BACKGROUND/AIMS: HCV NS5A appears to play an important role in HCV resistance to IFN-alpha but the molecular mechanism is not fully elucidated. Most studies regarding the involvement of signal transducer and activator of transcription 1 (STAT1) in inhibition of IFN-alpha signaling by NS5A were performed in non-hepatic cell lines and their relevance may be debatable. METHODS: We analyzed the effects of NS5A on IFN-alpha signaling through STAT1 phosphorylation in three hepatocyte-derived cell lines, Hep3B, J5 and Huh7. Interaction of NS5A and STAT1 was also investigated with co-immunoprecipitation and confocal microscopy. RESULTS: IFN-alpha induces STAT1 activation in Hep3B cells in a dose- and time-dependent manner. Transient or stable NS5A expression inhibits STAT1 phosphorylation in a dose-dependent manner in hepatocyte-derived cell lines, whereas the levels of STAT1 phosphorylation remain unchanged in non-hepatocyte HeLa and COS7 cells despite increasing amounts of NS5A. The NS5A may interact with STAT1, specifically, the N-terminal 488 amino acids of STAT1. Furthermore, NS5A inhibits activation of interferon-stimulated gene factor 3 (ISGF3) and interferon-stimulated response element (ISRE)-driven gene expression, as demonstrated by electrophoretic mobility shift assay and luciferase assay, respectively. CONCLUSIONS: NS5A may interact with STAT1 and inhibit IFN-alpha signaling through suppression of STAT1 phosphorylation specifically in hepatocyte-derived cells.
BACKGROUND/AIMS: HCV NS5A appears to play an important role in HCV resistance to IFN-alpha but the molecular mechanism is not fully elucidated. Most studies regarding the involvement of signal transducer and activator of transcription 1 (STAT1) in inhibition of IFN-alpha signaling by NS5A were performed in non-hepatic cell lines and their relevance may be debatable. METHODS: We analyzed the effects of NS5A on IFN-alpha signaling through STAT1 phosphorylation in three hepatocyte-derived cell lines, Hep3B, J5 and Huh7. Interaction of NS5A and STAT1 was also investigated with co-immunoprecipitation and confocal microscopy. RESULTS: IFN-alpha induces STAT1 activation in Hep3B cells in a dose- and time-dependent manner. Transient or stable NS5A expression inhibits STAT1 phosphorylation in a dose-dependent manner in hepatocyte-derived cell lines, whereas the levels of STAT1 phosphorylation remain unchanged in non-hepatocyte HeLa and COS7 cells despite increasing amounts of NS5A. The NS5A may interact with STAT1, specifically, the N-terminal 488 amino acids of STAT1. Furthermore, NS5A inhibits activation of interferon-stimulated gene factor 3 (ISGF3) and interferon-stimulated response element (ISRE)-driven gene expression, as demonstrated by electrophoretic mobility shift assay and luciferase assay, respectively. CONCLUSIONS: NS5A may interact with STAT1 and inhibit IFN-alpha signaling through suppression of STAT1 phosphorylation specifically in hepatocyte-derived cells.
Authors: Arup Banerjee; Budhaditya Mazumdar; Keith Meyer; Adrian M Di Bisceglie; Ratna B Ray; Ranjit Ray Journal: J Virol Date: 2011-02-23 Impact factor: 5.103