Hepatitis C Virus Replic and Mech of Interferon Resistan

Diabetes, Digestive, Kidney Diseases

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Abstract

Detailed knowledge about the life cycle of hepatitis C virus has been hampered by the lack of a robust cell culture system. The recent development of a replicon system is a major breakthrough. However, the replicon does not produce infectious virions. The purpose of this project is to develop a model system for production of infectious virions. To generate a DNA expression plasmid capable of directing the production of HCV RNA genome, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5? and 3? ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated in parallel as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural (core, E1, E2) and nonstructural (NS5A) proteins were detected by immunofluorescence and Western blot. RNase protection assays showed the presence of both positive- and negative-strand HCV RNA. Sequence analysis of the 5? and 3? ends provided further evidence of viral replication in this system. Sucrose density gradient centrifugation of the culture medium revealed the co-localization of HCV RNA and structural proteins in a fraction corresponding to the density of 1.16g/ml, which is the putative density of HCV virions. Electron microscopy demonstrated the presence of viral particles of about 50 nm in diameter in the peak fraction. The level of HCV RNA in the culture medium was as high as 10 million copies per ml. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show any evidence of viral replication, assembly and release. This system appears to support the production and secretion of high-level HCV virions. This model further extends the repertoire of tools available for the study of HCV biology, particularly the process of viral assembly and release. Response to interferon in patients infected with HCV has been variable. Recent studies suggested a region, termed IFN sensitivity determining region (ISDR) in the HCV NS5A gene, that is associated with resistance to interferon. The NS5A has also been shown to be a phosphoprotein, probably playing an important role in viral replication and viral-host interaction. Because of the functional importance of NS5A, our laboratory is conducting experiments to characterize its function and identify cellular factors that are the functional targets of this HCV gene product. We screened for HCV NS5A interacting proteins by using the yeast two-hybrid system and studied the functional consequence of this interaction. Several independent clones containing SH3 domains were isolated with Bin1, a tumor suppressor with pro-apoptotic properties, being the most frequently identified clone. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding, in vivo co-immunoprecipitation and confocal microscopy. Deletion and mutation analyses indicated that the SH3 binding motif of HCV NS5A and SH3 domain of Bin1 are essential for interaction. Human hepatoma (HepG2) cells lacking expression of Bin1 undergo apoptosis upon infection with adeno-Bin1. Bin1-induced apoptosis was inhibited in HepG2 cells expressing wild type NS5A but not NS5A mutant with mutations in the SH3 binding motif. Infectious HCV genome containing mutations in the SH3 binding motif was not infectious in chimpanzee. Our results indicate that this interaction is implicated in productive HCV infection and may contribute to the pathogenesis of hepatocellular carcinoma. In addition, the NS5A PxxP motif may represent a novel target for antiviral development. Additional experiments are under way to address the functional significance of this interaction. More than 50% of HCV-infected patients are resistant to alpha interferon combination therapy. Gamma interferon exerts potent activity against HCV replication in the replicon system but fails to exhibit efficacy in HCV-infected patients. To better understand the nature of interferon therapy, we examined the dynamic responses to IFN-alfa and -gamma in the chimpanzee model in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) from naive chimpanzees and healthy human donors were treated with human interferon (hIFN)-alfa and -gamma. Quantitative real-time PCR was performed to evaluate the expression of IFN-stimulated genes (ISGs). The overall dose-dependent and time-related response patterns were comparable in human and chimpanzee. However, both IFNs induced greater ISG expression in humans than in chimpanzees, suggesting that chimpanzees respond less well to hIFN in vitro. Furthermore, naive and HCV chronically infected chimpanzees were treated with IFN-alfa (10 million IU) and IFN-gamma (400 mcg). These doses are 3-4 times higher than those for human use to compensate for the lower efficacy of hIFNs in chimpanzees. ISG expression was analyzed in PBMCs and liver biopsies. Although PBMCs from both naive and HCV-infected animals had a rapid response to each drug (peaking at 8 hours post-treatment), the response in HCV-infected chimpanzees was lower (~4 fold) than that in naive chimpanzees. hIFN-alfa appeared to induce a transient elevation of ALT and a surprising concomitant increase (>5-fold) in viremia in HCV-infected chimpanzees. Interestingly, both drugs induced a strong ISG response in the livers of naive chimpanzees, but not at all in those of HCV-infected animals. In conclusion, these data indicate a deficiency of response, particularly in hepatocytes, to hIFNs in HCV-infected chimpanzees. Further study on the mechanism of IFN pathway blockage by HCV infection in chimpanzee may provide novel insights into the clinical issue of nonresponse to IFN therapy.