title: HeLa cells are phenotypically limiting in cyclin E/CDK2 for efficient human papillomavirus DNA replication.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
authors.forename: B Y
authors.initials: BY
authors.lastname: Lin
authors.forename: T
authors.initials: T
authors.lastname: Ma
authors.forename: J S
authors.initials: JS
authors.lastname: Liu
authors.forename: S R
authors.initials: SR
authors.lastname: Kuo
authors.forename: G
authors.initials: G
authors.lastname: Jin
authors.forename: T R
authors.initials: TR
authors.lastname: Broker
authors.forename: J W
authors.initials: JW
authors.lastname: Harper
authors.forename: L T
authors.initials: LT
authors.lastname: Chow
mesh_terms: D042846:CDC2-CDC28 Kinases; D002457:Cell Extracts; D019927:Cyclin E / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D027383:Papillomaviridae / Q000378:metabolism*; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D002457:Cell Extracts; D019927:Cyclin E; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; D009856:Oncogene Proteins, Viral; D014764:Viral Proteins; C084762:oncogene protein E1, Human papillomavirus type 18; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases