Mapping the transcriptional transactivation function of simian virus 40 large T antigen

Abstract

T antigen is able to transactivate gene expression from the simian virus 40 (SV40) late promoter and from several other viral and cellular promoters. Neither the mechanisms of transactivation by T antigen nor the regions of T antigen required for this activity have been determined. To address the latter point, we have measured the ability of a set of SV40 large T antigen mutants to stimulate gene expression in CV-1 monkey kidney cells from the SV40 late promoter and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter. Transactivation, although reduced, was retained by an N-terminal 138-amino-acid fragment of T antigen. Mutants with alterations at various locations within the N-terminal 85 amino acids transactivated the RSV LTR promoter less well than did wild-type T antigen. Most of these were also partially defective in their ability to transactivate the SV40 late promoter. Two mutants with lesions in the DNA-binding domain that were unable to bind to SV40 DNA were completely defective for transactivation of both promoters, while a third mutant with a lesion in the DNA-binding domain which retained origin-binding activity transactivated both promoters as well as did wild-type T antigen. Only a low level of transactivation was seen with mutant T antigens which had lesions in or near the zinc finger region (amino acids 300 to 350). Mutations which caused defects in ATPase activity, host range/helper function, binding to p53, binding to the retinoblastoma susceptibility protein, or nuclear localization had little or no effect on transactivation. These results suggest that the N-terminal portion of T antigen possesses an activation activity. The data are consistent with the idea that the overall conformation of T antigen is important for transactivation and that mutations in other regions that reduce or eliminate transactivation do so by altering the conformation or orientation of the N-terminal region so that its ability to interact with various targets is diminished or abolished.

Association of p300 and CBP with Simian Virus 40 Large T Antigen

“…This activity may be important at the beginning of SV40 infection of certain differentiated cells because it may help to remove the proliferation block imposed by differentiation-inducing transcription factors such as myoD (21,22) or C/EBP␣ (8,52). Activation of transcription is a well-known activity of T (20,61), and the c-jun gene (18) may represent an important target. After overcoming growth arrest, stimulation of the expression of c-jun and other immediate-early genes may be essential to the initiation of cell cycle progression, culminating finally in cellular DNA synthesis and viral replication.…”

Adding an Rb-binding site to an N-terminally truncated simian virus 40 T antigen restores growth to high cell density, and the T common region in trans provides anchorage-independent growth and rapid growth in low serum concentrations

“…Alterations in at least four regions of T antigen diminish one or more transforming activities. T antigens with small deletions or multiple amino acid substitutions within the T common region cannot form dense foci on continuous cell lines (23,30,31,34,36,39,49) or primary rat embryo fibroblasts (30). Genetic alteration of the region encompassing the Rb/p107/p130-binding site (amino acids 101 to 118) prevents dense focus formation (20,37,42) and anchorage-independent growth (5,43).…”

The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex

“…SV40 Tag has been shown to associate directly with the tumor suppressor proteins p53 and Rb (reference 15 and references cited therein) and the enhancer-binding protein TEF-1 (3,19) as well as with TBP. The amino-terminal 121 or 138 amino acid residues of Tag are sufficient for weak (i.e., two-to threefold) transactivation in vivo (51,65). Amino acid residues 101 to 249 of Tag are sufficient to activate transcription 20-fold in a cell-free transcription system (3).…”