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Tan SL, editor. Hepatitis C Viruses: Genomes and Molecular Biology. Norfolk (UK): Horizon Bioscience; 2006.

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Hepatitis C Viruses: Genomes and Molecular Biology.

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Chapter 13HCV Regulation of Host Defense

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Abstract

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

Introduction

Exposure to HCV typically leads to persistent infection associated with a chronic disease course. The ability of HCV to mediate persistent, life-long infection in its human host is linked to the evasive nature of the virus to thwart the host immune system and to resist the antiviral actions of IFN-based therapy. Molecular studies of HCV-host interactions have revealed several levels of immune regulation and evasion directed by HCV protein products. This chapter provides an overview of the virus-host interface of these regulatory processes and their impact on HCV replication and persistence.

Innate Intracellular Immune Defenses

In response to virus infection, signaling pathways within mammalian cells direct a variety of intracellular events that generate an antiviral state directly within the infected cell. This antiviral response, termed the ‘host response” to virus infection, represents our first line of immune defense against virus infection. If this response is successful, exposure to the virus will render a self-limiting, abortive infection. It is the hepatic host response that imposes initial immune defenses against HCV infection (Gale, Jr., 2003). The host response is triggered when the infected cell recognizes a molecular signature within the invading virus. This signature, known as a pathogen-associated molecular pattern (PAMP), is typically a physical characteristic of the virus that is recognized and engaged by specific PAMP receptor proteins within the host cell (O’Neill, 2004). The PAMP/PAMP receptor interaction initiates signaling cascades that induce the expression of antiviral effector genes (Sen, 2001). For RNA viruses, protein and nucleic acid products of infection comprise an array of PAMP signatures that can engage specific PAMP receptors, including Toll-like receptors (TLRs) and nucleic acid binding proteins (Fig. 1; Iwasaki and Medzhitov, 2004; Cook et al., 2004). The HCV RNA contains specific PAMP signatures, including poly-uridine motifs and stem-loop double-stranded RNA (dsRNA) structures within its single-stranded RNA genome (Tuplin et al., 2002; Simmonds et al., 2004). HCV RNA is sufficient to induce the host response in cultured hepatocyte-derived cell lines (McCormick et al., 2004; Sumpter et al., 2005). The product of retinoic acid inducible gene I (RIG-I), which has been defined as a dsRNA PAMP receptor, is critical for host response signaling induced by HCV RNA (Yoneyama et al., 2004; Sumpter et al., 2005). In hepatocytes, the independent signaling pathways of RIG-I and Toll-like receptor 3 (TLR3) direct the host response to virus infection (Li et al., 2005a).

Fig. 1. Triggering the host response to HCV infection.

Fig. 1

Triggering the host response to HCV infection. HCV triggers the host response through the process pathogen-associated molecular pattern (PAMP)/PAMP receptor engagement. Certain RNA motifs within the HCV genome have PAMP attributes that activate the host (more...)

PAMP/PAMP receptor engagement signals the activation of a variety of transcription factors in the infected hepatocyte. PAMP-driven transcription factor activation results in the immediate expression of host response genes (Fig. 2; Malmgaard, 2004). Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) are triggered in response to virus infection, and each are activated upon cellular recognition of the HCV PAMP (Au et al., 1995; Lin et al., 1999; Fredericksen et al., 2001; Richmond, 2002; Prabhu et al., 2004). Their activation proceeds through PAMP-responsive signaling pathways of the cell that promote their nuclear translocation and transactivation functions. Other IRF family members, including IRF-5 and IRF-7, are essential components of the host response to virus infection (Barnes et al., 2001; Kawai et al., 2004), though the specific role of each in HCV infection has not been characterized. Virus-induced signaling events that activate ATF-2 and further direct chromatin remodeling contribute to the building of a virus-triggered enhancesome on the IFN-β promoter. The IFN-β enhanceosome includes IRF-3 and NF-κB, which produce a transcriptional response resulting in IFN-β expression and secretion from the infected cell (Sen, 2001). NF-κB activation and function is central to the chemokine and proinflammatory cytokine response to virus infection, which functions side by side with IFN to modulate the ensuing adaptive immune response (Tai et al., 2000). Secreted levels of IFN-β drive autocrine and paracrine signaling processes by binding the IFN-α/β receptors of the infected cell and local surrounding tissue, respectively. This results in activation of the Jak-STAT pathway. Here, receptor-associated Jak and Tyk1 protein kinases phosphorylate signal transducer and activator of transcription (STAT) proteins on critical serine and tyrosine residues to confer STAT activation, STAT association with IRF-9, and nuclear localization of the resulting ISGF3 transcription factor complex. ISGF3 is the central transcription factor that promotes high level expression of the interferon stimulated genes (ISGs) by binding to the IFN-stimulated response element (ISRE) within the ISG promoter/enhancer region. IFN binding to the IFN α/β receptor and signaling of the Jak-STAT pathway drives a second wave of transcriptional activity initiated by virus infection and denoted by the expression of ISGs.

