Hepatitis C virus (HCV) causes chronic infection in a large proportion of infected individuals leading to hepatitis, liver cirrhosis and hepatocellular carcinoma. The type 1a (H77C) HCV is the most prevalent strain in North America, for which no tissue culture has been reported to date. The goal of this research is to develop a robust cell culture system for the H77C (type 1a) HCV, which makes infectious virus using a unique approach. The hypothesis that increasing the translation efficiency of the HCV internal ribosome entry site (IRES) by manipulating either the HCV IRES sequence (or structure) or cellular factors (that otherwise attenuate viral translation) may lead to increased replication of the HCV genome and consequently higher viral titers in cell culture will be tested. The rationale for utilizing this strategy is two fold: First, increased synthesis of viral RNA polymerase and other viral non-structural proteins should directly affect the rate and extent of viral RNA synthesis. Secondly, higher quantities of viral proteins should block HCV host evasion response more completely allowing efficient viral replication to occur. A novel in vivo fluorescent protein (EBFP/EGFP)-based screen will be used to screen a randomized H77C HCV IRES library to identify mutations that significantly enhance viral protein synthesis without compromising viral RNA replication. The stronger mutant IRES elements identified by the screen will be used to substitute the weaker wt H77C IRES element in the full-length infectious H77C clone. The efficiency of these mutant full-length constructs in directing viral protein and RNA synthesis as well as producing infectious H77C virus particle will be tested in Huh-7.5 cells by using biochemical and immunological techniques. The manipulation of cellular factors that attenuate viral RNA translation will involve silencing of the antiviral response protein, PKR (dsRNA-activated protein kinase) by SiRNA against PKR. The effect of PKR knockdown on viral protein and RNA synthesis will be determined by established biochemical techniques. Finally, the effect of PKR silencing on production of infectious virus by H77C RNA containing stronger IRES mutants will be determined. These studies should lead to development of a robust type 1a HCV tissue culture system amenable to molecular genetic analysis and virus-host interaction and improved strategies for future development of antiviral therapeutics against the type 1 a virus. ? ? ?
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