role in the regulation of HCV replication [51]. In support of this view, a kinase inhibitor reduced the levels of p58 activated replication of wildtype replicons but inhibited the replication of adaptive mutants, suggesting that the ratio between p58 and p56, the basally phosphorylated form, is crucial for the control of RNA amplification [52]. As described previously, replicons with adaptive mutations that reduce p58 levels do not assemble and release virus particles, suggesting that NS5A might also have a role in the assembly of virus particles and, hence, in the decision of whether amplified plus strand RNA returns to the ribosome for a further round of genome replication or enters the assembly pathway for the formation of infectious virus. Unlike all other NS proteins, NS5A exhibits some of its functions in trans, which is consistent with its potential role as an activator or repressor of one or more cellular pathways. However, there are still major gaps that need to be filled to enhance our understanding of NS5A function. For example, although it is known that serines are the primary targets for both basal and hyperphosphorylation, the locations of the serines on the polypeptide are still uncertain. Moreover, identification of the kinases involved in the modification of this protein will be essential to better investigate the possible function of NS5A in the regulation of RNA amplification and assembly of virus particles. Finally, we must determine the nature of the physiologically relevant cellular factors that affect NS5A activity. Although the list of NS5A binding partners is large [53], the interaction with human vesicle-associated membrane protein-associated protein A (hVAP-A) is of particular interest because it is regulated by NS5A phosphorylation [54,55]. HVAP-A binds to p56 but not to the hyperphosphorylated p58. Ectopic expression of truncated hVAP-A fragments inhibits viral replication, which is consistent with a possible role in HCV replication [56]. The protein is localized in membranes of the ER-Golgi system and has a role in intracellular vesicle trafficking. Perhaps NS5A hijacks hVAP-A to modulate the switch from replication to assembly. Does HCV inhibit the IFN response? The most important clinical problem in HCV biology concerns the failure of IFN-a-based therapies in w50% of patients infected with HCV1b, the most prevalent genotype in the world. The solution is most likely to be complex because IFN-a has pleiotropic effects on virusinfected cells and on lymphocytes that participate in the adaptive immune response that is necessary to clear viral infections. Earlier studies invoked a role for NS5A in the selection of IFN-resistant virus during antiviral therapy. It was based on an apparent correlation between certain sequence variations in a small region of NS5A (ISDR, Figure 1) and the response to IFN therapy [57]. These initial claims were corroborated by results suggesting binding of NS5A to PKR, a factor known to have a role in the cellular stress response [58]; however, subsequent studies pointed to a more complex scenario. For example,
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HCV replicons derived from IFN ‘resistant’ and ‘sensitive’ HCV isolates appear to be equally sensitive to IFN-a in Huh7 cells, suggesting that the IFN resistance observed in patients might be controlled, at least in part, by cellular factors [59]. The genotype-specific factors that control the outcome of IFN therapy, however, still remain elusive. Recent investigations into the innate immune response in the HCV system were driven by new information concerning the components of signal transduction pathways that are used by cells in ‘sensing’ pathogens and also about mechanisms used by many viruses to antagonize these pathways. Cells sense pathogens with the help of pattern recognition receptors and intracellular proteins that activate signal transduction pathways, leading to the expression of IFN-b and other stress-induced genes [60,61]. One of the major pathways involved in the recognition of viral pathogens is that of the Toll-like receptor 3 (TLR3). This pathway leads to the activation of the kinases TBK1 and IKK3, which in turn, activate the latent transcription factors NFkB and IRF3, which are required for the transcription of IFN-b (Figure 3). A cytoplasmic helicase with a caspase recruitment domain – RIG-I – signals into this pathway by a still unknown mechanism following its activation, presumably by viral RNA [62].
