Characterization of a HCV NS5A protein derived from a patient with hepatoma

A cDNA encoding hepatitis C virus NS5A protein was isolated from the serum of a patient with hepatocellular carcinoma. The NS5A ^HCC was localized in both the cytoplasmic and nuclear fractions of Huh-7 cells. Immunoprecipitation and electrophoresis experiments showed four major phosphorylated species of NS5A ^HCC, p58, p56, p53, and p50. Two mutants (NS5A ^HCC-NLSmt and NS5A ^HCC-TSmt) carrying mutations on the putative nuclear localization signal were engineered. NS5A ^HCC-NLSmt was localized exclusively in the cytoplasm, whereas some forms of NS5A ^HCC-TSmt can be transported into the nucleus. These NS5A ^HCC mutant proteins were capable of transactivating c-fos and SV40 promoters. However, the transactivation efficiency was not dependent on its capability of nuclear localization. Subsequently, interaction between NS5A ^HCC mutants and Grb2 was studied. While capable of transactivating oncogenic promoters, NS5A ^HCC-TSmt could not interact with Grb2. Our results suggested that other cytosolic pathways independent of Grb2-mediated mechanisms were involved in the transactivation activity of HCV NS5A.