HeLa Cells Are Phenotypically Limiting in Cyclin E/CDK2 for Efficient Human Papillomavirus DNA Replication

• Published in: The Journal of biological chemistry 2000-03, Vol.275 (9), p.6167-6174
• Main Authors: Lin, Biing Yuan, Ma, Tianlin, Liu, Jen-Sing, Kuo, Shu-Ru, Jin, Ge, Broker, Thomas R., Harper, J.Wade, Chow, Louise T.
• Format: Article
• Language: English
• Subjects: AE1 protein; CDC2-CDC28 Kinases; cdk2 protein; cdk3 protein; Cell Extracts; cyclin E; Cyclin E - metabolism; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; DNA Replication; DNA-Binding Proteins - metabolism; E1 protein; HeLa Cells; Human papillomavirus 18; Humans; Oncogene Proteins, Viral - metabolism; Papillomaviridae - metabolism; Plasmids; Protein-Serine-Threonine Kinases; Replication Origin; Simian virus 40 - genetics; Transfection; Viral Proteins - metabolism; Virus Replication
• ISSN: 0021-9258; 1083-351X
• 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.