Hepatitis C virus (HCV) has a positive strand RNA genome that codes for a polyprotein that is processed co-translationally and post-translationally into three structural and at least seven nonstructural (NS) proteins. To investigate the function of NS5A, a recombinant vaccinia virus was constructed in which the NS5A gene was cloned under the control of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells. In addition, the NS5A gene was also cloned under the control of cytomegalovirus (CMV) early promoter. The NS5A expressed in monkey kidney (CV-1) cells was located predominantly in the cytoplasm. Using immunohistochemical analysis, the subcellular distribution of NS5A in liver biopsy samples from chronic HCV-infected patients was also found to be in the cytoplasm. However, the NS5A protein has a stretch of positively charged domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-terminal half of the protein. To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in which regions containing the NLS were fused to the N-terminus of the E. coli beta-galactosidase (E. coli beta-Gal). The expression of the fusion proteins in CV-1 cells resulted in their nuclear localization, indicating that the putative NLS is functional in targeting the heterologous protein, E. coli beta-Gal, to the nucleus, although the native NS5A is retained in the cytoplasm.