Regulation of Viral Transcription Units by SV40 T-Antigen
We have investigated the ability of SV40 T-antigen to trans-activate the SV40 late and the Adenovirus E2 promoters. Transcriptional control signals required for T-antigen trans-activation of the SV40 late promoter include T-antigen binding site II and the SV40 72-bp repeats. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences (mp 5171–272) results in quantitative removal of limiting trans-acting factors required for late gene expression in COS-1 cells. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the 72-bp repeats dramatically reduces the competition efficiency, suggesting a physical interaction between proteins binding to the separate regulatory domains. Transfection experiments have been performed in ts2 COS cells, which express the ts 1609 SV40 T-antigen. Transfection at the non-permissive temperature (40°C) resulted in a 5- to 10-fold reduction in SV40 late promoter activity compared to the permissive temperature (33°C), suggesting that trans-activation of the SV40 late promoter requires continued expression of T-antigen.
SV40 T-antigen trans-activates the Ad E2 promoter as effectively as does the Ad E1A protein. While 79 bp of upstream sequences are required for basal, E1A or T-antigen stimulated E2 promoter function, our experiments indicate that cellular factors which mediate stimulation by T-antigen and E1A are different. The sequences between -75 and -30 contain two imperfect 14 bp repeats separated by 16 bp. Using chemically synthesized DNA fragments containing the inverted repeat, we demonstrate that E1A efficiently induces transcriptional activity when these sequences are inserted in either orientation upstream of a heterologous promoter. Similar results were obtained using SV40 T-antigen. This suggests that target sequences for both E1A and T-antigen trans-activation of the E2 promoter are located between -85 and -29 and function in an orientation-independent fashion.
KeywordsMigration Phenol Titration Sarcoma Oligomer
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