Hepatitis C virus–cell interactions and their role in pathogenesis

Clin Liver Dis 7 (2003) 67 – 88

Hepatitis C virus–cell interactions and their role in pathogenesis Stephen J. Polyak, PhD Departments of Laboratory Medicine and Microbiology, University of Washington, Box 359690, 325 9th Avenue, Seattle, WA, 98104-2499, USA

At the time a virus infects a cell, it encounters a hostile environment that is unsuited to its survival and propagation. A successful virus therefore must assume control of the cell to promote conditions favorable for an infectious cycle. For persistent viral infections, this control of the host must facilitate longterm (and sometimes attenuated) replication. The genes and gene products the virus brings into the cell not only are required to promote the replication of large quantities of progeny viruses, but they also must interact with or perturb host cell proteins that are required for viral replication or aimed at the virus’ demise. Thus, the study of virus – host interactions provides insight into mechanisms of virus persistence, antiviral resistance, and pathogenesis.

The medical significance of hepatitis C virus infection As covered elsewhere in this issue, hepatitis C virus (HCV) infects an estimated 3% or 170 million of the world’s population and causes an estimated 476,000 deaths per year caused by complications of end– stage liver disease. Of those acutely infected with HCV, most develop chronic infection. Moreover, 70% of patients with chronic viremia develop histological evidence of chronic liver disease. Chronic infection results in a spectrum of liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Hepatitis C is the most frequent indication for liver transplantation [1,2] (see article by Rosen in this issue for more detailed information). In Europe, approximately 9 million people are infected with HCV, 5 million in the European Union, with a variable prevalence ranging from 0.5% in Northern Europe to 2% in Southern Europe. The prevalent genotype in Europe is 1b (47%), followed by 1a (17%), 3 (16%), 2 (13%), and other genotypes (7%) [3]. In the United States, about 1.8% of the general

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population or 4 million people are infected. About 57% of cases involve genotype 1a, 17% genotype 1b, 14% genotype 2, 7% genotype 3, 0.9% genotype 4, and 3.2% genotype 6 [4,5]. The prevalent genotype in Japan is type 1b.

Acute hepatitis C virus infection As alluded to previously, progression to chronic hepatitis C is the norm for the most acutely infected patients. A growing research area involves the characterization of virus and host factors associated with clearance of acute HCV infection. An impediment to these studies is the difficulty in diagnosing acute HCV infection, because the initial manifestations are often subclinical. The establishment of networks to identify and manage healthcare workers with occupational exposure to HCV has greatly facilitated research on acute HCV [6,7], however. Studies of acutely infected patients have demonstrated that broad, multi-specific cytotoxic T lymphocyte (CTL) immune responses are associated with HCV clearance [6,8,9]. Similar findings also have been made during experimental infection of chimpanzees [10]. Recently, two mechanisms have been described whereby HCV – host interactions appear to influence the progression of acute to chronic hepatitis C. The generation of viruses with mutations in CTL epitopes has been shown to permit acute HCV to persist by escape from the cell-mediated immune response [11]. Moreover, the ability of the HCV NS5A protein to interact with the interferon (IFN) -induced cellular protein, RNA-activated protein kinase (PKR), correlates with progression from acute to chronic HCV infection [12]. Understanding the factors involved in clearance of acute HCV will continue to be an important area of research.

Antiviral therapy for hepatitis C virus Recombinant antiviral IFN-a has been used for more than a decade as a treatment of chronic hepatitis C, despite the fact that most patients still fail to have sustained responses (ie, clearance of viremia) [13] (see article in this issue by McHutchison and Fried for more detailed information). More recent therapies including IFN plus ribavirin [14 – 16], pegylated IFN [17,18], and pegylated IFN plus ribavirin [19], result in significant improvements in sustained response rates. Nonetheless, HCV viremia is not eradicated in more than 60% of treated patients. This is particularly evident in patients with genotype 1a or 1b HCV infections. This is clearly a problem in areas where genotype 1 is common such as in North America, Europe, and Japan. In this light, HCV seems resistant to IFN antiviral therapy. As a consequence, much attention has been focused on characterizing HCV – host interactions that contribute to resistance to IFN therapy. Elucidation of the mechanisms of antiviral resistance is important for improving therapy for chronic hepatitis C. An additional benefit from studies of this nature is that insights into viral pathogenesis often are uncovered simultaneously.

