Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

Abstract

Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region Ti) efficiently bound TBP. TEF-1 bound neither region Ti nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383. The Gal4 fusions demonstrated that no region of T antigen could activate a promoter containing Gal4-binding sites, suggesting that T antigen does not contain an activation domain of the type defined by this assay. However, the Gal4 fusion proteins maintained their ability to activate promoters known to be activated by wild-type T antigen. The fusion with region Ti, which binds only TBP, modestly activated the SV40 late promoter and the simple TEF-1/TATA promoter. Region T5, which binds both TBP and TEF-1, activated each of these promoters to levels equivalent to that of wild-type T antigen. The correlation between the binding of both TEF-1 and TBP and the ability to mediate wild-type levels of transcriptional activation suggests that T antigen causes activation through direct interactions with multiple factors in the transcription complex.The simian virus 40 (SV40) early-gene product large T antigen is a potent viral oncoprotein that interacts with a number of cellular proteins known to affect cell growth and gene expression. These interactions may account for the wide variety of functions attributed to T antigen (reviewed in references 14, 28, and 36). One of these functions is the transcriptional activation of both viral and cellular promoters, which was first detected through studies of activation of the SV40 late promoter (Fig. 1A) (7, 24). Subsequently, large T antigen was shown to be a promiscuous transcriptional activator because of its effect on many viral and cellular promoters (1,17,31,42, 52). This activation of cellular promoters may be as...

SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription.

“…On the other hand, none of the TAFs associated with GST-small t (lanes t). Our laboratory and others have also demonstrated a direct interaction between TBP and large T antigen (Gruda et al 1993;Johnston et al 1996; data not shown). In summary, our results indicate that the association of large T antigen with SL1 is mediated by specific protein-protein contacts with TAFI48 , TAFI110, and TBP.…”

“…Several studies have indicated that large T antigen can regulate cell growth, in part, by binding and inactivating the tumor suppressor gene products pRB and p53 (Hinds and Weinberg 1994;Nevins 1994). In addition, large T antigen can transactivate cellular genes, and several studies have indicated that this process is mediated by specific proteinprotein interactions with multiple components of the transcriptional machinery (Gruda et al 1993;Berger et al 1996;Damania and Alwine 1996;Johnston et al 1996). These alterations in the transcriptional activity of specific cellular genes most likely play an important role in the growth-promoting activity induced by large T antigen.…”

`Gain of function' phenotype of tumor-derived mutant p53 requires the oligomerization/nonsequence-specific nucleic acid-binding domain

“…Because wild-type p53 inhibits and mutant p53 activates di erent promoters to varying extents Ginsberg et al, 1991;Lechner et al, 1992;Santhanam et al, 1991;Subler et al, 1992), it was not possible to use an internal control such as pSVbGal or RSVbGal (Ludes-Meyers et al, 1996). A similar situation has been recognized for SV40 T antigen-mediated regulation of promoters (Gruda et al, 1993). Therefore, multiple independent experiments (three or more) were done to determine the standard deviations.…”

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen

“…In contrast, mutant T176-708 was completely inactive in both assays. The region spanning amino acids 128 ± 175 overlaps part of region X (spanning amino acids 131 ± 259), which has been suggested to be required for induction of DNA synthesis by T antigen (Dobbelstein et al, 1992) and for interaction with several cellular proteins including the TATA-binding protein and the transcriptional activator TEF-1 (Gruda et al, 1993).…”