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Amplification of bovine papillomavirus type 1 (BPV-1) DNA in growth-arrested mouse cell cultures appears to mimic the process of induction of vegetative BPV-1 DNA synthesis in cells of the stratum spinosum in productively infected bovine warts. In both cases, cells permissive for viral DNA amplification express large amounts of viral E2 protein which accumulates within the cell nucleus. Whereas in latently infected virus-transformed cells truncated transcriptional repressor forms of E2 predominate, our previous studies have demonstrated that the full-length E2 transcriptional trans-activator protein is preferentially expressed during the period of maximal BPV-1 DNA amplification in growth-arrested cell cultures. To investigate the role of the full-length E2 gene in the induction of viral DNA amplification in this experimental viral replication system we have used a mutant BPV-1 genome (BPVE2-ts1) containing an E2 gene which is temperature-sensitive (ts) for transcriptional trans-activation. This mutant genome has also been shown to be ts for stable viral plasmid DNA replication and for the induction of cell transformation. We show here that viral DNA amplification was not severely impaired when BPVE2-ts1-transformed cells were tested at the restrictive temperature, indicating that the transcriptional trans-activating function of E2 was not essential for viral DNA amplification in division-arrested cells and, moreover, that the trans-activation and replication functions of E2 were separable. Consistent with this hypothesis, amplification of the BPVE2-ts1 genome at the restrictive temperature was still associated with the accumulation of large amounts of nuclear E2 antigen, showing that the mutation did not disrupt nuclear transport or render the E2 protein highly unstable. Furthermore, C127 cells harbouring ts E2 and full-length E1 expression constructs supported transient plasmid replication of a BPV origin vector at the restrictive temperature. These observations imply that E2 functions primarily as a viral replication factor in the vegetative phase of BPV-1 DNA replication, and suggest a fundamental difference in the genetic regulation of stable BPV-1 plasmid DNA replication in mitotic cells and viral DNA amplification in postmitotic cells.

journal of general virology

Evidence That the Transcriptional Trans-activating function of the Bovine Papillomavirus Type 1 E2 Gene is not Required for Viral DNA Amplification in Division-arrested cells