Mapping the transcriptional transactivation function of simian virus 40 large T antigen.
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 promoter, 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 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.
Zhu
J Y
JY
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Rice
P W
PW
Chamberlain
M
M
Cole
C N
CN
D000251:Adenosine Triphosphatases / Q000235:genetics; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000276:immunology; D015967:Gene Expression Regulation, Viral; D016296:Mutagenesis; D010449:Peptide Mapping; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D000251:Adenosine Triphosphatases