Hepatitis C virus (HCV) NS5A protein transcriptionally modulates a number of cellular genes. Since there is no evidence of binding of NS5A protein to DNA, it is likely to exert its activity in concert with cellular factor(s). In this study, we have identified a specific interaction of HCV NS5A with homeodomain protein PTX1 of human origin by a yeast two-hybrid interacting cloning system. The authenticity of this interaction was verified by mammalian two-hybrid assay, in vivo co-immunoprecipitation analysis, and from a colocalization study. Recently, murine PTX1 (mPTX1) has been shown to repress virus-induced murine interferonA4 promoter activity. Interferon-à alone or together with ribavirin is the only available therapy for HCV-infected patients. Therefore, we examined whether coexpression of NS5A and human PTX1 (hPTX1) proteins modulate human IFN-à promoter activity. An in vitro reporter assay by transfection of HepG2 cells with NS5A suggested an activation of IFN-à promoter to approximately 20-fold upon Newcastle disease virus (NDV) infection. Under similar experimental conditions, hPTX1-activated IFN-à prompter to approximately sevenfold, unlike mPTX1. However, cotransfection of NS5A and hPTX1 displayed a lower interferon promoter activity, probably for physical association between these two proteins. Subsequent study demonstrated that activation of IFN promoter by NS5A is associated with an increased expression of IRF-3. Further analysis revealed that ectopic expression of NS5A in HepG2 cells enhances endogenous IFN-à secretion and MxA expression upon induction with NDV. However, exogenous expression of hPTX1 did not significantly alter NS5A-mediated function in the stable transfectants. Taken together, these results suggested that the level of endogenous hPTX1 is not sufficient to block the function of NS5A for augmentation of virus-mediated IFN activity in HepG2 cells.