Hepatitis C Virus NS5A Protein Down-regulates the Expression of Spindle Gene Aspm through PKR-p38 Signaling Pathway

Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G(2)/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G(2)/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma.