EFFECTS OF MUTATIONS WITHIN THE SV40 LARGE T-ANTIGEN ATPASE P53 BINDING DOMAIN ON VIRAL REPLICATION AND TRANSFORMATION/

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles i n productive infection or virus-induced cell transformation. The carbo xy-terminal portion of T antigen includes a domain that carries the nu cleotide binding and ATPase activities of the protein, as well as sequ ences required for T antigen to associate with the cellular tumor supp ressor p53. Consequently this domain functions both in viral DNA repli cation and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nin e mutants with single or multiple substitutions between amino acids 40 2 and 430, a region thought to be important for both the p53 binding a nd ATPase functions. The mutants were examined for the ability to prod uce infectious progeny virions, replicate viral DNA in vivo, perform i n trans complementation tests, and transform established cell lines. T wo of the mutants exhibited a wildtype phenotype in all these tests. T he remaining seven mutants were defective for plaque formation and vir al DNA replication, but in each case these defects could be complement ed by a wild-type T antigen supplied in trans. One of these replicatio n-defective mutants efficiently transformed the REF52 and C3H10T1/2 ce ll lines as assessed by the dense-focus assay. The remaining six mutan ts were defective for transforming REF52 cells and transformed the C3H 10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce incr eased levels of p53.