Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

Abstract

The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and Cll cells (CV-1 cell lines transformed with UVirradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC1l. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys --Arg at amino acid 516. pC1l has two mutations, one predicting a change of Pro -* Ser at residue 522, and another predicting a Pro --Arg change at amino acid 549. The two Cll mutations were separated from each other to form two distinct viral genomes in pCllA and pC11B. pC2, pCll, pC11A, and pCllB are able to transform both primary and established rodent cell cultures. The Cll and C1lA T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen.The multifunctional large T antigen of simian virus 40 (SV40) is essential in both the lytic cycle of the virus and the oncogenic transformation of cells by SV40. T antigen, encoded by the SV40 A gene, is expressed early in the lytic pathway and functions in numerous processes throughout the infectious cycle. It is required for the initiation of viral DNA replication (59), autoregulates early viral RNA synthesis (1, 51, 52, 61), stimulates late viral transcription (30;

A specific signature of Merkel cell polyomavirus persistence in human cancer cells

“…The mutations reported by Shuda et al (2) occur within the helicase part of the large T antigen of MCpyV and result in replication incompetence. This finding is reminiscent of early observations made by Gluzman and colleagues (8)(9)(10). That group reported that replication-defective SV40 virus DNA revealed an elevated transformation potential for various cell types.…”

Interactions Required for Binding of Simian Virus 40 T Antigen to the Viral Origin and Molecular Modeling of Initial Assembly Events

“…Subsequent molecular modeling studies indicate that a loop in T-ag centered on the highly basic ( 511 KKHLNKR 517 ) motif is situated opposite the boundary regions. That this motif might play an important role in DNA replication is supported by previous mutagenesis studies (53), for instance, those that characterized the Lys5163Arg mutation (42). While this conservative T-ag mutant bound to the core origin, it was defective in replication.…”

T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

“…Similar results have been seen in vitro and in vivo for transformation studies of animal polyomaviruses. Gluzman and colleagues (25)(26)(27) reported enhanced transforming activity for defective SV40 with mutations disrupting viral replication and helicase activity. In vitro MuPyV-transformed cell lines lack LT protein expression required for viral replication but express MT and ST oncoproteins that do not encode the T antigen helicase domain (28).…”