Lin B Y, Ma T, Liu J S, Kuo S R, Jin G, Broker T R, Harper J W, Chow L T
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
J Biol Chem. 2000 Mar 3;275(9):6167-74. doi: 10.1074/jbc.275.9.6167.
Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Purified cyclin E/CDK2 or cyclin E/CDK3 complex, but not other cdks, partially complemented HeLa cell extracts. Cyclin E/CDK2 expression vectors also enhanced transient HPV replication in HeLa cells. HeLa cell-derived HPV-18 E1 protein is truncated at the carboxyl terminus but can associate with cyclin E/CDK2. This truncated E1 was replication-incompetent and inhibited cell-free HPV replication. These results indicate that HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient HPV replication, most likely due to sequestration by the endogenous, defective HPV-18 E1 protein. Further analyses of the regulation of HPV E1 and HPV replication by cyclin E may shed light on the roles of cyclin E/CDK2 in cellular DNA replication.
人乳头瘤病毒（HPV）含起始点的质粒在人乳头瘤病毒起始点识别蛋白E2和复制起始蛋白E1存在的情况下，能在人293细胞或细胞提取物中高效复制，而源自宫颈癌的HPV - 18阳性HeLa细胞或细胞提取物对HPV DNA复制的支持能力较差。我们最近发现，HPV - 11 E1通过一个RXL基序与细胞周期蛋白/细胞周期蛋白依赖性激酶（cdk）复合物相互作用，并且是这些激酶的底物。该基序或候选cdk磷酸化位点中的E1突变在复制过程中受损，这表明cdks在HPV复制中发挥作用。我们现在证明，HeLa细胞中的一种限制活性是细胞周期蛋白E/CDK2。纯化的细胞周期蛋白E/CDK2或细胞周期蛋白E/CDK3复合物，而非其他cdks，能部分补充HeLa细胞提取物。细胞周期蛋白E/CDK2表达载体也增强了HeLa细胞中HPV的瞬时复制。源自HeLa细胞的HPV - 18 E1蛋白在羧基末端被截断，但仍能与细胞周期蛋白E/CDK2结合。这种截断的E1无复制能力，并抑制无细胞体系中的HPV复制。这些结果表明，HeLa细胞在细胞周期蛋白E/CDK2方面在表型上限制了HPV的有效复制，最有可能是由于被内源性的、有缺陷的HPV - 18 E1蛋白所隔离。对细胞周期蛋白E对HPV E1和HPV复制调控的进一步分析，可能会揭示细胞周期蛋白E/CDK2在细胞DNA复制中的作用。