Fig. 2. Signaling the host response to HCV infection.

Fig. 2

Signaling the host response to HCV infection. 1. Viral PAMP (HCV RNA) binding to RIG-I, TLR3 or other PAMP receptors results in the phosphorylation and activation of IRF-3 by the TBK1 or IKKɛ protein kinases. The dimer of phospho-IRF-3 translocates (more...)

ISG Products Have Antiviral Functions

The human genome encodes hundreds of ISGs (Der et al., 1998). The ISG products direct regulatory functions that control virus infection. ISGs have been shown to interrupt HCV replication through processes that include suppression of viral protein synthesis and synthesis inhibition of the viral negative strand replicative intermediate (Guo et al., 2001; Shimazaki et al., 2002; Wang et al., 2002; Prabhu et al., 2004). The main feature resulting from the secretion of IFN from infected cells into the local tissue is the paracrine induction of a tissue-wide host response that blocks cell to cell spread of the virus. Many components of the host response pathways are themselves ISGs, and though expressed basally at a low level that facilitates surveillance and response to virus infection, their abundance will increase in response to IFN signaling. In the liver, paracrine signaling of IFN serves to enhance overall responsiveness of cellular signaling pathways to potentiate the host response to infection in a tissue-wide manner. IFN signaling thus provides an amplification loop to further promote the host response against HCV (Foy et al., 2005). Recent studies have shown that the amplification loop is dependent upon the transcriptional activity of IRF-7 (Honda et al., 2005). IRF-7 is an ISG and its expression in the liver is IFN-dependent (Smith et al., 2003; Honda et al., 2005). IRF-7 promotes the expression of the various IFN-α subtypes, wherein IFN-α production and secretion mediate further amplification of the host response and prolonged IFN production (Honda et al., 2005). IFN-based therapy for HCV infection exploits these actions of IFN-α to limit HCV replication and spread (Fig. 2) (McHutchison and Patel, 2002). IFN-α also signals the maturation of immune effector cells, antigen presenting cells and dendritic cells, and it potentiates the production of other proinflammatory cytokines by resident hepatic cells to indirectly modulate cell-mediated defenses and adaptive immunity (Biron, 1999). Viral triggering and control of the host response may therefore define cellular permissiveness for HCV RNA replication and influence the outcome of infection.

Cross-Talk Between Innate and Adaptive Immune Defenses

IFN-α is a potent immunomodulator that influences the onset of the cellular immune response and adaptive immunity to HCV infection. IFN-α modulates natural killer (NK) cell activity toward lysis of infected target cells by promoting NK cell activation and proliferation, and supporting their survival through the induction of IL-15 production (Biron, 1999; Loza and Perussia, 2004). Activated NK cells produce IFN-γ (Shoukry et al., 2004). In the HCV-infected liver, NK cell homing and local secretion of IFN-γ may serve to limit HCV replication directly. Indeed, IFN-γ has been shown to mediate direct antiviral effects against HCV RNA replication in vitro (Frese et al., 2002), and its production by immune effector cells in the liver associates with viral clearance in the chimpanzee model of HCV infection (Thimme et al., 2001; Su et al., 2002). IFN-α also influences the maturation of dendritic cells and modulates their presentation of viral antigens (Colonna et al., 2004; Barth et al., 2005). Antigen presentation by dendritic cells under the influence of IFN-α contributes to the differentiation of CD4 T cells toward the Th1 phenotype and importantly, a Th1-predominate response is associated with clearance of HCV infection (Shoukry et al., 2004). IFN-α production during HCV infection may therefore indirectly contribute toward directing the maturation of CD4 T cells to the Th1 phenotype. Dendritic cells are also involved in the cross priming and IFN-γ production of CD8 T cells. In this context the co-stimulatory signals presented at the time of antigen cross presentation to CD8 T cells can determine whether the cells are cross primed for a cytotoxic response or cross-tolerized for anergy (Cooper et al., 1999; Shoukry et al., 2004). TLR3 plays an essential role in cross priming by dendritic cells, in which it signals the production of IFN-α triggered by viral PAMPs within products of phagocytosis (Schulz et al., 2005). IFN-α produced by the dendritic cell in this manner induces the expression of co-stimulatory molecules and cytokines to promote the cross priming and activation of CD8 cells, thus linking host response signaling processes to induction of the adaptive immune response. The link between innate, IFN-induced antiviral pathways and the adaptive immune response is not entirely understood and further studies are required to define the linkage of these processes with the outcome of HCV infection.

