Keith Meyer

Transcriptional Repression of p53 Promoter by Hepatitis C Virus Core Protein

Our previous results have suggested that the putative core protein of hepatitis C virus (HCV) transcriptionally regulates cellular and viral genes, inhibits cisplatin and c-myc-mediated apoptotic cell death under certain conditions, and transforms primary rat embryo fibroblast cells with a cooperative oncogene. Because HCV appears to cause hepatocellular carcinoma, we evaluated the regulatory role of the HCV core protein on p53, a well known tumor suppressor gene, by an in vitro transfection assay. HCV core protein repressed transcriptional activity of the p53 promoter when tested separately in COS7 and HeLa cells. Deletion mutational analysis of the HCV core gene indicated that the regulatory domain involved in the repression of p53 transcriptional activity is located around amino acid residues 80-122 encompassing a putative DNA binding motif and two major phosphorylation sites. Results from this study suggest that the putative core protein may have an important biological role in the promotion of cell growth by repressing p53 transcription, and this appears to be consistent with certain earlier observations about HCV core moving into the nucleus.

Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein

The genomic region encoding the hepatitis C virus (HCV) core protein was cloned into a mammalian expression vector to study its role on the transcriptional regulation of cellular proto-oncogene and viral promoters. Using a transient transfection assay in human hepatocellular carcinoma (HepG2) cells, we demonstrate that the HCV core protein activates the human c-myc, Rous sarcoma virus long terminal repeat (LTR), and simian virus 40 (SV40) early promoters; and suppresses the c-fos promoter and human immunodeficiency virus type 1 (HIV-1) LTR activity. The transcriptional regulation of cellular proto-oncogenes by the HCV core protein suggests possible involvement of the core protein in the deregulation of normal hepatocyte growth and hepatocarcinogenesis.

HEPATITIS C VIRUS NS5A PROTEIN MODULATES CELL CYCLE REGULATORY GENES AND PROMOTES CELL GROWTH

The phosphoprotein NS5A of hepatitis C virus has recently been suggested to control PKR protein kinase for resistance to interferon. To investigate other functions of NS5A, studies were initiated on the regulation of transcription of important cellular genes and of cell growth by this protein. The results suggested that NS5A protein represses transcription of the cell cycle regulatory gene p21WAF1, while it activates the human proliferating cell nuclear antigen gene in murine fibroblasts and human hepatoma cells. Furthermore, introduction of NS5A into murine fibroblasts (NIH3T3) promoted anchorage-independent growth and tumour formation in nude mice. Thus, NS5A appears to exhibit a role in cell growth regulation.

Hepatitis C Virus Genotype 1a Growth and Induction of Autophagy

ABSTRACT We have previously reported that immortalized human hepatocytes (IHH) support the generation of infectious hepatitis C virus (HCV) genotype 1a (clone H77). In the present study, we have investigated the growth of HCV genotype 1a (clone H77) through serial passages and accompanying changes in IHH in response to infection. Eleven serial passages of HCV genotype 1a (clone H77) in IHH were completed. Virus replication was ascertained from the presence of HCV-specific sequences, the detection of core antigen, the virus genome copy number, and the virus titer in IHH culture fluid. Electron microscopy suggested that HCV infection induces autophagic vacuole formation in IHH. Fluorescence microscopy displayed localization of autophagic markers, microtubule-associated protein-1 light chain-3 and Apg5, on the vacuoles of HCV-infected hepatocytes. Taken together, our results suggested that HCV genotype 1a (clone H77) can be serially passaged in IHH and that HCV infection induces an autophagic response in hepatocytes.

HEPATITIS C VIRUS CORE PROTEIN REPRESSES P21WAF1/CIP1/SID1 PROMOTER ACTIVITY

Hepatitis C virus (HCV) often causes a prolonged and persistent infection, and an association between hepatocellular carcinoma (HCC) and HCV infection has been noted. Recent experimental evidence using a cloned genomic region suggests that the putative core protein of HCV has numerous biological properties and is implicated as a viral factor for HCV mediated pathogenesis. WAF1/Cip1/Sid1 (p21) is the prototype of a family of proteins that inhibit cyclin-dependent kinases (CDK) and regulate cell cycle progression in eukaryotic cells. In this study, we have observed that the HCV core protein represses the transcriptional activity of the p21 promoter when tested separately by an in-vitro transient expression assay using murine fibroblasts (NIH3T3), human hepatocellular carcinoma (HepG2), and human cervical carcinoma (HeLa) cells. A deletion analysis of the p21 promoter suggested that the HCV core responsive region is located downstream of the p53 binding site. A gel mobility shift analysis showed that the HCV core protein does not bind directly to p21 regulatory sequences. Thus, the HCV core protein appears to act as an effector in the promotion of cell growth by repressing p21 transcription through unknown cellular factor(s).

