ABSTRACT
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.