IFN-γ Activation of the Antiviral Response

IFN-γ may serve to complete the signaling loop between infected hepatocytes and immune effector cells. The IFN-γ receptor is expressed by hepatocytes and in most other tissues. In the liver, IFN-γ produced by infiltrating NK cells and activated T cells, where it can bind its receptor in paracrine fashion to induce a hepatic IFN-γ response. IFN-γ receptor binding leads to phosphorylation of STAT1 through the receptor-bound Jak1 and Jak2 protein kinases (Stark et al., 1998). The phosphorylated STAT then forms a homodimer that translocates to the cell nucleus and acts on the gamma-activated sequence elements of target genes to promote their transcription (Der et al., 1998). In hepatocytes the genes under the control of the GAS element have significant overlap with those expressed in response to IFN α/β and under control of the ISRE (Cheney et al., 2002). GAS elements are also found within genes whose products are involved in antigen processing and presentation, immune effector action and apoptosis, thus deriving a variety of antiviral actions. A role for GAS element genes in host defense against HCV is supported by studies of the chimpanzee model for HCV infection, where the high expression of IFN-γ responsive genes in the liver has associated with the resolution of acute infection in the chimpanzee model (Su et al., 2002). Together with IFN-α, the actions of IFN-γ provide addition levels of host defense cross-talk that serves to limit HCV infection.

Hepatic Defenses Are Triggered by HCV

Functional genomic analyses from cohorts of patients infected with HCV have shown that infection associates with a hepatic gene expression profile marked by ISGs whose expression levels vary widely among patients (Smith et al., 2003). These observations indicate that HCV can induce and regulate the hepatic host response to infection. Gene profiling studies of HCV-infected chimpanzees have demonstrated that acute resolving HCV infection is associated with a host response characterized by high level hepatic ISG expression (Bigger et al., 2001). In similar studies, “outcome predictor” gene sets were identified by the overall hepatic expression level of certain ISGs and virus-responsive genes. This gene set was accurately defined as those virus-responsive genes whose high expression associated with low viral load and effective viral clearance but whose low expression correlated with progression to chronic HCV infection (Su et al., 2002). Like many virus-responsive genes and ISGs, the various products of the “outcome predictor” gene set are known to interact with components of T cell immunity, again demonstrating the complex cross-talk that goes on during the host response to infection and between parameters of innate and adaptive immunity. These observations demonstrate that the hepatic host response is triggered during HCV infection but differentially regulated in association with disease course, and that HCV mediates persistence through strategies to regulate and/or evade the antiviral actions of this response.

Triggering the Host Response to HCV Infection

The processes by which HCV initiates and controls the host response have been addressed in cell culture models of HCV RNA replication and viral protein expression. Genome-length or specific subgenomic fragments of HCV RNA are sufficient to trigger IFN-β expression and production when introduced into cultured human hepatoma cells (Fredericksen et al., 2001; McCormick et al., 2004), and this effect has been attributed in part to the 5′ and 3′ nontranslated region (NTR) of the HCV genome. These regions encode a series of highly conserved stem-loop/dsRNA structures, and the 3′ NTR includes a variable length poly u region (Tuplin et al., 2002). In cultured cells the HCV NTRs present PAMP structures that serve as potent agonists of the host response. This suggests that during infection these RNA motifs are recognized and engaged by PAMP receptor(s) that trigger the host response (Sumpter et al., 2005).

