Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3.
Among the hepatotropic viruses, hepatitis C virus (HCV) is considered to be the leading cause of liver disease in humans, affecting approximately 2% of the world population. HCV-encoded nonstructural protein 5A (NS5A) is a 56-58-kDa phosphoprotein, which is produced from the processing of viral polyprotein. The potential mechanism(s) by which NS5A is able to influence key cellular processes are largely unknown. In this study, we investigated the functional properties of NS5A. In vivo co-immunoprecipitation and pull-down assays demonstrated that NS5A forms a heteromeric complex with TATA box binding protein (TBP) and tumor suppressor protein p53. Mutants of TBP and p53 showed reduced binding to NS5A. To determine the functional relevance of these associations, we found that NS5A inhibits the binding of both p53 and TBP to their DNA consensus binding sequences in vitro. NS5A also inhibited the p53-TBP and p53-excision repair cross complementing factor 3 (ERCC3) protein-protein complex formation. Furthermore, NS5A repressed the p53 regulated p21 (WAF1) promoter and a synthetic promoter containing multiple p53 responsive DNA elements binding sites in HCT116 p53(+/+) cell line. p53-mediated transcriptional activation from both promoters was reduced approximately 3-5-fold following expression of NS5A. Taken together, these results suggest that NS5A may exert its influence on key cellular processes by functional associations with p53 and TBP. This could explain one of the possible mechanism(s) by which NS5A is able to exert its effect on cellular gene expression and cell growth regulation.
Qadri
Ishtiaq
I
Department of Medicine, Division of Gastroenterology and Hepatology, Hepatobiliary Center, University of Colorado Health Sciences Center, B-145, 4200 E., 9th Avenue, Denver 80262, USA. Ishtiaq.qadri@uchsc.edu
Iwahashi
Mieko
M
Simon
Francis
F
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D002846:Chromatography, Affinity; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016174:Hepacivirus; D006801:Humans; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D035181:TATA-Box Binding Protein; D016159:Tumor Suppressor Protein p53; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase