title: T-antigen of sv40 blocks p53 transactivation but not p53 specific binding to DNA.
abstract: The p53 is a DNA binding phosphoprotein that can act as a transcriptional activator through high affinity DNA binding sequences (HBS). The large T antigen (LT-ag) of SV40 virus can bind p53 and their association is considered important for transforming activities of the virus. In this study, we investigated the effects of LT-ag on transcriptional transactivating function of p53 using cotransfection assays and DNA-binding 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 transactivation by wild-type p53. The LT-ag was unable to inhibit transactivation of a reporter gene containing a similar promoter (TK). The LT-ag mutants defective for binding to p53, failed to inhibit transactivation. The LT-ag inhibited the transactivation of a CAT reporter gene containing the GAL4-DNA recognition sequences by the p53 protein which was fused to the heterologous DNA binding domain (amino acids 1-147 of GAL4) in cotransfected cells showing that inhibition of p53 activities by LT-ag was not restricted to the p53 HBS-dependent reporter. LT-ag failed 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 analysis showed that in SKOV3, nuclear localization of wild type p53 was 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 able to bind to the HBS. These results suggest that LT-ag of SV40 preferentially binds the transcriptionally active p53, preventing it from transactivating through p53-HBS; the transcriptionally inactive p53 in these cells can still bind p53-HBS.
authors.affiliation: UNIV CALIF LOS ANGELES,CEDARS SINAI MED CTR,SCH MED,DEPT MED,DIV HEMATOL ONCOL,LOS ANGELES,CA 90048. UNIV CALIF LOS ANGELES,CEDARS SINAI MED CTR,SCH MED,DEPT PATHOL,LOS ANGELES,CA 90048.
authors.forename: Y
authors.initials: Y
authors.lastname: Imai
authors.forename: A
authors.initials: A
authors.lastname: Chumakov
authors.forename: C
authors.initials: C
authors.lastname: Miller
authors.forename: J
authors.initials: J
authors.lastname: Said
authors.forename: H
authors.initials: H
authors.lastname: Koeffler