CHARACTERIZATION OF THE NUCLEAR-LOCALIZATION SIGNAL AND SUBCELLULAR-DISTRIBUTION OF HEPATITIS-C VIRUS NONSTRUCTURAL PROTEIN NS5A

Hepatitis C virus (KCV) has a positive strand RNA genome that codes fo r a polyprotein that is processed co-translationally and post-translat ionally into three structural and at least seven nonstructural (NS) pr oteins. To investigate the function of NS5A, a recombinant vaccinia vi rus was constructed in which the NS5A gene was cloned under the contro l of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells. In add ition, the NS5A gene was also cloned under the control of cytomegalovi rus (CMV) early promoter. The NS5A expressed in monkey kidney (CV-1) c ells was located predominantly in the cytoplasm. Using immunohistochem ical analysis, the subcellular distribution of NS5A in liver biopsy sa mples from chronic HCV-infected patients was also found to be in the c ytoplasm. However, the NS5A protein has a stretch of positively charge d domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-ter minal half of the protein. To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in w hich regions containing the NLS were fused to the N-terminus of the E. coli beta-galactosidase (E. coli beta-Gal). The expression of the fus ion proteins in CV-1 cells resulted in their nuclear localization, ind icating that the putative NLS is functional in targeting the heterolog ous protein, E. coli beta-Gal, to the nucleus, although the native NS5 A is retained in the cytoplasm.