SV40 EARLY-TO-LATE SWITCH INVOLVES TITRATION OF CELLULAR TRANSCRIPTIONAL REPRESSORS

We have purified factors from HeLa cell nuclear extracts that bind to the transcriptional initiation site of the SV40 major late promoter (SV40-MLP). The resulting fraction consists predominantly of three proteins, collectively called initiator-binding protein of SV40 (IBP-s) with electrophoretic mobilities of approximately 45-55 kD. Gel mobility-shift and DNase I-protection analyses indicate that each of these three proteins associates with high affinity to sequences located at the initiation site and 55 bp downstream of it. IBP-s-binding sites with lower affinities are located at +5 and +30. Addition of purified IBP-s to a cell-free transcription system represses transcription from the SV40-MLP, but not the SV40 early promoter. SV40 mutants lacking the two strongest IBP-s-binding sites (1) are not repressed by the addition of IBP-s in vitro, (2) overproduce late RNA (relative to wild-type SV40) at low, but not high, template copy number in vitro, and (3) exhibit increased levels of late RNA at early, but not late, times after transfection into CV-1 cells. Therefore, IBP-s is a cellular repressor of transcription of the SV40-MLP that may, in large part, be responsible for the replication-dependent component of the early-to-late shift in SV40 gene expression. Partial amino acid sequence data obtained from the approximately 55-kD component of IBP-s indicate that it is hERR1, an orphan member of the steroid-thyroid hormone receptor superfamily. These findings suggest simple molecular mechanisms by which hormones may modulate expression of viral late genes. We speculate that activation of expression of the late genes of other viruses may occur by similar mechanisms.