Hepatitis C Virus Core Protein Upregulates Serine Phosphorylation of Insulin Receptor Substrate-1 and Impairs the Downstream Akt/Protein Kinase B Signaling Pathway for Insulin Resistance

ABSTRACT Chronic hepatitis C virus (HCV) infection has a significantly increased prevalence of type 2 diabetes mellitus (T2DM). Insulin resistance is a critical component of T2DM pathogenesis. Several mechanisms are likely to be involved in the pathogenesis of HCV-related insulin resistance. Since we and others have previously observed that HCV core protein activates c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase, we examined the contribution of these pathways to insulin resistance in hepatocytes. Our experimental findings suggest that HCV core protein alone or in the presence of other viral proteins increases Ser312 phosphorylation of the insulin receptor substrate-1 (IRS-1). Hepatocytes infected with cell culture-grown HCV genotype 1a or 2a displayed a significant increase in the Ser473 phosphorylation status of the Ser/Thr kinase protein kinase B (Akt/PKB), while Thr308 phosphorylation was not significantly altered. HCV core protein-mediated Ser312 phosphorylation of IRS-1 was inhibited by JNK (SP600125) and phosphatidylinositol-3 kinase (LY294002) inhibitors. A functional assay also suggested that hepatocytes expressing HCV core protein alone or infected with cell culture-grown HCV exhibited a suppression of 2-deoxy-d-[3H]glucose uptake. Inhibition of the JNK signaling pathway significantly restored glucose uptake despite HCV core expression in hepatocytes. Taken together, our results demonstrated that HCV core protein increases IRS-1 phosphorylation at Ser312 which may contribute in part to the mechanism of insulin resistance.

Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein

ABSTRACT We have previously shown that hepatitis C virus (HCV) core protein modulates multiple cellular processes, including those that inhibit tumor necrosis factor alpha (TNF-α)-mediated apoptosis. In this study, we have investigated the signaling mechanism for inhibition of TNF-α-mediated apoptosis in human hepatoma (HepG2) cells expressing core protein alone or in context with other HCV proteins. Activation of caspase-3 and the cleavage of DNA repair enzyme poly(ADP-ribose) polymerase were inhibited upon TNF-α exposure in HCV core protein-expressing HepG2 cells. In vivo protein-protein interaction studies displayed an association between TNF receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD), suggesting that the core protein does not perturb this interaction. A coimmunoprecipitation assay also suggested that HCV core protein does not interfere with the TRADD-Fas-associated death domain protein (FADD)-procaspase-8 interaction. Further studies indicated that HCV core protein expression inhibits caspase-8 activation by sustaining the expression of cellular FLICE (FADD-like interleukin-1β-converting enzyme)-like inhibitory protein (c-FLIP). Similar observations were also noted upon expression of core protein in context to other HCV proteins expressed from HCV full-length plasmid DNA or a replicon. A decrease in endogenous c-FLIP by specific small interfering RNA induced TNF-α-mediated apoptotic cell death and caspase-8 activation. Taken together, our results suggested that the TNF-α-induced apoptotic pathway is inhibited by a sustained c-FLIP expression associated with the expression of HCV core protein, which may play a role in HCV-mediated pathogenesis.

Hepatitis C virus NS5A protein protects against TNF-α mediated apoptotic cell death

Abstract Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179–1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-α induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-α mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-α induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-α mediated apoptotic cell death.

Hepatitis C virus inhibits cell surface expression of HLA-DR, prevents dendritic cell maturation, and induces interleukin-10 production.

ABSTRACT Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune responses against HCV antigens. HCV proteins have been shown to affect various intracellular events and modulate immune responses, although the precise mechanisms used to mediate these effects are not fully understood. In this study, we have examined the effect of HCV proteins on the modulation of major histocompatibility complex (MHC) class II expression and other functions important for antigen presentation in humans. Expression of an HCV1-2962 genomic clone (HCV-FL) in human fibrosarcoma cells (HT1080) inhibited gamma interferon (IFN-γ)-induced upregulation of human leukocyte antigen-DR (HLA-DR) cell surface expression. Furthermore, inhibition of promoter activities of MHC class II transactivator (CIITA), IFN-γ-activated site (GAS), and HLA-DR was observed in IFN-γ-inducible HT1080 cells expressing HCV-FL by in vitro reporter assays. Exposure of human monocyte-derived dendritic cells (DCs) to cell culture-grown HCV (HCVcc) genotype 1a (clone H77) or 2a (clone JFH1) significantly inhibited DC maturation and was associated with the production of IL-10. Furthermore, DCs exposed to HCVcc were impaired in their functional ability to stimulate antigen-specific CD4-positive (CD4+) and CD8+ T-cell responses. Taken together, our results indicated that HCV can have direct and/or indirect inhibitory effects on antigen-presenting cells, resulting in reduction of antigen-specific T-cell activation. These effects may account for or contribute to the low overall level of immunogenicity of HCV observed in chronically infected patients.

Characterization of antibodies induced by vaccination with Hepatitis C virus envelope glycoproteins

Abstract Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 were used with MF59 adjuvant as a candidate vaccine for a phase 1 safety and immunogenicity trial. Ten of 41 vaccinee serum samples displayed a neutralization titer of ⩾1:20 against vesicular stomatitis virus (VSV)-HCV pseudotype, 15 of 36 serum samples tested had a neutralization titer of ⩾1:400 against human immunodeficiency virus (HIV)-HCV pseudotype, and 10 of 36 serum samples tested had a neutralization titer of ⩾1:20 against cell culture-grown HCV genotype 1a. Neutralizing serum samples had increased affinity levels and displayed >2-fold higher specific activity levels to well-characterized epitopes on E1/E2, especially to the hypervariable region 1 of E2.