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The modus operandi for studying HCV –host interactions usually involves overexpression of HCV RNA and proteins in cell culture and assessment of the effects on host cell metabolism and signal transduction pathways. It is therefore possible that the level of HCV protein expression in cellular systems is higher than in the HCV-infected liver, raising questions about the biological relevance of the results. To this end, some studies use regulated expression systems that facilitate HCV protein analysis at more physiological concentrations. Importantly, in other research arenas, similar studies have permitted the dissection of molecular mechanisms and the development of specific inhibitors of various pathways. In some cases, the inhibitors have found their way into the clinic, such as the PKC inhibitor CGP41251 [20], the BCR-ABL tyrosine kinase inhibitor STI571 [21], and the natural inhibitor of angiogenesis, angiostatin [22]. Thus, the study of HCV – host interactions also may open up new avenues for therapeutic intervention through targeted disruption or promotion of virus –host protein complexes.

Virus – host interactions during the hepatitis C virus life cycle For HCV, virus – cell interactions can be grouped into three broad categories: entry, replication, and assembly/release. These events are summarized in Fig. 1. In the initial stage of the infection, HCV binds to and enters the target cell. This virus – cell interaction may be mediated by at least two putative cellular receptors for HCV, the low-density lipoprotein receptor, LDLR or CD81. The HCV envelope glycoproteins, E1 and E2, are responsible for binding the cell surface receptors. E1 and E2 are believed to be present on the virion surface in a noncovalently associated complex. The complex is formed during virus assembly, which occurs at the endoplasmic reticulum [23]. HCV particles in human sera are complexed with antibodies and low-density lipoproteins (LDL) [24,25]. Agnello et al demonstrated that HCV – LDL complexes are capable of entry into various human liver cell lines [26]. Although the precise molecular events involved in LDLR-mediated entry await further characterization, up-regulation of LDLR using chemicals such as lovastatin appear to enhance HCV entry in this model system. If LDLR turns out to be a bona fide cellular receptor for LDL-complexed HCV, then the lack of LDLR and the use of LDL-free HCV-infected serum might explain the generalized lack of reproducibility and poor success of serum-based HCV culture systems. The fact that HCV in patient serum is complexed not only with LDL but with antibodies also may influence the outcome of serum-based HCV infection systems, if Fc receptormediated endocytosis is operative. Abrigani et al demonstrated that the HCV E2 glycoprotein bound the tetraspanin molecule, CD81, on human B cells [27]. E2 was shown to bind CD81 present on B cells and various human cell lines including hepatoma Huh7 cells, suggesting that the E2-CD81 interaction is biologically relevant in getting HCV into the liver. Thus, CD81 originally was touted as a receptor for HCV. Several subsequent studies, however, indicated that this interpretation may have been premature, because there seems to be no correlation between the sequence

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Fig. 1. The HCV life cycle. In the first step, HCV binds to and enters cells. This step may be mediated by interaction of HCV glycoproteins with CD81 and LDLR. HCV then is uncoated in acidic endosomes (H+) where replication occurs. Replication involves multiple virus – virus and virus – host interactions that regulate HCV polyprotein production and processing, and genome (+RNA) versus antigenome synthesis. HCV proteins accumulated in the endoplasmic reticulum (ER) and Golgi apparatus, where assembly and release of progeny viruses, occur. Abbreviations: RdRp, RNA dependent RNA polymerase; Rep, HCV replicase complex.

