HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding.
OBJECTIVE
To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.


METHODS
p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter. Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.


RESULTS
Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.


CONCLUSIONS
HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.
Gong
Guo-Zhong
GZ
Center for Liver Diseases, Second Xiangya Hospital, Central South University, 86 Renmin Zhong Road, Changsha 410011, Hunan Province, China. guozhong_gong@hotmail.com
Jiang
Yong-Fang
YF
He
Yan
Y
Lai
Li-Ying
LY
Zhu
Ying-Hua
YH
Su
Xian-Shi
XS
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000494:pharmacology*; D012324:RNA-Dependent RNA Polymerase
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004247:DNA; D016159:Tumor Suppressor Protein p53; D017361:Viral Nonstructural Proteins; C493956:CDKN1A protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase