Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A.
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.
Ide
Y
Y
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City 66160-7421, USA.
Zhang
L
L
Chen
M
M
Inchauspe
G
G
Bahl
C
C
Sasaguri
Y
Y
Padmanabhan
R
R
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics; D000882:Haplorhini; D016174:Hepacivirus / Q000737:chemistry*; D006526:Hepatitis C / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012328:RNA Viruses / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase