Stephen J. Polyak

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

Elevated Levels of Interleukin-8 in Serum Are Associated with Hepatitis C Virus Infection and Resistance to Interferon Therapy

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.

Identification of hepatoprotective flavonolignans from silymarin

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-α-induced NF-κB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 μM, except for isosilybin B, which was toxic to cells above 10 μM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 μM. Silymarin suppressed TNF-α activation of NF-κB dependent transcription, which involved partial inhibition of IκB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Subversion of Cell Signaling Pathways by Hepatitis C Virus Nonstructural 5A Protein via Interaction with Grb2 and P85 Phosphatidylinositol 3-Kinase

ABSTRACT Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.

Multiple Effects of Silymarin on the Hepatitis C Virus Lifecycle

Silymarin, an extract from milk thistle (Silybum marianum), and its purified flavonolignans have been recently shown to inhibit hepatitis C virus (HCV) infection, both in vitro and in vivo. In the current study, we further characterized silymarins antiviral actions. Silymarin had antiviral effects against hepatitis C virus cell culture (HCVcc) infection that included inhibition of virus entry, RNA and protein expression, and infectious virus production. Silymarin did not block HCVcc binding to cells but inhibited the entry of several viral pseudoparticles (pp), and fusion of HCVpp with liposomes. Silymarin but not silibinin inhibited genotype 2a NS5B RNA‐dependent RNA polymerase (RdRp) activity at concentrations 5 to 10 times higher than required for anti‐HCVcc effects. Furthermore, silymarin had inefficient activity on the genotype 1b BK and four 1b RDRPs derived from HCV‐infected patients. Moreover, silymarin did not inhibit HCV replication in five independent genotype 1a, 1b, and 2a replicon cell lines that did not produce infectious virus. Silymarin inhibited microsomal triglyceride transfer protein activity, apolipoprotein B secretion, and infectious virion production into culture supernatants. Silymarin also blocked cell‐to‐cell spread of virus. Conclusion: Although inhibition of in vitro NS5B polymerase activity is demonstrable, the mechanisms of silymarins antiviral action appear to include blocking of virus entry and transmission, possibly by targeting the host cell. HEPATOLOGY 2010

Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4+CD25+FoxP3+CD127low regulatory T cells, contraction of CD4+ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species.

Impaired T‐cell responses in chronic hepatitis C virus (HCV) patients have been reported to be associated with the establishment of HCV persistent infection. However, the mechanism for HCV‐mediated T‐cell dysfunction is yet to be defined. Myeloid‐derived suppressor cells (MDSCs) play a pivotal role in suppressing T‐cell responses. In this study we examined the accumulation of MDSCs in human peripheral blood mononuclear cells (PBMCs) following HCV infection. We found that CD33+ mononuclear cells cocultured with HCV‐infected hepatocytes, or with HCV core protein, suppress autologous T‐cell responses. HCV core‐treated CD33+ cells exhibit a CD14+CD11b+/lowHLADR−/low phenotype with up‐regulated expression of p47phox, a component of the NOX2 complex critical for reactive oxygen species (ROS) production. In contrast, immunosuppressive factors, arginase‐1 and inducible nitric oxide synthase (iNOS), were not up‐regulated. Importantly, treatment with an inactivator of ROS reversed the T‐cell suppressive function of HCV‐induced MDSCs. Lastly, PBMCs of chronic HCV patients mirror CD33+ cells following treatment with HCV core where CD33+ cells are CD14+CD11b+HLADR−/low, and up‐regulate the expression of p47phox. Conclusion: These results suggest that HCV promotes the accumulation of CD33+ MDSC, resulting in ROS‐mediated suppression of T‐cell responsiveness. Thus, the accumulation of MDSCs during HCV infection may facilitate and maintain HCV persistent infection. (HEPATOLOGY 2012)

Hepatitis C virus induces oxidative stress, DNA damage and modulates the DNA repair enzyme NEIL1

Background and Aims:  Hepatitis C virus (HCV)‐induced chronic inflammation may induce oxidative stress which could compromise the repair of damaged DNA, rendering cells more susceptible to spontaneous or mutagen‐induced alterations, the underlying cause of liver cirrhosis and hepatocellular carcinoma. In the current study we examined the induction of reactive oxygen species (ROS) resulting from HCV infection and evaluated its effect on the host DNA damage and repair machinery.