Chiang C M, Dong G, Broker T R, Chow L T
Department of Biochemistry, School of Medicine and Dentistry, University of Rochester, New York 14642.
J Virol. 1992 Sep;66(9):5224-31. doi: 10.1128/JVI.66.9.5224-5231.1992.
Replication of human papillomavirus type 11 (HPV-11) DNA requires the full-length viral E1 and E2 proteins (C.-M. Chiang, M. Ustav, A. Stenlund, T. F. Ho, T. R. Broker, and L. T. Chow, Proc. Natl. Acad. Sci. USA 89:5799-5803, 1992). Using transient transfection of subgenomic HPV DNA into hamster CHO and human 293 cells, we have localized an origin of replication (ori) to an 80-bp segment in the upstream regulatory region spanning nucleotide 1. It overlaps the E6 promoter region and contains a short A + T-rich segment and a sequence which is homologous to the binding site of the bovine papillomavirus type 1 (BPV-1) E1 protein in the BPV-1 ori. However, unlike the BPV-1 ori, for which half an E2-responsive sequence (E2-RS) or binding site suffices, an intact binding site is essential for the HPV-11 ori. Replication was more efficient when additional E2-RSs were present. The intact HPV-11 genome also replicated in both cell lines when supplied with E1 and E2 proteins. Expression vectors of transcription repressor proteins that lack the N-terminal domain essential for E2 transcriptional trans activation did not support replication in collaboration with the E1 expression vector. Rather, cotransfection with the repressor expression vectors inhibited ori replication by the E1 and E2 proteins. These results demonstrate the importance of the N-terminal domain of the E2 protein in DNA replication and indicate that the family of E2 proteins positively and negatively regulates both viral DNA replication and E6 promoter transcription.
人乳头瘤病毒11型（HPV - 11）DNA的复制需要全长病毒E1和E2蛋白（C.-M. 蒋、M. 乌斯塔夫、A. 斯滕 Lund、T.F. 何、T.R. 布罗克和L.T. 周，《美国国家科学院院刊》89:5799 - 5803，1992年）。通过将亚基因组HPV DNA瞬时转染到仓鼠CHO细胞和人293细胞中，我们已将复制起点（ori）定位到上游调控区中跨越核苷酸1的一个80 bp片段。它与E6启动子区域重叠，包含一个短的富含A + T的片段以及一个与牛乳头瘤病毒1型（BPV - 1）ori中BPV - 1 E1蛋白结合位点同源的序列。然而，与BPV - 1 ori不同，对于BPV - 1 ori而言，半个E2反应序列（E2 - RS）或结合位点就足够了，而完整的结合位点对于HPV - 11 ori是必不可少的。当存在额外的E2 - RS时，复制效率更高。当提供E1和E2蛋白时，完整的HPV - 11基因组在这两种细胞系中也能复制。缺乏E2转录反式激活所必需的N端结构域的转录抑制蛋白表达载体，与E1表达载体共同作用时不支持复制。相反，与抑制蛋白表达载体共转染会抑制E1和E2蛋白对ori的复制。这些结果证明了E2蛋白的N端结构域在DNA复制中的重要性，并表明E2蛋白家族对病毒DNA复制和E6启动子转录具有正负调控作用。