The nature of at least one HCV RNA PAMP receptor was revealed through studies of the Huh7-derived (human hepatoma) cell line, termed Huh7.5. This cell line does not mount a host response to virus infection or transfected HCV RNA and it is highly permissive for HCV RNA replication (Blight et al., 2002; Sumpter et al., 2005). Complementation studies identified RIG-I as an HCV RNA PAMP receptor that binds HCV dsRNA motifs and signals the activation of IRF-3 and NF-κB. This was shown to induce IFN-β expression and onset of the host response (Sumpter et al., 2005). RIG-I is an RNA helicase and a member of the DEx/D box RNA helicase family. It contains amino-terminal regions of homology to the caspase activation and recruitment domain (CARD) (Yoneyama et al., 2004). RIG-I signaling is mediated by the CARD homology motifs while the helicase domain imparts PAMP recognition, RNA-binding and regulation of signaling (Yoneyama et al., 2004). Signaling by RIG-I directs a host response that suppresses HCV RNA replication (Sumpter et al., 2005; Foy et al., 2005). The permissiveness of Huh7.5 cells for HCV RNA replication has been attributed to a point mutation in the RIG-I CARD motifs that ablated downstream IRF-3 phosphorylation and NF-κB activation (Sumpter et al., 2005). Virus-induced signaling by RIG-I and the resulting host response may therefore influence the outcome of HCV infection. TLR3 is a dsRNA PAMP receptor that signals a host response after its engages the PAMP ligand (Fig. 2) (Alexopoulou et al., 2001). TLR3 signals the activation of IRF-3 and NF-κB through MyD88-independent processes that require the Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) protein (Yamamoto et al., 2003). Genetic and biochemical studies have now defined prominent roles for the RIG-I and TLR3 pathways in signaling the host response to virus infection in hepatocytes (described below), though the role of each in natural HCV infection remains to be evaluated.

Protein products of infection may also stimulate the host response to HCV. Expression of the HCV NS5A protein induces cellular stress signaling pathways to activate STAT3 (Gong et al., 2001). Similar to the IFN α/β receptor signaling, STAT3 promotes gene expression through processes that involve the Jak-STAT pathway (Sarcar et al., 2004). This results in a gene expression profile that includes ISGs and proinflammatory cytokines that may influence the overall level of HCV RNA replication (Zhu et al., 2003). Moreover, the HCV core protein has been shown to activate the catalytic activity of protein kinase R (PKR), an ISG effector component of the host response to virus infection (Delhem et al., 2001). PKR is a dsRNA binding protein. Its activation is induced by binding to dsRNA PAMPs, resulting in eukaryotic initiation factor 2 phosphorylation and in inhibition of local protein synthesis. Active PKR also signals the DNA binding activity of NF-κB and induces IRF-1 transcription-effector action (Williams, 1999). Activation of PKR by the HCV core protein is most likely attributed to the 5′ NTR viral RNA binding property of the core protein (Tanaka et al., 2000), which would provide the PKR activator dsRNA PAMP required for kinase activation. Cell interaction with virus particles may also trigger signaling events that induce IFN production. HCV pseudo particle binding to dendritic cells has been shown to mediate particle uptake and dendritic cell activation (Barth et al., 2005). Since dendritic cell subsets constitute a major source of IFN production during viral infection, HCV modulation of dendritic cell function may influence HCV infection by modulation local or systemic IFN levels (Colonna et al., 2004).