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Figure 3. Interferon (IFN) signal transduction pathway. The figure shows a model for the Toll-like receptor 3 (TLR3) and RIG-I pathways that are required for the induction of IFN-b through the tank binding kinase-1 (TBK1) and IkB kinase 3 (IKK3) and the canonical IKKabg complex leading to the activation of interferon regulatory factor 3 (IRF3) and NFkB, respectively [61]. The adaptor TIR domain-containing adaptorinducing IFN (TRIF) and an unknown protein required for RIG-I signalling are indicated as targets for cleavage by the NS3–4A protease. The two caspase recruitment domains (CARD) and the RNA helicase domain of RIG-I are indicated (black and grey rectangles, respectively) [62]. www.sciencedirect.com
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Evidence for a role of HCV in the suppression of the TLR3 pathway was obtained with the help of Sendai virus. Although Sendai virus infections lead to the rapid expression of IFN-b in normal Huh7 cells, infections of some Huh7 cell lines expressing HCV replicons did not induce such a response [63]. Thus, HCV replication blocked the signal transduction pathway normally activated by Sendai virus in infected cells. Inhibition of HCV replication with IFN-a or viral protease inhibitors reversed the block to Sendai virus induced IFN-b expression, demonstrating that it was caused by the virus and not by a defective cellular pathway. Genetic experiments revealed that enzymatically active NS3–4A protease was sufficient for the observed inhibition, suggesting that it cleaves a protein in the TLR3 pathway. The TIR domain-containing adaptor-inducing IFN-b (TRIF), a TLR3 adaptor protein, and RIG-I were identified as potential targets for the HCV protease [64,65]. The human TRIF protein has an NS3–4A cleavage site and TRIF levels appeared to be reduced in cells expressing HCV replicons, compared to control cells. However, it is probable that TRIF did not have a role in Huh7 cells because they are deficient in TLR3 expression and inhibition of TRIF expression with RNAi did not prevent IFN-b expression after Sendai virus infection [64]. Instead, RIG-I-dependent activation of the IFN-b promoter was suppressed in Huh7 cells expressing NS3–4A protease. The cellular target for the protease is not yet known. Because RIG-I and other known members of the TLR3 pathway did not appear to be substrates, ongoing investigations to fill this gap might uncover a novel branch of the TLR3 pathway that has a role in the activation of IFN-b. So far, all the evidence for a role of the HCV protease in the regulation of the stress response was derived under selected conditions with the help of tissue culture cells; this raises the question of whether a similar inhibition occurs under natural conditions in infected hepatocytes. DNA microarray analyses of liver tissue from experimentally infected chimpanzees revealed that HCV replication was associated with the induction of IFN-stimulated genes (ISGs) [66]. Induction of ISG is not universally associated with hepatitis virus infections because it was not observed during HBV infections [67]. Several observations suggested that hepatocytes were the cells expressing ISGs. First, ISG induction began as early as two days post-infection before lymphocytes infiltrated the liver. Second, the levels of induction were high, suggesting that the majority of cells in the liver expressed ISGs. Third, an independent immunohistochemical study with tissue sections from HCV-infected human patients demonstrated that expression of at least one ISG, MxA, occurred in hepatocytes [68]. Notably, in this study an inverse correlation was found between the levels of MxA expression in hepatocytes and the outcome of IFN therapy in patients. At first glance, it seems that these results are in apparent conflict with the observations described previously. However, it is important to consider that the factors controlling activation and repression of an innate immune response must be balanced to guarantee survival of hosts and pathogens. Nevertheless, these results pose
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the question of how HCV replication could occur in the presence of an antiviral state, considering that IFN-a is a potent inhibitor of viral replication. Perhaps these results could explain why HCV RNA and proteins are barely detectable in infected livers. One possibility is that, following an infection, HCV RNA replication ensues until the concentration of viral products triggers the induction of an innate response (i.e. through the activation of RIG-I), which in turn leads to an inhibition of viral replication. Thus, HCV and ISG RNA levels in an infected hepatocyte would be predicted to oscillate in phases that are offset with respect to each other during the course of the infection. In this model, the role of the protease would be to delay activation of a RIG-I dependent antiviral response and, hence, provide a means to establish a balance between host and pathogen. Future perspectives The challenge for HCV research is twofold. First, basic research needs to uncover the salient features that set hepaciviruses apart from the two other genera of the Flaviviridae family. Chief among these features are the identification of the receptor(s) that control tissue specificity and the viral and cellular factors that regulate replication. The possibility of producing and passing infectious HCV in tissue culture cells has just provided a major boost towards the accomplishment of these goals. Identification of the phosphorylation sites in NS5A that have a role in the regulation of HCV replication and the kinase(s) that are responsible for these protein modifications will be essential to enhance our understanding about the regulation of HCV replication. Identification of the receptor and kinase could be exploited for the development of new antivirals that might overcome the problem of resistance and meet the second challenge to cure patients who fail IFN-based therapies or cannot tolerate its debilitating side effects. Along this line, testing the hypothesis of a possible correlation between ISG expression and IFN therapy outcome should receive high priority. Many host factors that bind HCV RNA or proteins have already been identified. The task ahead of us is to incorporate them into a plausible model for the HCV life cycle. Given the rapid progress over the past few years, we can be optimistic that these and other challenges can eventually be met and HCV might become the first example of a chronic viral infection that can be cured with a rational approach. Acknowledgements I would like to thank Kerry Campbell and Jared Evans for suggestions and critical reading of the manuscript and acknowledge NIH and the Commonwealth of Pennsylvania for financial support.