of CD81 from various species and susceptibility of cell lines to HCV infection. For example, E2 binds to tamarin CD81 more tightly than human CD81 in vitro and on cell lines, yet tamarins are not susceptible to HCV infection [28,29]. More recent studies using cells from Tupeia belangeri (tree shrew) suggest an alternative receptor for HCV. Tupeia hepatocytes support HCV binding and infection, and this seems to be independent of CD81 [30]. Furthermore, Baumert’s group has demonstrated that the binding of HCV virus-like proteins (containing core, E1 and E2) bind to human cell lines independent of CD81 and LDLR [31]. These studies suggest that there are additional cell surface molecules that mediate HCV entry into cells. In other viral infections, there are precedents for this concept. For example, HIV uses CD4 and chemokine receptors [32] for virus entry, while herpesviruses (HSV) use herpes virus entry mediator (HVEM; a member of the tumor necrosis factor [TNF] receptor family), heparin sulfate, and nectins 1 and 2 (members of the immunoglobulin superfamily) [33,34]. For HSV, multiple viral glycoproteins are required during binding, fusion, and entry. Specifically, glycoproteins gB and gC mediate high affinity interaction with cell

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surface heparin sulfate, followed by low-affinity interaction of gD with HVEM, nectin 1, or nectin 2 [33,34]. Interaction of gD with any of the receptors seems to trigger fusion. HCV may use E1 and E2 during virus entry, because it has been reported that E1 may interact with LDLR [35], while E2, as described previously, binds to CD81. Thus, HCV entry likely involves E1 and E2 proteins and multiple cell surface molecules. Differential usage of these molecules may be required for HCV entry into nonhepatocyte cells such as lymphocytes, monocytes, and bone marrow [36 – 45] and also may influence HCV-associated pathology. Even if CD81 is not involved in HCV entry directly, CD81 engagement by HCV virions theoretically could affect immune cell activation functions. Indeed, cross-linking of CD81 with monoclonal antibodies or immobilized E2 inhibited NK cytotoxic activity and IFN-g production [46] and granule release, proliferation, and activated mitogen – activated protein kinase (MAPK) signaling cascades [47]. Cross-linking of CD81 also seems to make T cells hyper-responsive in interleukin (IL)-2, IFN-g, and IL-4 production [48]. These studies suggest that the accessory interaction of HCV with CD81 may affect the development of innate and acquired immunity to HCV. HCV infection is not a prerequisite for these biological effects on the immune system. Thus, cell function may be altered as immune cells ‘‘sample’’ the microenvironment through HCV –host interactions that are limited to molecules on the cell surface. As shown in Fig. 1, after HCV enters the cell, it is uncoated. This may occur in acidic endosomes, because lysosomotropic agents that raise the pH of endosomes inhibit the entry of pseudotyped E1 – and E2 – containing VSV particles [35]. Although this effect may be caused by the vesicular stomatitis virus (VSV) backbone used in this study, many other viruses including arenaviruses, Hantaan virus, hepatitis A virus, and Semliki Forest virus, require low pH for viral entry [49 – 53]. Most notably, entry of the flavivirus St. Louis Encephalitis virus into mosquito cells requires low pH [54]. In theory, HCV interaction with other cell surface molecules may be a postreceptor-binding event that directs internalized HCV into the acidic endosome, reinforcing the concept that multiple interactions between HCV glycoproteins and cellular membrane proteins are involved in HCV entry. Following uncoating, the HCV RNA is translated, and the polyprotein is processed proteolytically into the 10 viral proteins. The first third of the polyprotein encodes the structural proteins that form the three-dimensional architecture of the virus. They include the core protein, two envelope proteins (E1, E2), and a protein of unknown function (P7). An additional open reading frame in the core gene recently was described, although the role of this protein in HCV replication and pathogenesis remains to be determined [55]. Nonstructural proteins are derived from the remaining two-thirds of the polyprotein and have functions in HCV replication. They include NS2, NS3 (a serine protease/helicase), NS4A, NS4B, NS5A, and NS5B (the RNA – dependent RNA polymerase) [56]. Successful viral protein translation and genome replication requires the coordination of multiple viral and cellular proteins. For instance, polypyrimidine tract binding protein (PTB) has been shown to bind to the core coding region and 30 UTR [57,58]. PTB

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binding to the core region inhibits HCV translation, which is relieved if RNA transcripts contain the 30UTR. These data suggest that PTB binding to HCV RNA may facilitate the switch from HCV polyprotein translation to negative strand replication. More recent studies suggest that the La antigen, an important cellular translation factor, may also interact with the 50 and 30 UTRs [59,60]. The previous examples underscore the reliance of HCV on cellular factors for successful replication. Continued research into this area likely will uncover other HCV – host interactions that may be suitable targets for therapeutic drug discovery. Specificity is achievable if the engineered molecule can be directed to the HCV – host interaction, thereby disrupting only those cells that contain replicating virus.