Regulation and Evasion of the Host Response by HCV

HCV utilizes a variety of strategies to regulate and evade the host response. Studies of the HCV/host interface have revealed PAMP-responsive signaling pathways that impart IRF-3 activation, IFN α/β receptor signaling, and ISG effector action as major sites of control and evasion of the host response (Katze et al., 2002). The HCV NS3/4A protease has been identified as an antagonist of virus-induced IRF-3 activation and IFN-β expression. NS3/4A mediates a block to IRF-3 activation triggered either by endogenous replicating HCV replicon RNA or by exogenous virus infection of cells harboring a replicating HCV genomic RNA (Foy et al., 2003). The IRF-3 blockade was been attributed to the NS3/4A protein complex, which mediates a block to virus-induced IRF-3 phosphorylation, resulting in retention of IRF-3 in an inactive, cytoplasmic-bound state. NS3 is a bifunctional enzyme, and it encodes serine protease within its amino-terminal domain and a RNA helicase within its carboxyl-terminal domain (Reed and Rice, 1998). Importantly, the NS3/4A complex constitutes the essential viral protease, and it releases the nonstructural proteins from the HCV polyprotein during virus replication (De Francesco and Steinkuhler, 2000). The helicase activity of NS3 is not required for the control of IRF-3 activation but the NS3/4A protease activity is required. This suggests that HCV blocks IRF-3 activation through NS3/4A proteolysis of essential host cell proteins that confer PAMP signaling (Foy et al., 2003). NS3/4A regulation of RIG-I signaling has been identified as a causal link of the IRF-3 phosphorylation blockade directed by NS3/4A. These studies showed that NS3/4A blocks the host RIG-I pathway through the protease-dependent disruption of CARD-homology domain signaling that is normally induced upon RIG-I binding to the HCV RNA PAMP ligand. The NS3/4A block to RIG-I signaling additionally ablates virus activation of NF-κB (Foy et al., 2005). This dual regulation of IRF-3 and NF-κB indicates that NS3/4A must target and cleave common factor(s) involved in IRF-3 and NF-κB activation but the nature of such factors have not been defined.

TLR3 signaling is also targeted and regulated by NS3/4A. In this case NS3/4A protease activity has been shown to cleave the TRIF adaptor protein between amino acids C372 and S373 (Li et al., 2005b). This cleavage site is homologous with the HCV NS5A/B polyprotein cleavage site. Structure/function studies show that NS3/4A recognizes TRIF through binding of a proline-rich region adjacent to the site of cleavage (Ferreon et al., 2005). TRIF is essential for signaling by TLR3 but it does not play a role in RIG-I pathway, nor does TRIF cleavage by NS3/4A provide a mechanism of RIG-I pathway regulation, which must occur through cleavage of yet addition cellular targets (Foy et al., 2005; Li et al., 2005a). NS3/4A cleavage of TRIF prevents TLR3 signaling, thus blocking IRF-3 and NF-κB activation and preventing IFN production (Fig. 2) (Li et al., 2005b). The targeting of the RIG-I and TLR3 pathways by NS3/4A allows HCV to evade two major arms of IFN production and host defense. This has been further validated through pharmacologic studies that used a peptidomimetic active site NS3 protease inhibitor to evaluate the requirement for protease activity in the regulation of host response signaling by NS3/4A. Treatment of cells that expressed wild type, functional NS3/4A showed that the protease inhibitor effectively removed the blockade to RIG-I and TLR3 signaling imposed by HCV, thereby restoring virus-induced IRF-3 phosphorylation/activation and the activation NF-κB (Foy et al., 2003; Foy et al., 2005). Protease inhibitor treatment of cells also protected TRIF from cleavage by NS3/4A (Li et al., 2005b).

What are the implications resulting from HCV disruption of RIG-I or TLR3 signaling? First, viral control of RIG-I and TLR3 serves to attenuate major pathways of IFN production by infected cells and tissues. Second, many of the components of these pathways are IFN-responsive and though expressed at low levels, their expression is increased after exposure of cells and tissues to IFN α/β. The IFN-responsiveness of these factors provides an amplification loop to enhance the strength and length of the host response. The signaling blockade imposed by NS3/4A breaks this IFN amplification loop (Foy et al., 2005) and may limit the level and diversity of ISG expression induced in response to IFN therapy. Third, HCV attenuation of the host response and IFN production is expected to cause alterations in antigen presentation by the affected hepatic tissue, potentially leading to inefficient activation of cytolytic T cells and an inability of the adaptive immune response to clear HCV-infected hepatocytes by disrupting the cross talk between the host response and the adaptive immune response. This may provide a causal link between high level intrahepatic ISG expression and a vigorous T cell response to diverse viral epitopes, both of which are associated with viral clearance and inversely correlate with chronic infection (Su et al., 2002; Shoukry et al., 2004). Fourth, IRF-3 has been ascribed proapoptotic and tumor suppressor functions (Heylbroeck et al., 2000; Duguay et al., 2002). A prolonged block to IRF-3 activation might disrupt these actions and cause a tumorigenic phenotype within infected cells. This could provide a link between chronic HCV and hepatocellular carcinoma (Liang and Heller, 2004). Finally, the blockade of NF-κB function may interfere with a variety of chemokines and cytokine genes whose expression is dependent on NF-κB and serve to drive the inflammatory response to virus infection (Zhu and Liu, 2003; Foy et al., 2005). HCV regulation of NF-κB may therefore contribute to the systemic immune defects associated with HCV infection.