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32 Otto, G.A. and Puglisi, J.D. (2004) The pathway of HCV IRES-mediated translation initiation. Cell 119, 369–380 33 Hellen, C.U. and Sarnow, P. (2001) Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593–1612 34 Lohmann, V. et al. (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 35 Behrens, S.E. et al. (1998) Characterization of an autonomous subgenomic pestivirus RNA replicon. J. Virol. 72, 2364–2372 36 Khromykh, A.A. and Westaway, E.G. (1997) Subgenomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 71, 1497–1505 37 Bukh, J. et al. (2002) Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 99, 14416–14421 38 Kato, T. et al. (2003) Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817 39 Cohen, J. (2005) Virology. Culture systems for hepatitis C virus in sight at last. Science 308, 1539–1541 40 Yi, M. and Lemon, S.M. (2003) 3 0 nontranslated RNA signals required for replication of hepatitis C virus RNA. J. Virol. 77, 3557–3568 41 Yi, M. and Lemon, S.M. (2003) Structure–function analysis of the 3 0 stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication. RNA 9, 331–345 42 You, S. et al. (2004) A cis-acting replication element in the sequence encoding the NS5B RNA-dependent RNA polymerase is required for hepatitis C virus RNA replication. J. Virol. 78, 1352–1366 43 Lee, H. et al. (2004) cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome. J. Virol. 78, 10865–10877 44 Friebe, P. et al. (2005) Kissing-loop interaction in the 3 0 end of the hepatitis C virus genome essential for RNA replication. J. Virol. 79, 380–392 45 Cheng, J.C. et al. (1999) Specific interaction between the Hepatitis C Virus NS5B RNA polymerase and the 3 0 end of the viral RNA. J. Virol. 73, 7044–7049 46 Wang, G.H. and Seeger, C. (1993) Novel mechanism for reverse transcription in hepatitis B viruses. J. Virol. 67, 6507–6512 47 Rieder, E. et al. (2000) Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation. J. Virol. 74, 10371–10380 48 Friebe, P. and Bartenschlager, R. (2002) Genetic analysis of sequences in the 3 0 nontranslated region of hepatitis C virus that are important for RNA replication. J. Virol. 76, 5326–5338 49 Tellinghuisen, T.L. et al. (2004) The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 279, 48576–48587 50 Tellinghuisen, T.L. et al. (2005) Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435, 374–379
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51 Blight, K.J. et al. (2000) Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1975 52 Neddermann, P. et al. (2004) Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture. J. Virol. 78, 13306–13314 53 Macdonald, A. and Harris, M. (2004) Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502 54 Tu, H. et al. (1999) Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263, 30–41 55 Evans, M.J. et al. (2004) Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication. Proc. Natl. Acad. Sci. U. S. A. 101, 13038–13043 56 Gao, L. et al. (2004) Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J. Virol. 78, 3480–3488 57 Enomoto, N. et al. (1995) Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J. Clin. Invest. 96, 224–230 58 Gale, M.J., Jr. et al. (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 59 Guo, J.T. et al. (2001) Effect of alpha interferon on the hepatitis C virus replicon. J. Virol. 75, 8516–8523 60 Boehme, K.W. and Compton, T. (2004) Innate sensing of viruses by Toll-like receptors. J. Virol. 78, 7867–7873 61 Akira, S. and Takeda, K. (2004) Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499–511 62 Yoneyama, M. et al. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737 63 Foy, E. et al. (2003) Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300, 1145–1148 64 Foy, E. et al. (2005) Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc. Natl. Acad. Sci. U. S. A. 102, 2986–2991 65 Li, K. et al. (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl. Acad. Sci. U. S. A. 102, 2992–2997 66 Bigger, C.B. et al. (2001) DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J. Virol. 75, 7059–7066 67 Wieland, S. et al. (2004) Genomic analysis of the host response to hepatitis B virus infection. Proc. Natl. Acad. Sci. U. S. A. 101, 6669–6674 68 MacQuillan, G.C. et al. (2002) Intrahepatic MxA and PKR protein expression in chronic hepatitis C virus infection. J. Med. Virol. 68, 197–205
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information.
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Salient molecular features of hepatitis C virus revealed Christoph Seeger Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
Hepatitis C virus (HCV) is a positive strand RNA virus with a narrow host and tissue tropism. It ranks among the most significant of human pathogens, causing inflammation, scarring and cancer of the liver. Recent investigations have shed light on some of the salient molecular features of this virus. These include a requirement for CD81 (a tetraspanin transmembrane protein for viral entry), a novel mechanism for the initiation of RNA synthesis, phosphorylation of a viral protein in the regulation of RNA amplification and virus assembly and, finally, a viral protease suppressing activation of the innate immune response in infected cells.
