Hepatitis C virus (HCV) causes the death of over 10,000 Americans annually. Despite its major impact on public health, relatively little is known about the replication or pathogenesis of this virus. Recently, the PI demonstrated an interaction between the viral NS5A protein and growth factor receptor-bound protein 2 (Grb2), a cellular adapter protein that mediates growth factor-induced signaling. The PI's hypothesis is that NS5A contributes to HCV pathogenesis by perturbing cellular signaling, potentially culminating in the short-circuiting of the interferon-signaling cascade. The PI proposes to examine the specificity of the effects associated with NS5A-Grb2 interactions by performing a systematic analysis of downstream pathways associated with Grb2 function utilizing epidermal growth factor (EGF) and IFN treated cell lines inducibly expressing wild-type NS5A and NS5A mutants. As an alternative the PI will examine these regulatory events in cells infected with NS5A expressing vaccinia viruses. The PI intends to analyze the activation state of the kinases involved in these pathways as well as the phosphorylation state of the kinase substrates and resultant transcription regulatory effects with a particular focus on possible involvement of the Jak/Stat pathway and the deregulation of IFN induced gene transcription. The PI will more globally examine the effects of NS5A on cellular gene expression in cells treated with types I and II IFN. As a complement to the more targeted approach described in the first aim, cDNA microarrays will be used to identify potentially novel cellular pathways and genes whose expression patterns are altered in response to regulated expression of NS5A. Glass slides containing PCR cDNA products representing ultimately over 45,000 known and unknown human genes, will be interrogated with poly (A)+ RNA prepared from wild-type and mutant NS5A expressing cell lines. Finally, the PI will use a biochemical purification strategy (including protein microsequencing by tandem mass spectrometry) to identify and subsequently clone the responsible cellular kinase. The PI's recent determination of Serine 2194 as the major NS5A phosphorylation site will facilitate in vitro and in vivo confirmatory studies that should verify the biological relevance and identification of the NS5A protein kinase. Identification of the kinase will likely help decipher the detailed molecular pathways uncovered from these studies. These studies may contribute to the identification of novel cellular gene targets for anti-HCV therapeutics.
