Ranjit Ray

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 physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner.

ABSTRACT We have previously demonstrated that hepatitis C virus (HCV) NS5A protein promotes cell growth and transcriptionally regulates the p21/waf1 promoter, a downstream effector gene of p53. In this study, we investigated the molecular mechanism of NS5A-mediated transcriptional repression of p21/waf1. We observed that transcriptional repression of the p21/waf1 gene by NS5A is p53 dependent by using p53 wild-type (+/+) and null (−/−) cells. Interestingly, p53-mediated transcriptional activation from a synthetic promoter containing multiple p53 binding sites (PG13-LUC) was abrogated following expression of HCV NS5A. Additional studies using pull-down experiments, in vivo coimmunoprecipitation, and mammalian two-hybrid assays demonstrated that NS5A physically associates with p53. Confocal microscopy revealed sequestration of p53 in the perinuclear membrane and colocalization with NS5A in transfected HepG2 and Saos-2 cells. Together these results suggest that an association of NS5A and p53 allows transcriptional modulation of the p21/waf1 gene and may contribute to HCV-mediated pathogenesis.

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).

Knockdown of Autophagy Enhances the Innate Immune Response in Hepatitis C Virus―Infected Hepatocytes

The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV‐induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of the innate immune system against viral infection. In this study, we found that knockdown of autophagy‐related protein beclin 1 (BCN1) or autophagy‐related protein 7 (ATG7) in immortalized human hepatocytes (IHHs) inhibited HCV growth. BCN1‐ or ATG7‐knockdown IHHs, when they were infected with HCV, exhibited increased expression of interferon‐β, 2′,5′‐oligoadenylate synthetase 1, interferon‐α, and interferon‐α–inducible protein 27 messenger RNAs of the interferon signaling pathways in comparison with infected control IHHs. A subsequent study demonstrated that HCV infection in autophagy‐impaired IHHs displayed caspase activation, poly(adenosine diphosphate ribose) polymerase cleavage, and apoptotic cell death. Conclusion: The disruption of autophagy machinery in HCV‐infected hepatocytes activates the interferon signaling pathway and induces apoptosis. Together, these results suggest that HCV‐induced autophagy impairs the innate immune response. (HEPATOLOGY 2011;53:406‐414)

Safety and Immunogenicity of HCV E1E2 Vaccine Adjuvanted with MF59 Administered to Healthy Adults

BACKGROUND Hepatitis C virus (HCV) causes chronic liver disease that often leads to cirrhosis and hepatocellular carcinoma. In animal studies, chimpanzees were protected against chronic infection following experimental challenge with either homologous or heterologous HCV genotype 1a strains which predominate in the USA and Canada. We describe the first in humans clinical trial of this prophylactic HCV vaccine. METHODS HCV E1E2 adjuvanted with MF59C.1 (an oil-in-water emulsion) was given at 3 different dosages on day 0 and weeks 4, 24 and 48 in a phase 1, placebo-controlled, dose escalation trial to healthy HCV-negative adults. RESULTS There was no significant difference in the proportion of subjects reporting adverse events across the groups. Following vaccination subjects developed antibodies detectable by ELISA, CD81 neutralization and VSV/HCV pseudotype neutralization. There were no significant differences between vaccine groups in the number of responders and geometric mean titers for each of the three assays. All subjects developed lymphocyte proliferation responses to E1E2 and an inverse response to increasing amounts of antigen was noted. CONCLUSIONS The vaccine was safe and generally well-tolerated at each of the 3 dosage levels and induced antibody and lymphoproliferative responses. A larger study to further evaluate safety and immunogenicity is warranted.

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.

ISG56 and IFITM1 proteins inhibit hepatitis C virus replication

ABSTRACT Hepatitis C virus (HCV) often leads to persistent infection. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients. We have observed previously that HCV infection induces IFN-β production in immortalized human hepatocytes (IHH) as early as 24 h after infection, although virus replication is not inhibited. To gain insights on possible countermeasures of virus for the suppression of host antiviral response, the cellular transcriptional profiles of ISGs were examined after various treatments of IHH. The majority of ISGs were upregulated in IFN-treated IHH from the level for mock-treated cells. However, the comparison of ISG expression in IFN-treated IHH and IFN-pretreated, HCV genotype 2a-infected IHH indicated that virus infection suppresses the upregulation of a subset of effector molecules, including ISG56 and IFITM1. Similar results were observed for HCV-infected Huh7 cells. Subsequent study suggested that the exogenous expression of ISG56 or IFITM1 inhibits HCV replication in IHH or Huh7 cells, and the knockdown of these genes enhanced HCV replication. Further characterization revealed that the overexpression of these ISGs does not block HCV pseudotype entry into Huh7 cells. Taken together, our results demonstrated that ISG56 and IFITM1 serve as important molecules to restrict HCV infection, and they may have implications in the development of therapeutic modalities.