T-ANTIGEN OF SV40 BLOCKS P53 TRANSACTIVATION BUT NOT P53 SPECIFIC BINDING TO DNA

The p53 is a DNA binding phosphoprotein that can act as a transcriptio nal activator through high affinity DNA binding sequences (HBS). The l arge T antigen (LT-ag) of SV40 virus can bind p53 and their associatio n is considered important for transforming activities of the virus. In this study, we investigated the effects of LT-ag on transcriptional t ransactivating function of p53 using cotransfection assays and DNA-bin ding electrophoretic mobility shift assays. A reporter gene containing a minimal TK promoter and two copies of HBS for p53 was cotransfected with p53 and LT-ag expression vector into human SKOV3 cells (p53 non- expressor). The LT-ag inhibited in a dose-dependent fashion transactiv ation by wild-type p53. The LT-ag was unable to inhibit transactivatio n of a reporter gene containing a similar promoter (TK). The LT-ag mut ants defective for binding to p53, failed to inhibit transactivation. The LT-ag inhibited the transactivation of a CAT reporter gene contain ing the GAL4-DNA recognition sequences by the p53 protein which was fu sed to the heterologous DNA binding domain (amino acids 1-147 of GAL4) in cotransfected cells showing that inhibition of p53 activities by L T-ag was not restricted to the p53 HBS-dependent reporter. LT-ag faile d to inhibit GAL4-p53 fragment containing the transactivating, but non -LT-ag binding region of p53, showing the importance of LT-ag binding to p53 in order to restrict p53 transactivation. Immunohistochemical a nalysis showed that in SKOV3, nuclear localization of wild type p53 wa s unaffected by coexpressed LT-ag. Gel shift analysis determined that nuclear extract from cells cotransfected with p53 and LT-ag expression vectors contained p53 not associated with LT-ag; this free p53 was ab le to bind to the HBS. These results suggest that LT-ag of SV40 prefer entially binds the transcriptionally active p53, preventing it from tr ansactivating through p53-HBS; the transcriptionally inactive p53 in t hese cells can still bind p53-HBS.