BOVINE PAPILLOMAVIRUS TYPE-1 E2 TRANSCRIPTIONAL REGULATORS DIRECTLY BIND 2 CELLULAR TRANSCRIPTION FACTORS, TFIID AND TFIIB

The bovine papillomavirus type 1 (BPV-1) E2 translational open reading frame encodes three proteins that regulate viral transcription and DN A replication: the E2 transcriptional activator (EZTA), the E2 transcr iptional repressor (E2TR) and the E8/E2 transcriptional repressor (E8/ E2TR). E2TA is a strong activator of papillomaviral promoters and is r equired for viral DNA replication. E2TR and E8/E2TR inhibit the activi ties of E2TA but also possess weak transactivational properties of the ir own. Two components of the cellular transcription apparatus, TFIID and TFIIB, have previously been shown to associate with other viral an d cellular transcriptional activators. We present evidence here that E 2TA, the full-length E2 open reading frame gene product, directly bind s both of these transcription factors in vitro. Glutathione S-transfer ase E2TA (GST-E2TA) fusion protein bound in vitro-synthesized TATA-box -binding protein (TBP), a component of TFIID, and in vitro synthesized TFIIB. Likewise, GST-E2TA bound TFIID and TFIIB present in a nuclear extract from the human cervical cancer-derived cell line, HeLa. The bi nding of GST-E2TA to TBP and TFIIB required no additional mammalian fa ctors, as shown by direct binding of GST-E2TA to bacterially synthesiz ed recombinant TBP and recombinant TFIIB. The domain of EZTA required for its interaction with both TBP and TFIIB was localized to the C ter minus of E2TA, a region also present in E2TR and E8/E2TR. This domain lies within the region of E2TA previously shown to confer cooperative DNA binding by E2TA and TBP and overlaps with the region of E2TA requi red for DNA binding and dimerization. Our findings, taken in context w ith previous studies, lead us to conclude that (i) cooperative DNA bin ding by E2 proteins and TBP is likely mediated by the direct binding o f E2 proteins to TBP, (ii) the weak transcriptional transactivation by E2TR and E8/E2TR may result as a consequence of direct TBP and TFIIB binding by these proteins, and (iii) TBP and/or TFIIB binding may be r equired but is not sufficient for E2TA's strong transactivational acti vity.