HCV Regulation of IFN Signaling

The overall low response rate of HCV to IFN therapy, particularly among patents with genotype 1 HCV infection (McHutchison et al., 2002), provides clinical evidence that HCV can effectively evade IFN actions in vivo. Many studies have focused on defining the molecular mechanisms by which HCV evades and resists IFN actions. These studies have shown that HCV protein expression associates with inhibition of STAT1 function, and can occur independently of STAT tyrosine phosphorylation (Heim et al., 1999). Analysis of transgenic mice that express HCV proteins in their hepatocytes showed that the regulation of STAT1 occurs below the level STAT tyrosine phosphorylation, resulting in a defective hepatic IFN response (Blindenbacher et al., 2003). STAT dysfunction might be attributed to protein phosphatase 2A (Fig. 3), which confers STAT1 hypomethylation and complex formation the protein inhibitor of activated STAT1 (PIAS). This was found to prevent STAT1 assembly into the ISGF3 complex and to attenuate ISG expression (Duong et al., 2004). The mechanism by which protein phosphatase 2A triggers these events is not known. Others have shown that expression of the HCV core protein associates with increased levels of suppressor of cytokine signaling (SOCS)-3 (Bode et al., 2003). SOCS3 belongs to a family of SOCS proteins that are negative regulators and inhibitors of Jak-STAT signaling. SOCS proteins mediate a classical negative feedback loop on IFN α/β receptor signaling events; SOCS-1 and SOCS-3 confer reduced levels of ISG expression (Alexander, 2002). While induction of SOCS-3 by the HCV core protein might impart evasion from IFN actions, it should be noted that the overall role of SOCS-3 in HCV infection is not known and it is unclear if expression of SOCS-3 by core is due to the potential for the core protein to stimulate IFN signaling events itself (Miller et al., 2004) or through induction of yet undefined signaling pathways that stimulate SOCS expression.

Fig. 3. HCV attenuation of IFN signaling.

Fig. 3

HCV attenuation of IFN signaling. IFN α/β Receptor signaling by IFN from autocrine/paracrine and therapeutic sources is subject to feedback inhibition by suppressor of cytokine signaling (SOCS) proteins. The HCV core protein can induce (more...)

Regulation of ISG Expression or Function

Molecular studies have linked HCV evasion of the host response and IFN therapy with various strategies directed by viral proteins to control ISG expression or function (Table 1). The HCV NS5A protein has been identified as an IFN antagonist. Several studies have shown that expression of NS5A alone can attenuate IFN-α actions and rescue the replication of IFN-sensitive viruses (Macdonald and Harris, 2004). Microarray analyses have shown that NS5A expression can confer a general attenuation of ISG expression, though the mechanism of this regulation was not defined (Geiss et al., 2003). NS5A induces interleukin (IL)-8 expression and secretion. This has implications for IFN therapy because IL-8 is a proinflammatory chemokine whose actions interfere with IFN (Fig. 3). The mechanism(s) of IL-8’s anti-IFN action may include attenuation of IFN signaling and ISG expression, and/or direct inhibition of select ISGs (Khabar et al., 1997). Serum IL-8 levels are found elevated in patients with chronic HCV, and NS5A has been shown to stimulate IL-8 production through transactivation of the IL-8 promoter, possibly involving NF-κB and AP-1 transcription factor activation by other cytokines (Polyak et al., 2001a, Polyak et al., 2001b). IRFs may also drive IL8 production when activated during virus infection (Casola et al., 2000). This latter point is another example that serves to demonstrate the link between innate antiviral and pro-inflammatory responses. In this case the link is exploited by NS5A as a means of evading the host response to HCV infection.

Table 1. Processes of ISG regulation or control by HCV.

Table 1

Processes of ISG regulation or control by HCV.

