The understanding of 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 has been a major breakthrough. However, the replicon does not produce infectious virions. The purpose of this project study is to develop a model system for production of infectious virions. We created an expression construct containing a known infectious HCV genotype 1b cDNA flanked by two ribozymes. The ribozymes were designed to generate the exact 5? and 3? ends of HCV. This construct was constructed into a mammalian expression system. HuH7 cells were co-transfected with the above plasmid and a plasmid expressing tTA transactivator to control the expression by tetracycline administration. Both HCV structural (core, E1, E2) and nonstructural (NS5A) proteins were detected by immunofluorescence in cells and Western blots of cell lysates. RNase protection assays showed the presence of both positive and negative strand viral RNA, suggesting viral replication. Culture media was subjected to sucrose density gradient centrifugation and analyzed for the presence of HCV particles by Taqman PCR, ELISA for HCV core protein, and Western Blot for E2. All three assays showed the same sharp peak corresponding to a sucrose density of 1.16g/cm3, which is consistent with the reported density of HCV virions. Levels of HCV RNA by Taqman PCR were as high as 1 million copies/ml of culture media. Electron microscopy demonstrated the presence of HCV virus-like particles (approximately 50 nm in diameter) in the peak fraction. This system appears to produce a high level of HCV particles that are secreted into the media. This model further extends the repertoire of tools available to study HCV biology and the effects of antivirals such as interferons. 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 are 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. Using the yeast two hybrid system, several independent clones that interact specifically with NS5A have been identified. One of the NS5A interactors is Bin1, which contains a SH3 domain. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding and in vivo co-immunoprecipitation assays in human hepatoma cells. Deletion and mutation analyses indicated the importance of the SH3 and SH3 binding domains in the interaction between Bin1 and NS5A. Bin1 is a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. Loss of Bin1 may promote malignancy by interfering with the apoptotic pathways. HepG2 cells lack expression of Bin1 and upon infection with adeno-Bin1, these cells undergo apoptosis, as determined by a variety of assays. Expression of the wild-type NS5A but not the mutant NS5A with mutations in the SH3-binding domain inhibits Bin1-induced apoptosis in HepG2 cells. Together, our results suggest that NS5A impairs Bin1-induced apoptosis and exerts its effect on cell growth regulation. Like many viruses encoding gene products interfering with apoptosis, the NS5A and Bin1 interaction may be important for the productive infection of HCV and may contribute to the pathogenesis of hepatitis C. Additional experiments are under way to address the functional significance of this interaction.
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