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Abstract

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication 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 nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

“…LT-Ag proteins encoded by SV40, BK virus, and JC virus have been shown to directly bind to p53 (15,(53)(54)(55)(56)(57)(58), whereas mouse polyomavirus LT-Ag does not bind p53 or inhibit p53-dependent transcription (59). To investigate whether the observed coprecipitation of MCPyV LT antigen with p53 is due to direct or indirect binding, we performed FACS-FRET experiments with living cells, as recently described (44), using different cell lines that ectopically express MCPyV LT or SV40 LT proteins and p53 as YFP or CFP fusion proteins.…”

p53 binds the nuclear matrix in normal cells: binding involves the proline-rich domain of p53 and increases following genotoxic stress

“…Indeed, free p53 has been reported in nuclear matrix fractions of SV40-transformed cells (Deppert and Haug, 1986). Mutants of SV40 large T that fail to interact with p53 also fail to bind the nuclear matrix (Deppert et al, 1989) and are transformation defective (Peden et al, 1989(Peden et al, , 1998. This raises the possibility that the sub-nuclear positioning of large T by p53 contributes towards the transforming potential of SV40 large T. In this context it is interesting to note that the association of large T with p300/CPB is also linked with its binding capacity for p53 and it has been suggested that p53 may mediate large T interaction with p300/CBP (Avantaggiati et al, 1996;Eckner et al, 1996;Lill et al, 1997).…”

Microbial Regulation of p53 Tumor Suppressor

“…In infected cells, viral protein (SV40 large T antigen [T-Ag]) binds p53 and inhibits p53-dependent transcription, resulting in accumulation of inactivated p53 protein [ 3 , 4 ]. Inhibition of p53 by large T-Ag is closely linked to the ability of the SV40 virus to induce tumorigenic transformation; SV40 mutants, which are defective in inhibition of p53, are also defective in cellular immortalization and transformation [ 5 , 6 ].…”