The NS5A and E2 proteins of HCV have both been identified as inhibitors of PKR (Gale, Jr. et al., 1998; Taylor et al., 1999; Noguchi et al., 2001). Inhibition of PKR may allow HCV to evade in part the translational-suppressive and signaling actions of PKR (Katze et al., 2002). This regulation is not universal and is subject to alteration through viral genetic variation, such that not all NS5A sequences have the ability to bind and inhibit PKR (Gimenez-Barcons et al., 2005). Thus, HCV evasion of PKR-independent processes of the host response must also contribute to HCV resistance to IFN. The product of the IFN-induced ISG56 gene (also known as IFIT1 or the 561 gene), p56, can suppress HCV RNA translation (Wang et al., 2002), and viral attenuation of ISG56 expression has been shown to associate with a level of resistance to HCV RNA replication from the antiviral actions of IFN in the HCV replicon cell culture model. ISG56 is both an ISG and an IRF-3-target gene (Grandvaux et al., 2002), and the IFN-resistant phenotype in this case associated with viral genetic adaptations that enhanced the NS3/4A blockade to IRF-3 signaling (Sumpter et al., 2004). This raises the possibility that HCV control of IRF-3 activation pathways may attenuate ISG expression by preventing the cross-talk of cellular pathways that converge on ISG promoter elements. Studies that have evaluated HCV interactions with the IFN-induced 2′–5′ oligoadenylate synthetase (OAS)/RNase L antiviral pathway have shown that HCV proteins interact with this pathway (Taguchi et al., 2004). When activated, this pathway directs RNase L, an endoribonuclease, to cleave the HCV genome RNA into nonfunctional products (Han and Barton, 2002). RNase L cleaves HCV RNA only at certain UU and UA dinucleotide sites (Han et al., 2004), and genotype 1 HCV sequences have overall fewer RNase L cleavage sites than HCV genotypes 2 or 3 (Han et al., 2004). This could provide a genetic mechanism for how, in part, HCV 1a and 1b infections resist IFN therapy (Table 1). However, as described above and owing to the pleiotropic nature of IFN effects mediated by the hundreds of ISGs, it is likely that HCV evades IFN action through multiple strategies to redirect ISG functions.

Viral Genetics Impact the Host Response to HCV Infection

The viral polymerase of HCV lacks proofreading function (Reed et al., 1998), and during persistent infection the error-prone virus replication generates a repertoire of highly related but genetically distinct viral variants or “quasispecies”. This provides a remarkable adaptive potential to HCV and has been implicated in evasion and control of the host response and IFN therapy (Farci, 2001). A hostile host environment may drive the outgrowth of HCV “evasion variants” from a preexisting quasispecies pool or through viral genetic adaptation. This idea is supported by in vivo studies that evaluated viral sequences from the E1 and E2 coding regions within patients with HCV infection. This work demonstrated viral genetic patterns that associated with infection outcome and in which the resolution of acute HCV infection consistently associated with an overall reduction in viral quasispecies complexity (Farci et al., 2000; Farci et al., 2002). In contrast, progression to chronic infection and resistance to IFN therapy associated with increased viral genetic complexity, suggesting that host immune pressure drives the outgrowth or selection of viral evasion variants able to persist and resist IFN action. Analysis of the HCV NS5A coding region has also identified specific domains that exhibit sequence variation in patients with differential outcomes to IFN therapy. Meta-analyses and long-term follow-up of these studies now provide support for NS5A sequence variation within a 40 aa “interferon sensitivity determining region” (ISDR) that associates with IFN therapy outcome (Enomoto et al., 1996, Pascu et al., 2004; Schinkel et al., 2004). The ISDR is within a genetically-flexible domain that is a major site of viral adaptations among HCV RNA replicons (Blight et al., 2000; Appel et al., 2005). Thus, ISDR variation may affect the HCV replication fitness and the host response to infection.

