Rank N M, Lambert P F
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
J Virol. 1995 Oct;69(10):6323-34. doi: 10.1128/JVI.69.10.6323-6334.1995.
The bovine papillomavirus type 1 (BPV-1) E2 translational open reading frame encodes three proteins that regulate viral transcription and DNA replication: the E2 transcriptional activator (E2TA), the E2 transcriptional repressor (E2TR) and the E8/E2 transcriptional repressor (E8/E2TR). E2TA is a strong activator of papillomaviral promoters and is required for viral DNA replication. E2TR and E8/E2TR inhibit the activities of E2TA but also possess weak transactivational properties of their own. Two components of the cellular transcription apparatus, TFIID and TFIIB, have previously been shown to associate with other viral and cellular transcriptional activators. We present evidence here that E2TA, the full-length E2 open reading frame gene product, directly binds both of these transcription factors in vitro. Glutathione S-transferase 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 binding of GST-E2TA to TBP and TFIIB required no additional mammalian factors, as shown by direct binding of GST-E2TA to bacterially synthesized recombinant TBP and recombinant TFIIB. The domain of E2TA required for its interaction with both TBP and TFIIB was localized to the C terminus 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 required for DNA binding and dimerization. Our findings, taken in context with previous studies, lead us to conclude that (i) cooperative DNA binding by E2 proteins and TBP is likely mediated by the direct binding of 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 required but is not sufficient for E2TA's strong transactivational activity.
1型牛乳头瘤病毒（BPV-1）的E2翻译开放阅读框编码三种调节病毒转录和DNA复制的蛋白质：E2转录激活因子（E2TA）、E2转录抑制因子（E2TR）和E8/E2转录抑制因子（E8/E2TR）。E2TA是乳头瘤病毒启动子的强激活因子，是病毒DNA复制所必需的。E2TR和E8/E2TR抑制E2TA的活性，但它们自身也具有较弱的反式激活特性。细胞转录装置的两个组成部分，即TFIID和TFIIB，先前已被证明可与其他病毒和细胞转录激活因子相互作用。我们在此提供证据表明，全长E2开放阅读框基因产物E2TA在体外可直接与这两种转录因子结合。谷胱甘肽S-转移酶E2TA（GST-E2TA）融合蛋白与体外合成的TATA盒结合蛋白（TBP，TFIID的一个组成部分）以及体外合成的TFIIB结合。同样，GST-E2TA与源自人宫颈癌的细胞系HeLa的核提取物中存在的TFIID和TFIIB结合。GST-E2TA与TBP和TFIIB的结合不需要额外的哺乳动物因子，这可通过GST-E2TA与细菌合成的重组TBP和重组TFIIB的直接结合得到证明。E2TA与TBP和TFIIB相互作用所需的结构域定位于E2TA的C末端，该区域在E2TR和E8/E2TR中也存在。该结构域位于先前已证明可赋予E2TA和TBP协同DNA结合能力的E2TA区域内，并且与DNA结合和二聚化所需的E2TA区域重叠。结合先前的研究，我们的发现使我们得出以下结论：（i）E2蛋白与TBP的协同DNA结合可能是由E2蛋白与TBP的直接结合介导的；（ii）E2TR和E8/E2TR的弱转录反式激活可能是由于这些蛋白直接与TBP和TFIIB结合所致；（iii）TBP和/或TFIIB的结合可能是必需的，但对于E2TA的强反式激活活性而言并不充分。
