title: Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle.
abstract: The study of human papillomavirus type 16 (HPV-16) replication has been impaired because of the lack of a cell culture system that stably maintains viral replication. Recently, cervical epithelial cell populations that stably maintain HPV-16 replicons at a copy number of approximately 1,000 per cell were derived from an HPV-16-infected patient (W12 cell clone 20863 [W12-E cells]). We used neutral/neutral and neutral/alkaline two-dimensional gel electrophoretic techniques to characterize HPV-16 DNA replication in these cells. When W12-E cells were maintained in an undifferentiated state mimicking the nonproductive stage of the life cycle, HPV-16 DNA was found to replicate primarily by theta structures in a bidirectional manner. The initiation site of HPV-16 DNA replication was mapped to approximately nucleotide 100, and the termination site was mapped to between nucleotides 3398 and 5990. To study the productive stage of HPV-16 DNA replication, W12-E cells were grown under culture conditions that promote differentiation of epithelial cell types. Under these conditions, where virus-like particles were detected, the mode of viral DNA replication changed from theta structure to what is apparently a rolling circle mode. Additionally, CIN 612-9E cells, which were derived from an HPV-31-infected patient and harbor HPV-31 extrachromosomally, exhibited the same switch in the mode of DNA replication upon induction of differentiation. These data argue that a fundamental switch in the mechanism of viral DNA replication occurs during the life cycle of the papillomavirus.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706, USA.
authors.forename: E R
authors.initials: ER
authors.lastname: Flores
authors.forename: P F
authors.initials: PF
authors.lastname: Lambert
mesh_terms: D002454:Cell Differentiation; D002467:Cell Nucleus / Q000648:ultrastructure; D002584:Cervix Uteri / Q000821:virology; D002999:Clone Cells; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D004847:Epithelial Cells; D004848:Epithelium / Q000648:ultrastructure / Q000821:virology; D005260:Female; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D027383:Papillomaviridae / Q000235:genetics / Q000502:physiology*; D013728:Terminator Regions, Genetic
chemical_list: D004279:DNA, Viral