Exogenous Induction of Antiviral Hepatic Defenses

Various studies have described an absence or only a low level expression of IFN α/β within liver tissue from patients with chronic HCV infection (Mihm et al., 2004). This lack of high IFN α/β gene expression within the HCV-infected liver provides indirect evidence that HCV imposes a blockade to IRF-3 activation in vivo and may explain why some patients with chronic infection do not express significant levels of hepatic ISGs. However, it fails to explain why other patients exhibit broad and abundant ISG expression despite only low level IFN α/β expression in the infected liver (Smith et al., 2003). The fact that hepatic ISG expression has associated with the level and extent of liver pathology (Smith et al., 2003) suggests that ISGs might be induced indirectly as a result of cellular stress from fibrosis and/or cirrhosis. ISGs are also induced through STAT3 signaling; studies have shown that STAT3 is activated by NS5A, and that STAT3 activation occurs concomitantly with HCV RNA replication (Gong et al., 2001; Waris et al., 2005). Viral activation of STAT3, possibly mediated through stress-responsive signaling events, may contribute to ISG expression during HCV infection (Fig. 4). ISGs may also be induced through TLR engagement exogenously by extracellular products of damaged tissue or viral replication. It is also noted that stress-induced cytokines, including TNF-α and IL-1 can trigger IRF-1 expression and transactivation function, resulting in a level of IFN-β production (Fujita et al., 1986). This could contribute to hepatic ISG expression even when the RIG-I or TLR3 pathways are blocked by the HCV NS3/4A protease. Exogenous immune effector cells that infiltrate the liver may also contribute to hepatic ISG expression. In particular, IFN production by tissue macrophages and dendritic cells that have infiltrated the infected tissue could lead to a paracrine IFN response and ISG expression during the processes of antigen cross-presentation in vivo (Schulz et al., 2005). By this model hepatic ISG levels would vary with the composition and extent of immune cell infiltration, which has been observed (Smith et al., 2003). Secretion of IFN-γ by T cells and NK cells that have infiltrated the infected liver also contributes to ISG expression but the pattern of expressed ISGs only partially overlaps with those induced by IFN α/β (Der et al., 1998).

Fig. 4. Processes of hepatic IFN production from exogenous sources.

Fig. 4

Processes of hepatic IFN production from exogenous sources. Various processes can result in hepatic ISG expression despite a block to the host response imposed by the actions of HCV proteins. In most of the examples shown, IFN will be produced from non-infected (more...)

The HCV/Host Interface Model of Viral Evasion

Fig. 5 depicts a model of the virus/host interface and host response regulation that forms a foundation for persistent HCV infection. The transmission event of HCV infection presents unique pressures for the virus to adapt to the new host environment. HCV adaptation to a new host will involve fine tuning of viral strategies to control and evade the host response to infection. The transmission event results in an acute infection that involves viral regulation of the host response though RIG-I, TLR3 and other host defense signaling pathways within the infected cell (Sumpter et al., 2005; Li et al., 2005a). Highly fit variants of HCV will mediate signaling interference at the virus/host interface. This involves the actions of the NS3/4A protease to block RIG-I and TLR3 signaling pathways. However, this regulation is influenced by viral genetic variation, and genetic distinctions among the many different quasispecies of HCV that are replicating in various cells at various times post-infection will result in differential levels of control and activation of this response. During acute infection the differential activation and control of the host response by viral genetic variants will lead to the production of IFN and ISG expression to mediate an antiviral state in the local hepatic tissue (Bigger et al., 2001). 15–25% of all exposures to HCV typically result in acute/resolving infection (McHutchison, 2004), indicating that a robust hepatic host response could provide protection against the replication and spread of HCV. The host response and the ensuing adaptive immune response present enormous pressures that will select for the outgrowth of viral quasispecies that can evade and successfully control the host response and immune defenses (Farci et al., 2000; Sumpter et al., 2004). HCV/host interactions at key sites of host defense signaling serve to suppress the host response, attenuate the actions of IFN therapy, and provide a solid foundation for persistent HCV infection. This model incorporates the important, variable and perhaps unpredictable aspect of viral adaptation and quasispecies selection within the evasion and control strategies by which HCV limits the host response to infection. Further studies to identify the viral genetic elements (including viral PAMPs and PAMP structure), signaling factors and ISG effectors that regulate the host response to infection will certainly increase our understanding of host defense and the HCV/host interface that controls infection outcome.

Fig. 5. HCV/host interactions regulate infection outcome.

Fig. 5

HCV/host interactions regulate infection outcome. The model is described in the text, and its shows a flow of events in which the virus/host interface and interactions within the host response will decide the fate of HCV infection. By this model, HCV (more...)

Acknowledgements

The Gale laboratory is supported by grants from the NIH, the Ellison Medical Foundation and the Burroughs Wellcome Fund. DS is supported by NIH training grant 5T32DK007745. M.G. is the Nancy C. and Jeffrey A. Marcus Scholar in Medical Research in Honor of Dr. Bill. S. Vowell.

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