The hepatitis C virus core and NS5A proteins As alluded to previously, HCV – host interactions have been investigated intensely despite the lack of a robust viral infection system. As a result, many laboratories have established model cell culture systems to study HCV –host interactions, with a major focus on the core and NS5A proteins. At first glance, these two viral proteins appear different in several aspects. The first is their position in the HCV genome; they are at opposite ends, with core being the first protein produced, while NS5A is penultimate. Second, the encoded proteins are different in size, with the core being 19 to 21 kDa and NS5A being 56 to 58 kDa. The third difference lies in the presumed roles of the proteins in the lifecycle of HCV. The HCV core is a structural protein that binds RNA and ribosomes [61] and presumably promotes HCV capsid formation, while NS5A is probably a component of the HCV polymerase complex [62] and as such, functions in HCV replication. The proteins share several similarities, however. One is in subcellular localization; the predominant forms of the proteins are found in the cytoplasm. This is illustrated in Fig. 2, which depicts immunofluorescent detection of NS5A (left panel) and core (right panel) in human cells. Both proteins are localized predominantly to the perinuclear area in the cytoplasm. This location of HCV proteins to this region of the cell is not unique. Indeed, all HCV proteins have been localized to a similar region within the cell [63 – 65]. The location within the cell to where HCV proteins localize corresponds to the endoplasmic reticulum (ER) and golgi apparatus. This is the region where HCV replication is believed to occur (see Fig. 1) and is consistent the subcellular localization of other flaviviruses including West Nile virus [66] and dengue virus [67]. The targeting of HCV protein expression to the ER membrane likely is facilitated by way of interaction of HCV proteins with cellular proteins. In this regard, NS5A and NS5B interact with hVAP-33, a cellular protein found primarily in the ER [68]. Core and NS5A proteins also share the similarity of colocalizing on cytoplasmic lipid droplets in hepatocytes [69] and having truncated forms that localize to the nucleus [70,71]. Recently, a small amphipathic alpha helix in the amino terminus of NS5A was shown to mediate retention of NS5A on cytoplasmic membranes [72]. The biological implications of the location of core and NS5A in the cell may relate

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Fig. 2. Localization of HCV proteins in human cells. NS5A (left panel) and core (right panel) proteins were expressed in human osteosarcoma and hepatocytes (Huh7) cells, respectively, and immunofluorescent detection of the proteins was performed. As can be seen, both proteins are located primarily in the cytoplasm, although small amounts of NS5A can be seen in the nucleus and core is associated with cytoplasmic lipid droplets.

to the proteins’ abilities to perturb several signal transduction pathways involved in cell cycle, antiviral, apoptotic, and lipid regulation.

HCV NS5A – host interactions For genotype 1a and 1b isolates, NS5A is 448 amino acids in length and is post-translationally modified by a series of phosphorylations on conserved serine residues, resulting in the production of phosphorylated and hyperphosphorylated forms of the protein [73 – 80]. Major sites for phosphorylation of NS5A on Ser-2194 and Ser-2321 have been described recently [81,82], and the cellular serine kinase(s) responsible for NS5A phosphorylation has been characterized partially [77,83,84]. The exact role of NS5A phosphorylation on protein function is not known, but the serines are conserved strictly in vivo, despite the fact that many other regions of NS5A mutate during chronic HCV infection [85]. Conversely, studies using HCV subgenomic replicons suggest that hyperphosphorylation of NS5A is not required for HCV replication [86]. Many studies on the effects of NS5A expression on cellular metabolism and physiology have been performed. During the course of these studies, NS5A has emerged as an important protein in the HCV life cycle. Fig. 3A summarizes some of the structure – function relationships for NS5A that are described in the proceeding section. In general, NS5A may directly regulate HCV replication through interaction with the HCV –encoded polymerase complex [62,87]. NS5A’s role as a direct modulator of viral replication also stems from observations that adaptive mutations in NS5A increase the replication of HCV subgenomic replicons [86,88,89]. Mutations in NS5A also accumulate during IFN therapy of patients