A major medical challenge Human hepatitis C virus (HCV) is a major blood-borne hepatitis virus that causes chronic infections in an estimated 170 million people, second only to human hepatitis B virus (HBV) [1]. Hepatitis, fibrosis, cirrhosis of the liver and hepatocellular carcinoma are common sequelae of HCV infections. As a consequence, HCV infections are the major cause for orthotopic liver transplantation [2]. Unfortunately, liver grafts are rapidly infected with residual virus and a second, usually more aggressive cycle of the disease takes its course [3]. Vaccines to prevent initial infections are available against HBV but not HCV. HCV stands for a heterogeneous group of positive strand RNA viruses that form the genus Hepacivirus of the Flaviviridae family of viruses. Hepaciviruses are divided into six principal genotypes and their subtypes, which can exhibit as much as 30% sequence diversity [4]. During the course of an infection, subtypes can exist as a quasispecies where the dominant population constantly changes in the face of an immune response directed against B- and T-cell epitopes in the viral polyprotein [5,6]. However, in contrast to HBV, HCV infections can now be cured with a interferon (IFN-a)-based therapy at a remarkable rate – between 50% and 90% depending on the genotype [7]. Unfortunately, the most common genotype – genotype 1 – is also the most refractory to therapy, resulting in a vast cohort of infected patients that are currently without hope of a cure. However, the cloning of HCV in the late 1980s spurred an enormous research effort aimed at understanding the intricacies of HCV and to develop better antiviral therapies [8]. Corresponding author: Seeger, C. ([email protected]). Available online 8 September 2005
This review highlights recent studies on the host and tissue tropism of HCV, the mechanism and regulation of HCV replication, and the regulation of the IFN response. For more detailed descriptions of clinical aspects of HCV infections, and many other investigations on the molecular biology of this virus, the reader is referred to previously published reviews [9,10]. Host and tissue tropism Broad species and tissue specificity is a hallmark of many viruses belonging to the Flaviviridae family. By contrast, HCV infections are restricted to humans and chimpanzees and target primarily hepatocytes. The molecular basis for this narrow tropism is not yet understood because information about the nature of the viral receptor is still incomplete. Several years ago, the tetraspanine transmembrane protein CD81 was identified as a candidate receptor because its large external loop (LEL) could bind to the envelope protein E2 of HCV1a in solution or when expressed on the surface of cells [11,12]. CD81 is expressed in many different cell types where it has a complex role in the reorganization of membranes in response to external stimulation [13]. Subsequent studies demonstrated that HIV particles carrying the HCV E1 and E2 envelope glycoproteins could infect certain human liver or hepatoma-derived cells expressing CD81 [14–18]. Infectivity of these pseudotypes was pH-dependent and could be neutralized with antibodies against E2 and CD81, as well as with a soluble form of CD81. Similarly, the infectivity of an HCV isolate produced in tissue culture cells (described below) could be inhibited with recombinant LEL or anti-CD81 antibodies [19–21]. Thus, these observations are consistent with the original observation predicting a direct interaction between the tetraspanin transmembrane protein and E2. The results also indicated that additional factors required for infectivity are expressed on the surface of hepatocyte-derived cells, in support of the hypothesis that HCV, like HBV, is a hepatotropic virus [17]. Besides CD81, several other candidate receptors have been identified. Among them is a second E2 binding partner, human scavenger receptor class B type I (SR-BI) [22]. SR-BI is expressed primarily in the liver, therefore, the E2–SR-BI interaction is consistent with the apparent tissue specificity of HCV. However, the significance of this interaction for viral infection has been questioned by the demonstration that antibodies against SR-BI could diminish binding of E2 or HCV particles to susceptible
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Upon entry and uncoating, the genome acts as mRNA for the translation of the viral polyprotein. Like with other plus strand RNA viruses, this step is not yet understood. However, it represents a remarkable event in cell biology because it requires that, during an infection, a single RNA molecule is transported to ribosomes with high efficiency. Translation of the polyprotein is directed by an internal ribosomal entry site (IRES, see text). Translation begins with the synthesis of the core protein followed by the two envelope glycoproteins E1 and E2 and the small p7 protein. They undergo cotranslational processing by cellular signal peptidases. In contrast to the structural genes, the remaining six non-structural proteins are processed by the viral NS3 protease with the exception of the NS2-NS3 site, which is cleaved by the NS2-NS3 cysteine protease. Some of the major biochemical and structural properties of the HCV polypeptides have been summarized in Figure 1 and were described in detail elsewhere (e.g. [10]). Translation is interrupted by an unknown signal that activates a reaction leading to the formation of a replication complex on membranes of the endoplasmic reticulum. As a consequence, the mRNA is transformed back to a genome that now acts as a template for the synthesis of minus strand RNA. Viral protein synthesis arrests until plus strands are synthesized and delivered to the ribosome for a new round of RNA amplification. Alternatively, the plus strands are assembled with the structural proteins into virus particles. As observed with other positive strand RNA viruses, plus strands are wtenfold more abundant than minus strands.
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Replication of the viral genome Following infection and uncoating, the HCV RNA transits from genome to mRNA (Box 1). A salient feature of HCV lies in the properties of the internal ribosomal entry site (IRES), which controls translation of the viral polyprotein (Figure 1). The IRES can engage 40S ribosomes in the absence of canonical translation factors, similar to a prokaryotic mRNA [29]. Binding of the IRES induces conformational changes in the 40S subunit that are believed to correctly position the AUG initiation codon at the P site of the ribosome [30,31]. The binary IRES–40S structure binds to eIF3 and the ternary eIF2–Met-tRNA– GTP complex to enable formation of the IRES–80S translation machinery [32]. Thus, in contrast to the
primary hepatocytes but could not inhibit infection of these cells [23]. Obviously, the HCV receptor story is still incomplete leading to the debate about the tropism of this virus. Many studies have provided evidence for the presence of HCV RNA in lymphocytes and other tissues. The question remains whether the virus replicates in these cells and whether they release infectious HCV into the bloodstream. Viral RNA levels are low, on average only a few copies per hepatocyte, thus, the demonstration of viral replication
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requires the selective detection of minus strands (intermediates of viral replication) that are present at tenfold lower levels compared with plus strands. Under conditions where such a distinction was possible with strand-specific PCR, minus strands were only detected in infected liver tissue but not in lymphocytes or other tissues of infected chimpanzees [24]. Moreover, under the same conditions, minus strands could not be detected in peripheral blood mononuclear cells (PBMC) from infected patients [24,25]. Nevertheless, studies providing support for HCV replication in extrahepatic tissues and immortalized lymphocytes continue to flare up and fuel the debate about tissue tropism of HCV (see references [26,27]). HCV RNA replication can occur in cells of non-hepatic origin and even in mouse cells, therefore, it is likely that host and tissue tropism is determined during an early step of the viral life cycle [28]. Thus, clarification of this important problem will require more detailed information about the viral receptor and the mechanism of entry.
Box 1. Model for the replication of HCV
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Figure 1. Physical and functional map of Hepatitis C virus (HCV). The open reading frame encodes four structural (core, envelope 1 and 2 and p7) and six non-structural proteins (NS2–5B). It is flanked by the 5 0 and 3 0 untranslated regions (UTRs). A more detailed description of the 3 0 UTR is provided in Figure 2. Asterisks denote proposed functions of p7 and NS4B. The detailed map of NS5A depicts the three major structural domains (D 1–3) with the helical anchor domain (H), the cysteine residues (C) forming the Zn2C binding domain, the interferon sensitivity determining region (ISDR) and two regions that are not essential for the replication of viral subgenomes in tissue culture cells (D). The amino acid sequence of a segment containing the most frequently observed mutations in adapted replicons (black spheres) is shown at the bottom of the figure. The numbering corresponds to the HCV1b isolate Con1 (AJ238799). Abbreviations: HVR, hypervariable region; Rep. Complex, replication complex; SL, stem-loop; VR, variable region. www.sciencedirect.com
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were defects in the assembly and secretion of virus and, most probably as a consequence of this, lack of infectivity and spread after intrahepatic inoculation into chimpanzees [37]. Recently, the generality of these findings has been challenged with an HCV clone belonging to genotype 2 that replicated with high efficiency in different cell lines without the requirement for adaptive mutations and permitted the production of infectious virus in tissue culture cells [19–21,38] (reviewed in Ref. [39]). However, unlike the other clones used to date, this isolate was obtained from a patient with fulminant hepatitis, a rare form of HCV-induced disease that results in massive liver damage with often fatal outcome. Although it is conceivable that this clone represents a cytopathic variant of a normally non-cytopathic virus with mutations that interfere with controlled amplification of viral RNA, infection of Huh7 cells and a chimpanzee did so far not support this view [19–21,38]. The value of the replicon system for a better understanding of the HCV life cycle has been underscored recently by the discovery of a novel signal near the 3 0 end of the NS5B coding region, which is required for RNA replication. Initial genetic studies revealed stringent requirements for the nucleotide sequence composition of the 3 0 untranslated regions (UTR) that can fold into three stem-loop (SL) structures [40,41] (Figure 2a). Comparative sequence analyses revealed the presence of several stem-loop structures overlapping the coding region of the polymerase that were conserved among HCV isolates [42–44]. Genetic
well-characterized type I and type II IRES element of poliovirus and encephalomyocarditis virus (EMCV), respectively, translation in HCV does not depend on eIF4G and eIF4A (reviewed in Ref. [33]). Although the significance of this unusual mechanism for HCV replication is not known, it might be required for efficient translation in hepatocytes that are normally in a growth arrested state (Go). Like translation, the formation of functional replication complexes also appears to depend on mechanisms that set HCV apart from other plus strand RNA viruses. In tissue culture cells, replication occurs only under selected conditions that are just beginning to be understood. The so-called ‘HCV replicon system’ was developed based on previous studies with West Nile virus (WNV) and bovine viral diarrhoea virus (BVDV), which demonstrated that the structural genes were not required for RNA replication and that subgenomic replicons carrying a neomycin resistance gene could be used to establish cell lines expressing replicons [34–36]. However, the scenario with HCV differed in a major way in that subgenomes prepared from available infectious HCV clones from genotype 1 generally replicated with extremely low efficiency or not at all in most cell lines used for transfection. Efficient replication required either mutations in the non-structural (NS) proteins or, alternatively, depended on cellular factors that are enriched in certain subclones of the hepatoma cell line Huh7 (reviewed in Ref. [10]). Notable features of full length replicons with adaptive mutations (a) SL VI
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Figure 2. Initiation of RNA replication. (a) The proposed secondary and tertiary structures at the 3 0 end of HCV RNA. Stem-loops (SL) VII–IV are located in the coding region of the polymerase (NS5B), whereas SLs III–I are present in the 3 0 UTR region. The two clusters of SLs are separated by the variable region (VR) and the poly(U/C) tract. The positions are numbered according to the sequence of HCV1b Con1 (AJ238799). The figure is not drawn to scale. (b) A model for the initiation of minus strand RNA, which predicts that the 3 0 UTR folds into a kissing-loop structure with the help of Watson–Crick base pairs that can form between the apical loops in SL V and SL II (step 1, [44]). The polymerase then binds to the tertiary structure to initiate minus strand RNA synthesis (steps 2 and 3). www.sciencedirect.com
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experiments revealed that the structure of at least one of these stem-loop structures (SL V, Figure 2) was essential for RNA replication. Importantly, it was demonstrated that the loop region of SL V can base-pair with the loop of SL II present in the 3 0 UTR and form a kissing-loop interaction [44] (Figure 2). Finally, biochemical studies indicated that the NS5B RNA polymerase binds with high affinity to SL V and adjacent sequences in the coding region of NS5B [43,45]. These results helped to refine the model for the initiation of minus strands, which predicts that the 3 0 end of HCV RNA is in a closed loop formation that aligns the binding site for the polymerase with the 3 0 end of the RNA to facilitate the initiation of minus strand RNA synthesis. It is not yet known whether RNA synthesis initiates at the 3 0 of the RNA or whether the polymerase copies a few nucleotides from an internal site and then translocates a short primer to the 3 0 end, essentially as it has been proposed for protein-primed replication in poliovirus and hepatitis B virus [46,47]. In this model, the poly (U/C) tract separating the two stem-loop clusters could act as a linker providing the flexibility necessary for the formation of the tertiary interaction between SL II and SL V. Consistent with such a model, deletion of the tract inhibited RNA replication, probably because it either prevented loop formation or caused premature termination of minus strand synthesis. This last phenomenon might be due to a steric problem created by the proximity of SL V, the binding site for the polymerase and the nascent minus strand (Figure 2b). In support of this possibility, artificially truncated poly(U/C) tracts expanded to their natural size during replication [48]. Thus, if the loop is too small, the polymerase might arrest, slip back and expand the loop until it can continue RNA synthesis. Does NS5A regulate HCV replication? A major question concerning the life cycle of HCV is how the persistence of viral RNA is controlled in infected cells. HCV is a non-cytolytic virus, therefore, RNA accumulation must reach a steady state following an initial exponential amplification phase. Although the mechanism regulating this process is not yet understood, it is tempting to speculate that the NS5A protein might be an important player. NS5A has three major domains that are connected by two protease sensitive hinge regions [49]. The N-terminal domain consists of an amphipathic a-helix proposed to act as a membrane anchor attached to a Zn2C binding site composed of four conserved cysteine residues. Mutations in any of the four residues inhibit HCV replication in tissue culture cells [49]. X-ray crystallographic analysis of the structure of this domain confirmed the presence of the Zn-coordination motif and revealed the presence of a disulfide bond [50]. However, because the cysteine residues required for proposed formation of disulfide bonds were not essential for replication in tissue culture cells, it is uncertain whether they have a role for NS5A function. The first hinge region is the target for many adaptive mutations that are necessary for the persistent replication of HCV replicons in Huh7 cells. The most commonly found adaptive mutations map to conserved serine residues and www.sciencedirect.com
