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. To generate a DNA expression plasmid capable of directing the production of HCV RNA genome, an infectious HCV genotype 1b cDNA was inserted between two ribozymes. The ribozymes were designed to generate the exact 5? and 3? ends of HCV. This construct was placed into a tet-off vector (pTHr). A second construct was also generated which was identical to pTHr, except for a mutation in the active site of the viral RNA dependent RNA polymerase (pTHrG9715A). Huh7 cells were transiently transfected and expression was controlled with tetracycline. Both HCV structural (core, E1, E2) and nonstructural (NS5A) proteins were detected by immunofluorescence in cells (pTHr more than pTHrG9715A) and Western blots of cell lysates (pTHr but not pTHrG9715A). RNase protection assays showed the presence of both positive and negative strand viral RNA with pTHr transfections, suggesting viral replication. Culture media of the transfected cells were separated by 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 of positivity corresponding to a sucrose density of 1.16g/ml, which is the putative density of HCV virions. Levels of HCV RNA by Taqman PCR were as high as 10 million/ml of culture media. Electron microscopy demonstrated the presence of HCV like particles (approximately 50 nm in diameter) in the peak fraction. Mutational analysis of the 5? and 3? UTRs of HCV RNA, extracted from the peak fraction of sucrose gradient by previously established methods (J Virol.78,7:3633-43) further provided evidence for viral replication in this system . The 5? G in the cDNA sequence, was replaced by an A or T in the viral RNA. A described variable base pair, in the stem loop 1 of the 3? UTR, was also detected in some of the samples. This system appears to produce a high level of HCV virions that are secreted into the tissue culture media. Infectivity is not yet demonstrated. This model further extends the repertoire of tools available for the study of HCV biology, and in particular may help understand hepatitis C virion assembly and release. RNA interference (RNAi) mediated by short interfering RNAs (siRNAs) has been widely used to inhibit the replication of viruses. To use RNAi accurately for this purpose, it is essential to determine the specificity of siRNA-mediated RNAi. Application of siRNA duplexes to interfere with the expression of a specific gene requires knowledge of target accessibility, highly effective delivery of siRNAs into the target cells, and possibly long-term stability of siRNA in some situations. In mammalian cells, siRNA molecules are thought to specifically silence gene expression without the activation of interferon response pathway. However, recent studies have indicated that some siRNA molecules are still capable of inducing the interferon pathway (Sledz et al., 2003). This is of major importance, as siRNAs are currently being explored for their potential therapeutic use in various viral infections. We have screened the whole genome of CON1 HCV for a suitable siRNA target and have defined one in the NS5B region that could suppress more than 90 % of HCV protein production in the CON1 replicon bearing cells. We have introduced a mutation at position 10 of the siRNA where it differs from our Cg1b strain and observed total abrogation of suppression. However, a change of nucleotide at position 19 of the siRNA target believed to be critical for siRNA function has no effect on suppression. Furthermore, we have observed the suppression of HCV replication up to 20 days post-transfection of the siRNA into the replicon bearing cells using the Durascribe kit to synthesize the siRNA. This duration of suppression is much longer than what was reported previously (<96h). Direct comparison of this siRNA with two other siRNA targets published previously with anti-HCV activity (Wilson et al., 2003; Randall et al., 2003) showed that this siRNA is more potent than the other two in inhibiting HCV replication. Expression profiles of a number of interferon-induced genes were monitored with a wide dose-range of siRNA during the course of suppression. We found no evidence of activation of the double-stranded RNA triggered gene expression with IFN-associated antiviral pathways. We have identified a novel potent siRNA target on the HCV genome that holds potential promise for design of siRNA-based therapy for hepatitis C. 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.
Showing the most recent 10 out of 38 publications