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Fig. 3. NS5A (A) and core (B) structure function relationships. The top panel of each figure depicts the position of the gene in the context of the HCV polyprotein, along with major phosphorylation sites. Below each figure are the regions of each protein that are required for various activities including cytoplasmic retention (cyto) [72], interferon sensitivity determining region (ISDR) [90], PKR binding domain (PKR) [135], nuclear localization signal (NLS) [70], Grb2 binding domain (Grb2) [92], variable domains 3 [149] and 4 [85] (V3, V4). The domains required for binding cellular proteins and RNAs also are indicated.

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with HCV [85,90] and in chimpanzees that develop chronic infection following experimental inoculation with HCV [91]. Although the mutations described in vitro are not found in NS5A genes isolated from patients (data not shown), the fact that NS5A accumulates mutation in vitro and in vivo suggests that NS5A is under selective pressure in diverse environments. NS5A also promotes a cellular environment favorable to HCV replication, a function of the protein usually taking the form of perturbation of a signal transduction pathway. For example, NS5A interacts with Grb2, an adaptor protein involved in MAPK signaling, resulting in inhibition of epidermal growth factor (EGF) signaling by way of reduced extracellular regulated kinase (ERK) 1 and ERK2 phosphorylation [92]. A follow-up study reported that recombinant vaccinia virus expressing NS5A inhibited double-stranded RNA – activated protein kinase (PKR) activity and the subsequent phosphorylation of the eukaryotic initiation factor 2 (eIF2a) [93]. In the same study, NS5A was shown to perturb the p38 MAPK pathway, which normally leads to eIF – 4E phosphorylation, suggesting that NS5A may regulate cellular translation to favor IRESmediated translation during HCV infection [93]. These studies also imply that NS5A can affect the control of cellular proliferation by affecting homeostasis of mRNA translation. In this regard, NS5A inhibits apoptosis in some [94,95], but not all systems [96]. Proposed mechanisms for NS5A inhibition of apoptosis include negative regulation of PKR [94], inhibition of TNF-a induced activation of NF-kB by way of direct interaction with TRAF2 [97], and induction of oxidative stress and activation of NF-kB [98]. Another study suggests NS5A is inhibitory to the cell cycle by way of perturbation of Cdk1/2 complexes [99]. Given the inherent variability of all clinical NS5A isolates [85], the ability of NS5A to localize in the nucleus and cytoplasm [100], and the different cellular systems used, it is not surprising that NS5A has been reported to have such diverse effects. Nonetheless, the modulation of cellular proliferation and apoptosis by NS5A may have implications for the pathogenesis of HCV-related liver damage. In this regard, NS5A-mediated inhibition of apoptosis is associated with transformation of NIH 3T3 cells and tumor formation in nude mice, suggesting that NS5A has an oncogenic potential [94]. Moreover, NS5A associates with apolipoprotein AI (apoAI) on the surface of lipid droplets in Huh7 cells [69]. Because steatosis or lipid accumulation in the liver is a frequent histological finding in chronic hepatitis C, the interaction of NS5A with apoAI may be involved in the pathogenesis of hepatic steatosis. Interestingly, NS5A is not the only HCV protein that interacts with cellular apolipoproteins, as the HCV core protein binds apoAII.

HCV core– host interactions Like NS5A, the HCV core protein also seems to be a major player in the regulation of cellular growth and HCV pathogenesis. Core expression in transgenic mice induces hepatic steatosis and hepatocellular carcinoma [101,102].