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are associated with a reduction in the levels of p58, the hyperphosphorylated form of NS5A, suggesting that modification of NS5A has an important role in the regulation of HCV replication [51]. In support of this view, a kinase inhibitor reduced the levels of p58 activated replication of wildtype replicons but inhibited the replication of adaptive mutants, suggesting that the ratio between p58 and p56, the basally phosphorylated form, is crucial for the control of RNA amplification [52]. As described previously, replicons with adaptive mutations that reduce p58 levels do not assemble and release virus particles, suggesting that NS5A might also have a role in the assembly of virus particles and, hence, in the decision of whether amplified plus strand RNA returns to the ribosome for a further round of genome replication or enters the assembly pathway for the formation of infectious virus. Unlike all other NS proteins, NS5A exhibits some of its functions in trans, which is consistent with its potential role as an activator or repressor of one or more cellular pathways. However, there are still major gaps that need to be filled to enhance our understanding of NS5A function. For example, although it is known that serines are the primary targets for both basal and hyperphosphorylation, the locations of the serines on the polypeptide are still uncertain. Moreover, identification of the kinases involved in the modification of this protein will be essential to better investigate the possible function of NS5A in the regulation of RNA amplification and assembly of virus particles. Finally, we must determine the nature of the physiologically relevant cellular factors that affect NS5A activity. Although the list of NS5A binding partners is large [53], the interaction with human vesicle-associated membrane protein-associated protein A (hVAP-A) is of particular interest because it is regulated by NS5A phosphorylation [54,55]. HVAP-A binds to p56 but not to the hyperphosphorylated p58. Ectopic expression of truncated hVAP-A fragments inhibits viral replication, which is consistent with a possible role in HCV replication [56]. The protein is localized in membranes of the ER-Golgi system and has a role in intracellular vesicle trafficking. Perhaps NS5A hijacks hVAP-A to modulate the switch from replication to assembly. Does HCV inhibit the IFN response? The most important clinical problem in HCV biology concerns the failure of IFN-a-based therapies in w50% of patients infected with HCV1b, the most prevalent genotype in the world. The solution is most likely to be complex because IFN-a has pleiotropic effects on virusinfected cells and on lymphocytes that participate in the adaptive immune response that is necessary to clear viral infections. Earlier studies invoked a role for NS5A in the selection of IFN-resistant virus during antiviral therapy. It was based on an apparent correlation between certain sequence variations in a small region of NS5A (ISDR, Figure 1) and the response to IFN therapy [57]. These initial claims were corroborated by results suggesting binding of NS5A to PKR, a factor known to have a role in the cellular stress response [58]; however, subsequent studies pointed to a more complex scenario. For example,
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HCV replicons derived from IFN ‘resistant’ and ‘sensitive’ HCV isolates appear to be equally sensitive to IFN-a in Huh7 cells, suggesting that the IFN resistance observed in patients might be controlled, at least in part, by cellular factors [59]. The genotype-specific factors that control the outcome of IFN therapy, however, still remain elusive. Recent investigations into the innate immune response in the HCV system were driven by new information concerning the components of signal transduction pathways that are used by cells in ‘sensing’ pathogens and also about mechanisms used by many viruses to antagonize these pathways. Cells sense pathogens with the help of pattern recognition receptors and intracellular proteins that activate signal transduction pathways, leading to the expression of IFN-b and other stress-induced genes [60,61]. One of the major pathways involved in the recognition of viral pathogens is that of the Toll-like receptor 3 (TLR3). This pathway leads to the activation of the kinases TBK1 and IKK3, which in turn, activate the latent transcription factors NFkB and IRF3, which are required for the transcription of IFN-b (Figure 3). A cytoplasmic helicase with a caspase recruitment domain – RIG-I – signals into this pathway by a still unknown mechanism following its activation, presumably by viral RNA [62].
