Transcriptional suppression of miR-181c by hepatitis C virus enhances homeobox A1 expression.

Hepatitis C virus (HCV)-induced chronic liver disease is one of the leading causes of hepatocellular carcinoma (HCC). The molecular events leading to HCC following chronic HCV infection remain poorly defined. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, and their deregulation is associated with different viral infections. In this study, we observed that HCV infection of hepatocytes transcriptionally downregulates miR-181c expression by modulating CCAAT/enhancer binding protein ß (C/EBP-ß). Reduced expression of the pri-miR-181c transcript was noted following HCV infection. In silico prediction suggests that homeobox A1 (HOXA1) is a direct target of miR-181c. HOXA1 is a member of the homeodomain-containing transcription factor family and possesses pivotal roles in normal growth, development, and differentiation of mammalian tissues. Our results demonstrated that HOXA1 expression is enhanced in HCV-infected hepatocytes. Exogenous expression of the miR-181c mimic inhibits HOXA1 and its downstream molecules STAT3 and STAT5, which are involved in cell growth regulation. Interestingly, overexpression of miR-181c inhibited HCV replication by direct binding with E1 and NS5A sequences. Furthermore, accumulation of HCV genotype 2a RNA with miR-181c was observed in an RNA-induced silencing complex in Huh7.5 cells. Our results provide new mechanistic insights into the role of miR-181c in HCV-hepatocyte interactions, and miR-181c may act as a target for therapeutic intervention. Importance: Chronic HCV infection is one of the major causes of end-stage liver disease, including hepatocellular carcinoma. An understanding of the molecular mechanisms of HCV-mediated hepatocyte growth promotion is necessary for therapeutic intervention against HCC. In this study, we have provided evidence of HCV-mediated transcriptional downregulation of miR-181c. HCV-infected liver biopsy specimens also displayed lower expression levels of miR-181c. We have further demonstrated that inhibition of miR-181c upregulates homeobox A1 (HOXA1), which is important for hepatocyte growth promotion. Exogenous expression of miR-181c inhibited HCV replication by directly binding with HCV E1 and NS5A sequences. Taken together, our results provided new mechanistic insights for an understanding of the role of miR-181c in HCV-hepatocyte interactions and revealed miR-181c as a potential target for therapeutic intervention.

Polypyrimidine tract binding protein (PTB) associates with intronic and exonic domains to squelch nuclear export of unspliced RNA.

Retention of unspliced pre-messenger RNA (pre-mRNA) in the nucleus is essential for cell survival. Available nuclear factors must recognize and discern between diverse export signals present in pre-mRNA to establish an export inhibitory complex. We found that polypyrimidine domains present in both intron and exon were important for export inhibition of a minigene transcript based on hepatitis B virus pregenomic RNA. Overexpression of PTB drastically reduced export and presence of RRM4 domain seemed critical. This inhibitory network overrode stimulation from an exonic export-facilitating element. We posit that binding of PTB to multiple loci on pre-mRNA regulates nuclear retention.

Hepatitis C virus NS2 protein inhibits DNA damage pathway by sequestering p53 to the cytoplasm.

Chronic hepatitis C virus (HCV) infection is an important cause of morbidity and mortality globally, and often leads to end-stage liver disease. The DNA damage checkpoint pathway induces cell cycle arrest for repairing DNA in response to DNA damage. HCV infection has been involved in this pathway. In this study, we assess the effects of HCV NS2 on DNA damage checkpoint pathway. We have observed that HCV NS2 induces ataxia-telangiectasia mutated checkpoint pathway by inducing Chk2, however, fails to activate the subsequent downstream pathway. Further study suggested that p53 is retained in the cytoplasm of HCV NS2 expressing cells, and p21 expression is not enhanced. We further observed that HCV NS2 expressing cells induce cyclin E expression and promote cell growth. Together these results suggested that HCV NS2 inhibits DNA damage response by altering the localization of p53, and may play a role in the pathogenesis of HCV infection.

Hepatitis C virus infection modulates expression of interferon stimulatory gene IFITM1 by upregulating miR-130A.

We have examined the underlying mechanism of hepatitis C virus (HCV)-mediated IFITM1 regulation. IFITM1 is a potential target of miR-130a. Our results demonstrated that miR-130a expression was significantly higher in HCV-infected hepatocytes and liver biopsy specimens than in controls. Introduction of anti-miR-130a in hepatocytes increased IFITM1 expression. Hepatocytes stably expressing IFITM1 reduced HCV replication. Together, these results suggested that HCV infection of hepatocytes upregulates miR-130a and that use of anti-miR-130a may have potential for restriction of HCV replication.

Hepatitis C virus upregulates Beclin1 for induction of autophagy and activates mTOR signaling.

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.