A small subclass of SV40 T antigen binds to the viral origin of replication

Abstract We examined the affinities of SV40 large T antigen for unique viral DNA sequences by binding SV40 Bst NI DNA fragments in extracts of infected or transformed cells, and then immunoprecipitating the T antigen-DNA complex. The G fragment, which spans the viral origin of replication ( ori ) was quantitatively bound to T antigen. A T-antigen-specific monoclonal antibody (Mcl 7), which recognized only 5%–10% of the T antigen from infected or transformed cells, immunoprecipitated the majority of the ori -binding activity. This suggests that only a minor subclass of wild-type T antigen is active in binding to the origin. C6 cells contain a replication-defective mutant T antigen that when tested in the DNA-binding immunoassay, showed no affinity for the ori fragment. Mcl 7 not only failed to immunoprecipitate ori binding in C6 cells, but also did not detect any labeled C6 T antigen whatever. Thus Mcl 7 recognizes an immunologically distinct subset of wild-type T antigen that comprises the origin-binding form of the viral protein, which is absent in the C6 T antigen population. Mcl 122, which recognizes a 53 kilodalton host protein that complexes with T antigen, immunoprecipitated ori -binding activity from extracts of infected or transformed cells, but not from C6 cells. Thus wild-type T antigen can bind ori sequences even when complexed to the host protein. These data suggest that T antigen consists of different subpopulations with different functions.