Schmidt H M, Steger G, Pfister H
Institut für Virologie der Universität zu Köln, Cologne, Germany.
J Virol. 1997 Oct;71(10):8029-34. doi: 10.1128/JVI.71.10.8029-8034.1997.
The promoter P7535 of human papillomavirus type 8 and the promoter P7185 of bovine papillomavirus type 1 are negatively regulated by viral E2 proteins via the promoter proximal binding sites P2 and BS1, respectively. Mutations of these E2 binding sites can reduce basal promoter activity. This suggests binding of a transcription-stimulating factor and may indicate that repression by E2 is due to competitive binding of viral and cellular proteins. A computer search revealed putative binding sites for core-binding factor (CBF; also referred to as PEA2, PEBP2, or AML), overlapping with P2 and BS1. Binding of recombinant CBF proteins to these sites was confirmed by band shift analysis. Competition of CBF and E2 protein for DNA binding was shown for both human papillomavirus type 8 and bovine papillomavirus type 1. The importance of CBF-E2 competition in E2-mediated repression could be demonstrated by comparing the E2 effect on P7185 activity in two cell lines containing different amounts of endogenous CBF. In cells with large amounts of CBF, E2 repressed P7185 wild-type constructs to the basal promoter activity of a mutant (50%) that could not bind this protein any more. In contrast, in a cell line containing small amounts of CBF, the promoter activities of constructs with wild-type and mutated CBF binding sites hardly differed and specific repression by E2 was not detectable.
人乳头瘤病毒8型的启动子P7535和牛乳头瘤病毒1型的启动子P7185分别通过启动子近端结合位点P2和BS1受到病毒E2蛋白的负调控。这些E2结合位点的突变可降低基础启动子活性。这表明有转录刺激因子的结合，并且可能表明E2的抑制作用是由于病毒蛋白和细胞蛋白的竞争性结合。计算机搜索揭示了与P2和BS1重叠的核心结合因子（CBF；也称为PEA2、PEBP2或AML）的假定结合位点。通过凝胶迁移分析证实了重组CBF蛋白与这些位点的结合。对于人乳头瘤病毒8型和牛乳头瘤病毒1型，均显示了CBF和E2蛋白在DNA结合上的竞争。通过比较E2对两种含有不同量内源性CBF的细胞系中P7185活性的影响，可以证明CBF-E2竞争在E2介导的抑制中的重要性。在含有大量CBF的细胞中，E2将P7185野生型构建体抑制到不再能结合该蛋白的突变体的基础启动子活性水平（50%）。相反，在含有少量CBF的细胞系中，具有野生型和突变型CBF结合位点的构建体的启动子活性几乎没有差异，并且未检测到E2的特异性抑制作用。