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Figure 3. Interferon (IFN) signal transduction pathway. The figure shows a model for the Toll-like receptor 3 (TLR3) and RIG-I pathways that are required for the induction of IFN-b through the tank binding kinase-1 (TBK1) and IkB kinase 3 (IKK3) and the canonical IKKabg complex leading to the activation of interferon regulatory factor 3 (IRF3) and NFkB, respectively [61]. The adaptor TIR domain-containing adaptorinducing IFN (TRIF) and an unknown protein required for RIG-I signalling are indicated as targets for cleavage by the NS3–4A protease. The two caspase recruitment domains (CARD) and the RNA helicase domain of RIG-I are indicated (black and grey rectangles, respectively) [62]. www.sciencedirect.com
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Evidence for a role of HCV in the suppression of the TLR3 pathway was obtained with the help of Sendai virus. Although Sendai virus infections lead to the rapid expression of IFN-b in normal Huh7 cells, infections of some Huh7 cell lines expressing HCV replicons did not induce such a response [63]. Thus, HCV replication blocked the signal transduction pathway normally activated by Sendai virus in infected cells. Inhibition of HCV replication with IFN-a or viral protease inhibitors reversed the block to Sendai virus induced IFN-b expression, demonstrating that it was caused by the virus and not by a defective cellular pathway. Genetic experiments revealed that enzymatically active NS3–4A protease was sufficient for the observed inhibition, suggesting that it cleaves a protein in the TLR3 pathway. The TIR domain-containing adaptor-inducing IFN-b (TRIF), a TLR3 adaptor protein, and RIG-I were identified as potential targets for the HCV protease [64,65]. The human TRIF protein has an NS3–4A cleavage site and TRIF levels appeared to be reduced in cells expressing HCV replicons, compared to control cells. However, it is probable that TRIF did not have a role in Huh7 cells because they are deficient in TLR3 expression and inhibition of TRIF expression with RNAi did not prevent IFN-b expression after Sendai virus infection [64]. Instead, RIG-I-dependent activation of the IFN-b promoter was suppressed in Huh7 cells expressing NS3–4A protease. The cellular target for the protease is not yet known. Because RIG-I and other known members of the TLR3 pathway did not appear to be substrates, ongoing investigations to fill this gap might uncover a novel branch of the TLR3 pathway that has a role in the activation of IFN-b. So far, all the evidence for a role of the HCV protease in the regulation of the stress response was derived under selected conditions with the help of tissue culture cells; this raises the question of whether a similar inhibition occurs under natural conditions in infected hepatocytes. DNA microarray analyses of liver tissue from experimentally infected chimpanzees revealed that HCV replication was associated with the induction of IFN-stimulated genes (ISGs) [66]. Induction of ISG is not universally associated with hepatitis virus infections because it was not observed during HBV infections [67]. Several observations suggested that hepatocytes were the cells expressing ISGs. First, ISG induction began as early as two days post-infection before lymphocytes infiltrated the liver. Second, the levels of induction were high, suggesting that the majority of cells in the liver expressed ISGs. Third, an independent immunohistochemical study with tissue sections from HCV-infected human patients demonstrated that expression of at least one ISG, MxA, occurred in hepatocytes [68]. Notably, in this study an inverse correlation was found between the levels of MxA expression in hepatocytes and the outcome of IFN therapy in patients. At first glance, it seems that these results are in apparent conflict with the observations described previously. However, it is important to consider that the factors controlling activation and repression of an innate immune response must be balanced to guarantee survival of hosts and pathogens. Nevertheless, these results pose
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the question of how HCV replication could occur in the presence of an antiviral state, considering that IFN-a is a potent inhibitor of viral replication. Perhaps these results could explain why HCV RNA and proteins are barely detectable in infected livers. One possibility is that, following an infection, HCV RNA replication ensues until the concentration of viral products triggers the induction of an innate response (i.e. through the activation of RIG-I), which in turn leads to an inhibition of viral replication. Thus, HCV and ISG RNA levels in an infected hepatocyte would be predicted to oscillate in phases that are offset with respect to each other during the course of the infection. In this model, the role of the protease would be to delay activation of a RIG-I dependent antiviral response and, hence, provide a means to establish a balance between host and pathogen. Future perspectives The challenge for HCV research is twofold. First, basic research needs to uncover the salient features that set hepaciviruses apart from the two other genera of the Flaviviridae family. Chief among these features are the identification of the receptor(s) that control tissue specificity and the viral and cellular factors that regulate replication. The possibility of producing and passing infectious HCV in tissue culture cells has just provided a major boost towards the accomplishment of these goals. Identification of the phosphorylation sites in NS5A that have a role in the regulation of HCV replication and the kinase(s) that are responsible for these protein modifications will be essential to enhance our understanding about the regulation of HCV replication. Identification of the receptor and kinase could be exploited for the development of new antivirals that might overcome the problem of resistance and meet the second challenge to cure patients who fail IFN-based therapies or cannot tolerate its debilitating side effects. Along this line, testing the hypothesis of a possible correlation between ISG expression and IFN therapy outcome should receive high priority. Many host factors that bind HCV RNA or proteins have already been identified. The task ahead of us is to incorporate them into a plausible model for the HCV life cycle. Given the rapid progress over the past few years, we can be optimistic that these and other challenges can eventually be met and HCV might become the first example of a chronic viral infection that can be cured with a rational approach. Acknowledgements I would like to thank Kerry Campbell and Jared Evans for suggestions and critical reading of the manuscript and acknowledge NIH and the Commonwealth of Pennsylvania for financial support.
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