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Moreover, transgenic mice expressing HCV structural and nonstructural proteins also develop hepatic steatosis and hepatocellular carcinoma [103]. In this model system, steatosis develops in animals expressing core, E1, and E2 proteins, while the additional expression of the nonstructural proteins is a predisposing factor for developing cancer. The association of core with apolipoprotein A2 on lipid droplets in hepatocytes may be involved in the pathogenesis of liver disease in transgenic animals [104], through altered very low density lipoprotein (VLDL) secretion [105] and by way of core-induced oxidative stress and mitochondrial injury [106]. As described previously, NS5A binds apoAI on cellular lipid droplets in human hepatocytes [69]. Therefore, the interaction of HCV proteins with cellular apolipoproteins in hepatocytes may be mechanistically involved in the development of steatosis. Core protein also has been shown to interact with the cytoplasmic tail of the TNF-a receptor and induce apoptosis [107,108]. Studies from other groups, however, have reported that the HCV core protein inhibits apoptosis, promotes cell growth, and can transform cells to a tumorigenic phenotype [109 – 111]. Similarly, contradictory results on the effects of expression of the HCV core protein on MAPK, p53, p21/WAF, and NF-kB have been reported [112 – 116]. The reasons for these discrepancies are not clear, although the cell lines used may affect the apoptotic responses observed [117], or differences in subcellular localization of the core protein might explain the dichotomous effects of the protein [71]. Disparties could also be due to sequence differences among core quasispecies isolates. In a recent study, core was shown to interact directly with NS5A, inducing caspase-mediated cleavage of NS5A and apoptosis of osteosarcoma cells [118], suggesting that it is important to evaluate the effects of a single HCV protein in the context of additional, or ideally, all HCV proteins. A summary of the structure-function relationships for core is presented in Fig. 3B. In summary, the HCV core and NS5A proteins engage in many interactions within the host cell that have a significant influence on HCV replication and pathogenesis, and on HCV antiviral resistance.

The interferon system Many different signal transduction pathways are engaged in the binding of cytokines and chemokines to their cognate receptors. In general, these pathways involve a series of phosphorylation-dependent protein – protein interactions and small molecule second messengers that culminate in the assembly of transcription factor complexes to induce genes that mediate the biological activity of the cytokine or chemokine. The STAT/JAK (signal transducers and activator of transcription/Janus-associated kinase) pathway is the principle pathway used by IFN-a. At the time IFN-a binds its receptor, two receptor-associated tyrosine kinases, Tyk2 and Jak1, become activated by phosphorylation and phosphorylate STAT-1 and STAT-2 on conserved tyrosine residues [119]. STAT-1 and STAT-2 combine with the p48 protein to form the transcription factor IFN-

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stimulated gene factor 3 (ISGF-3), which enters the nucleus, binds to the IFNstimulated response element (ISRE), inducing transcription of IFN-a– induced genes [119]. Three IFN-induced genes have been studied extensively, the 20 – 50 oligoadenylate synthetase (OAS)/RNAse L system, PKR, and myxovirus resistance proteins (Mx). The constitutive expression of the RNAse L and PKR pathways contribute to the IFN antiviral state as observed by the increased susceptibility of cells to viral infection from mice devoid of these enzymes [120 –122]. The ubiquitous RNase L is activated by 20 –50-oligoadenylate (2-5A); OAS synthesizes 2 – 5A from ATP in response to double-stranded RNA, such as the replicative intermediates of certain RNA viruses [123,124]. RNAse L is believed to degrade viral RNA and is implicated in the control of positive – stranded RNA viruses such as the picornavirus and encephalomyocarditis virus (EMCV). PKR is an IFN-inducible dsRNA-activated serine/threonine kinase. Its action is mediated through inhibition of protein synthesis caused by phosphorylation of eIF2a and has been partially implicated in IFN inhibition of EMCV [125,126]. Mx proteins mediate the inhibition of VSV and orthomyoxoviruses by way of inhibition of viral genome amplification and protein synthesis [127]. Many viruses including HIV, poliovirus, HSV, influenza virus, adenovirus and vaccinia virus encode RNAs or proteins that specifically inhibit the IFN system, either by inhibiting STAT/JAK signal transduction or by inhibiting the enzymatic activity of IFN –induced cellular proteins. HCV can be added to this list of viral inhibitors of the IFN system.

