Robert Steele

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 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)

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.

Hepatitis C Virus Upregulates Beclin1 for Induction of Autophagy and Activates mTOR Signaling

ABSTRACT Hepatitis C virus (HCV) induces autophagosome formation in infected human hepatocytes. We have previously reported that HCV exploits autophagic machinery in favor of virus growth and survival in host cells (S. Shrivastava et al., Hepatology 53:406–414, 2011); however, the mechanisms for autophagy induction is poorly understood. In the present study, we observed that HCV infection transcriptionally upregulates Beclin1, which forms complex with Vps34, the class III phosphatidylinositol 3-kinase, as a first step for autophagy initiation. Although Bcl-2 has an anti-autophagy effect by its association with Beclin1 in nutrient-deprived cells, our studies revealed that HCV-mediated autophagy occurs independent of Beclin1–Bcl-2 dissociation. Mammalian target of rapamycin (mTOR) is a positive regulator of cell growth and is recognized as an inhibitor of autophagy induction. Our results demonstrated that HCV infection enhances phospho-mTOR expression and its downstream target 4EBP1 activation, suggesting that mTOR is not a negative regulator of HCV-induced autophagy. On the other hand, HCV infection in autophagy-impaired cells reduced phospho-mTOR, mTOR, and phospho-4EBP1 expression. Together, these results suggested that HCV induces autophagy by upregulating Beclin1 and activates mTOR signaling pathway, which in turn may promote hepatocyte growth.

MBP-1 Upregulates miR-29b, Which Represses Mcl-1, Collagens, and Matrix Metalloproteinase-2 in Prostate Cancer Cells

c-myc promoter binding protein (MBP-1) is a multi-functional protein known to regulate expression of targets involved in the malignant phenotype. We have previously demonstrated that exogenous expression of MBP-1 inhibits prostate tumor growth, although the mechanism of growth inhibition is not well understood. We hypothesized that MBP-1 may modulate microRNA (miRNA) expression for regulation of prostate cancer cell growth. In this study, we demonstrated that exogenous MBP-1 upregulates miR-29b by 5-9 fold in prostate cancer cells as measured by real-time quantitative reverse transcription-PCR. Subsequent studies indicated that exogenous expression of miR-29b inhibited Mcl-1, COL1A1, and COL4A1. Further, a novel target with potential implications for invasion and metastasis, matrix metallopeptidase-2 (MMP-2), was identified and confirmed to be a miR-29b target in prostate cancer cells. Together our results demonstrated that exogenous expression of miR-29b regulates prostate cancer cell growth by modulating anti-apoptotic and pro-metastatic matrix molecules, implicating therapeutic potential of miR-29b for prostate cancer inhibition.

Small Interfering RNA Targeted to Hepatitis C Virus 5′ Nontranslated Region Exerts Potent Antiviral Effect

ABSTRACT Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. Interferon alone or together with ribavirin is the only therapy for HCV infection; however, a significant number of HCV-infected individuals do not respond to this treatment. Therefore, the development of new therapeutic options against HCV is a matter of urgency. In the present study, we have examined vectors carrying short hairpin RNA (shRNA) targeting the 5′ nontranslated conserved region of the HCV genome for inhibition of virus replication. Initially, three sequences were selected, and all three shRNAs (psh-53, psh-274, and psh-375) suppressed HCV internal ribosome entry site (IRES)-mediated translation to different degrees in Huh-7 cells. Next, we introduced siRNA into Huh-7.5 cells persistently infected with HCV genotype 2a (JFH1). The most efficient inhibition of JFH1 replication was observed with psh-274, targeted to the portion from subdomain IIId to IIIe of the IRES. Subsequently, Huh-7.5 cells stably expressing psh-274 further displayed a significant reduction in HCV JFH1 replication. The effect of psh-274 on cell-culture-grown HCV genotype 1a (H77) was also evaluated, and inhibition of virus replication and infectivity titers was observed. In the absence of a cell-culture-grown HCV genotype 1b, the effects of psh-274 on subgenomic and full-length replicons were examined, and efficient inhibition of genome replication was observed. Therefore, we have identified a conserved sequence targeted to the HCV genome that can inhibit replication of different genotypes, suggesting the potential of siRNA as an additional therapeutic modality against HCV infection.