Park P, Copeland W, Yang L, Wang T, Botchan M R, Mohr I J
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8700-4. doi: 10.1073/pnas.91.18.8700.
Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase alpha-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus E1 proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase alpha-primase complex to the viral replication origin.
乳头瘤病毒的持续感染涉及病毒DNA作为核质粒的维持，其复制需要宿主DNA聚合酶。通过使用来自啮齿动物细胞的可溶性提取物来探究细胞DNA聚合酶α-引发酶全酶的作用，这些提取物在病毒E1解旋酶和E2转录因子存在的情况下复制牛乳头瘤病毒1和人乳头瘤病毒6b DNA。针对聚合酶α的催化性180 kDa亚基的单克隆抗体在该系统中抑制DNA合成。向中和的提取物中添加纯化的人聚合酶α-引发酶全酶可恢复其DNA合成活性。E1的氨基末端424个氨基酸与p180聚合酶亚基形成特定的蛋白质复合物。可以用针对含有DNA聚合酶活性的E1的抗体分离免疫复合物。此外，这种聚合酶活性可被抗聚合酶α抗体中和。在该体外系统中未遇到允许性障碍，因为牛E1可以与小鼠和人类复制装置相互作用。尽管猿猴病毒40和多瘤病毒编码的大肿瘤抗原与乳头瘤病毒E1蛋白的一级序列同源性有限，但在一级蛋白质结构水平上功能基序的组织非常相似。除了它们的起源特异性DNA结合活性外，这些解旋酶中的每一种可能都有助于将细胞聚合酶α-引发酶复合物募集到病毒复制起点。