Hepatitis C virus inhibition of the interferon system Hepatitis C virus proteins have been shown to interact with and inhibit the IFN system at several levels. For example, expression of the entire HCV polyprotein inhibited IFN – induced STAT/JAK signaling in human U2-OS osteosarcoma cells [128]. HCV proteins also interact with and inhibit the antiviral functions of IFN – induced host cell proteins. The author’s group initially demonstrated that NS5A partially inhibits the antiviral actions of IFN [129], a finding that has been verified by several other laboratories [94,130 –133]. At present, two mechanisms for inhibiting the antiviral actions of IFN by NS5A have been described. NS5A has been shown to interact with PKR, inhibiting PKR kinase activity [134,135]. These findings provided a molecular explanation for the clinical observation that mutations within a small, 40 amino acid domain in NS5A, termed the IFN sensitivity determining region (ISDR), are associated with response to IFN therapy [90,136]. Gale et al showed that the ISDR was required for binding to PKR, and accumulation of mutations within the ISDR abrogated the interaction of NS5A with PKR. Because PKR induction and activation by IFN inhibit the translation of viral mRNAs, blockade of PKR activity is presumed to permit HCV replication to continue during IFN challenge. Enomoto’s and Gale’s findings were controversial, because other clinical and in vitro studies could not verify the

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association of ISDR mutations and response to therapy [137] or interaction of NS5A with PKR [130]. Some of these differences are most certainly attributable to methodological differences. Because the bulk of ISDR studies limit the analysis to the ISDR and PKR binding region, it is premature to make conclusions on the biological activity of NS5A, especially because it is not known a priori how mutations in other regions of NS5A affect the conformation of the ISDR and PKR binding domains. It is known, however, that NS5A is variable throughout the entire gene and between different patients [85], indicating that future studies should focus on the entire gene. Furthermore, recent meta-analyses of the ISDR clinical studies indicate that low sample size was the predominant reason why previous studies could not find an association between ISDR mutation and IFN response [138]. Thus, the association of the ISDR with IFN response seems to have been validated, and one mechanism by which this occurs involves targeted disruption of PKR. Hopefully, this will provide a modus vivendi for the field. Recently, a novel mechanism for inhibition of the IFN system by the NS5A protein was characterized. NS5A was shown to induce the expression of the proinflammatory chemokine IL-8, which was associated with inhibition of IFN action [139]. NS5A induced transcriptional activation of the IL-8 promoter, possibly involving the transcription factors NF-kB and AP-1. Because NS5A can activate transcription, and NS5A mutants lacking amino termini can enter the nucleus, the precise mechanisms of IL-8 induction await further characterization. Using cDNA microarray analysis, NS5A also was shown to increase the expression of IL-8 in human hepatocytes, thereby extending this finding to the natural reservoir for HCV infection [140]. Mechanistically, the IL-8 anti-IFN action in EMCV-infected cells was associated with decreased OAS activity in a manner that was independent of OAS gene expression [141], suggesting that inhibition of the IFN system occurs at a post-transcriptional level. The IL-8 suppressive action on OAS activity in the presence of IFN-a seems to be linked to the type of the virus, because the antiIFN action of IL-8 was not seen with VSV infection. It appeared that IL-8 had no dramatic effect on ISRE-driven 6 –16 gene expression, supporting the hypothesis that IL-8 apparently blocks the antiviral action of IFN-a at late, such as OAS activity, rather than early stage. This is also the case with HCV NS5A-induced inhibition of IFN antiviral action, whereas NS5A protein expression did not appear to affect IFN-mediated signaling in HeLa cells [139], although it is still possible that NS5A affects STAT/JAK signal transduction at some level. Interleukin-8 also stimulates the replication of several viruses including CMV, HIV, and adenovirus. Although the effect of IL-8 on HCV replication has not been reported, it is intriguing that HCV –infected Saudi Arabian patients have elevated IL-8 and TNF-a (a physiological inducer of IL-8) levels, with biochemical nonresponders to IFN therapy having highest serum levels [142]. Elevated IL-8 also is associated with other viral infections including HIV, rhinovirus, and cytomegalovirus [143]. Another study, however, arrived at exactly the opposite conclusion. Researchers found that elevated IL-8 levels are correlated with IFN response in French patients with HCV [144]. In this

