Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.