The Investigation Of Antiviral Innate Immnue Response In A Novel Infectious System For Hepatitis Virus

More than 500 million people are persistently infected with hepatitis C virus(HCV) and/or hepatitis B virus(HBV) and are at a risk of developing chronic hepatitis,cirrhosis, and liver cancer. The absence of robust cell culture systems for both viral infections limits the understanding of viral life cycle, innate immune response and pathogenesis required for the development of antiviral drugs. Although the prophylactic vaccine for HBV has been developed, there is no vaccine available for HCV. In contrast to most other viral infection, the hallmark of HCV infection is that the majority patients(up to 80%) will develop chronic infection after viral exposure.On the other hand, approximate 20% of HCV infected patients resolve acute HCV infection without treatment, indicating that innate immune responses are capable of controlling the outcome of HCV infection.Virus-induced production of type I IFNs and the subsequent expression of IFN-stimulated genes(ISGs) are central to antiviral defenses. The innate immune response to virus infection is activated when conserved pathogen-associated molecular patterns(PAMPs) generated during infection are recognized by specific pattern recognition receptors(PRRs), including RIG-I-like receptors(RLRs), Toll-like receptors(TLRs), Nod-like receptors(NLRs) and the protein kinase R(PKR). However,the interaction between HCV and human hepatic innate antiviral responses is unclear.In this study, we aim to develop an appropriate cell culture system for HCV and HBV infection, and basing on it, to explore the mechanisms of innate immune responses during viral infection.Firstly, we have successfully established a novel human hepatoma cell line,termed HLCZ01, which was derived from the well-differentiated hepatocellular carcinoma of a male patient. The morphology of the HLCZ01 cell line is similar to that of primary human hepatocyte(PHH). HLCZ01 cells express liver-specific genes, such as human albumin(ALB), α1-antitrypsin(AAT), hepatocyte nuclear factor 4(HNF4),cytochrome P450 3A4(CYP3A4), and micro RNA122(mi R-122). HLCZ01 cells also express HCV receptors, including human CD81, SR-BI, CLDN1, and OCLN.Secondly, the HLCZ01 cell line is capable to support the entire life cycle of both HCV and HBV. HCV derived from cell culture and clinical isolates both propagate in HLCZ01 cells. HLCZ01 cells also support HBV infection and replication, includingcell cultured virus and clinical isolates. HLCZ01 cells infected with HBV and HCV release infectious viruses and support viral reinfection. Interestingly, both HBV and HCV replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBs Ag and human CD81, respectively. The replication of HBV and HCV is inhibited by antiviral drugs. The HLCZ01 cell line is the first cell line that supports the infection of HCV/HBV clinical isolates and both viruses coinfection. The cell line provides a powerful tool for exploring the mechanisms of viral life cycle and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.Thirdly, HLCZ01 cells and PHH mount an innate immune response to virus infection. On the one hand, IFN-β and some antiviral ISGs, including G1P3, ISG12 a and 1-8U, could be induced in the cells during acute HCV infection. The propagation of HBV in HCV-infected HLCZ01 cells does not affect the induction of IFN-β and ISGs by HCV. On the other hand, HCV infection induces apoptosis of HLCZ01 cells and PHH in the late period, which is another way for host to clear virus. Knockdown of TRAIL could reduce HCV-triggered apoptosis of human hepatocytes. Silencing of RIG-I in the hepatocytes can inhibit IFN-β and TRAIL expression and block apoptosis of the cells, which facilitates HCV replication in the cells. IRF3, a downstream molecule of RIG-I, is also involved in TRAIL-mediated apoptosis of hepatocytes.Finally, mi R-942 regulates HCV-induced apoptosis of human hepatocytes by targeting ISG12 a. ISG12 a is a key mediator for control of TRAIL-dependent apoptosis triggered by HCV infection. Mi R-942 is a candidate negative regulator of ISG12 a predicted by bioinformatics search, which has been conformed in the study. Moreover,HCV infection decreases mi R-942 expression in the hepatocytes, and the expression of mi R-942 is inversely correlated with that of ISG12 a in both HCV-infected cells and liver biopsies. Overexpression or silencing of mi R-942 in HCV-infected cells can reduce or enhance viral-induced apoptosis, respectively. HCV infection also up-regulates the expression of Noxa, which contributes to ISG12a-mediated apoptosis of the hepatocytes.In summary, this study provides a novel human hepatoma cell line HLCZ01,which supports the entire life cycle of both HCV and HBV. Basing on HLCZ01 infection system, I have explored some essential mechanisms of host innate response to viral infection. RIG-I pathway plays a pivotal role in the processes of type I IFN induction and TRAIL-mediated apoptosis during HCV infection. Mi R-942 mediates Noxa-dependent apoptosis of HCV-infected hepatocytes via regulation of ISG12 a.