Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen
Journal of Virology
Mouse C3H 1OT1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small
... tion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 1OT1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 1OT1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40. The 5,243-bp genome of simian virus 40 (SV40) encodes two proteins produced early after infection, the 708-aminoacid large T antigen and the 174-amino-acid small t antigen, as well as three capsid proteins and an additional late protein, the agnoprotein, involved in capsid assembly. The large T antigen is a multifunctional nuclear phosphoprotein which has been shown to play central roles both in the lytic infection of monkey cells and in the abortive and transforming infections of rodent cells. Enzymatically, T antigen possesses ATPase activity, DNA and RNA helicase activities, and both specific and nonspecific DNA-binding activities (for reviews, see references 4 and 45). T antigen is also a transcriptional activator (5, 24). One of the targets for this activation is the SV40 late promoter.