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study, the sample size was low (N = 18) compared with the study on Saudi patients (N = 132), and the five patients who responded to IFN and then relapsed had highly elevated pretreatment IL-8 levels compared with sustained responders or nonresponders. In a follow-up study from the same group in which 47 patients with HCV were analyzed, it was shown that serum levels of TNF-a were highest in nonresponder patients [145], similar to the results of Polyak et al. In addition, different ELISA kits and IL-8 standards were used between the studies of Neuman and Polyak, so it is difficult to compare the actual levels of IL-8. The common theme in these studies, however, is that HCV infection produces elevations in inflammatory cytokines and chemokines such as TNF-a and IL-8. Clearly, additional prospective studies on patients with HCV from different countries need to be performed. The clinical studies described previously also underscore the need for additional characterization of the interaction of HCV with the IL-8 system. Although the precise mechanisms for the proviral properties of IL-8 await further study, one mechanism could involve IL-8’s anti-IFN effects. It is also possible that IL-8 promotes virus replication through IL-8 –initiated signal transduction events. In summary, the interaction of NS5A with the IL-8 system represents a novel mechanism of inhibition of the IFN system by the NS5A protein.

Stimulation of the interferon system by hepatitis C virus core It has been demonstrated that the HCV core protein activates the 2– 5 OAS gene [146] and PKR enzyme activity [147], two key IFN-induced genes that mediate the antiviral effects of IFN. This suggests that core interacts with the innate cellular antiviral response. The authors have found that the HCV core protein activates the IFN system on a more global level involving activation of the STAT/JAK pathway and IFN –stimulated gene promoter activity (Polyak et al, submitted). This effect of core is hypothesized to reflect activation of the innate immune system by the HCV core protein, which is likely to be the first viral protein that a cell encounters during acute infection. It is also possible that stimulation of the IFN system by the HCV core protein is required to balance the IFN inhibitory actions of other HCV proteins such as E2 [148] and NS5A [134,139] to provide fine tuning of HCV replication.

Hepatitis C virus assembly and egress As shown in Fig. 1, the final category of the HCV life cycle is virus maturation and release. This category is as yet an untapped research area, quite likely because so many researchers are trying to determine how the virus enters cells and replicates. The future will provide interesting developments in this important area of the HCV life cycle. This stage of the HCV life cycle likely

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involves multiple interactions between HCV and host cell proteins, which may be an area for development of novel classes of antiviral molecules.

Summary In summary, HCV –cell interactions include those directly involved with the HCV life cycle such as virus attachment, entry, and replication. Included within this broad area of research are the interactions of HCV proteins with the IFN system, cytokine and chemokine pathways such as IL-8, and various other cellular proteins and pathways. The plethora of contradictory and sometimes confusing accessory HCV – host interactions defies precise predictions of their role in HCV biology. It is clear that these virus – cell interactions affect HCV replication, antiviral resistance, persistence, and pathogenesis. Because HCV – host interactions are initiated immediately on infection, they are operative during acute HCV infection, whereby HCV interacts with innate cellular antiviral and immune systems. The magnitude and duration of these HCV – host interactions therefore may influence the development of acquired immunity. Because HCV exists as a quasispecies in all infected individuals, heterogeneity in biological responses to HCV – host interactions is predicted, revealing opportunities for the development of various genotypic and phenotypic prognostic indicators. With the model systems in place, these hypotheses can be tested. The challenge for the future is to determine if there is a hierarchical importance to these interactions, to delineate how these virus –cell interactions affect the patient infected with HCV, and to determine whether any of these interactions represents a target for therapeutic intervention.

Acknowledgments The author would like to thank Stuart Ray, MD and Christoph Sarrazin, MD for providing templates for Figs. 1 and 3, and David Gretch, MD, PhD for insightful discussions.

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