T ANTIGENS ENCODED BY REPLICATION-DEFECTIVE SIMIAN-VIRUS-40 MUTANT-DL1135 AND MUTANT-5080

We present a preliminary biochemical characterization of two simian vi rus 40 mutants that affect different T antigen replication functions, SV40 T antigen mutants dl1135 (Delta 17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transfo rm cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess i n more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculov irus system and purified them by immunoaffinity chromatography. With e ach of the purified proteins, we examined some of the biochemical acti vities of T antigen required for replication, viz, ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays, Consistent with previou s studies, we found that the 5080 protein is defective for multiple bi ochemical activities including ATPase, helicase, ori-specific unwindin g, and ATP-induced hexamerization. However, this mutant retains some s equence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one o f several mutants with an altered aminoterminal domain which can repli cate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affe cts a T antigen enzymatic function directly required far viral DNA syn thesis, dl1135 may alter an activity required to prepare the cell for viral replication.