CIS AND TRANS REQUIREMENTS FOR STABLE EPISOMAL MAINTENANCE OF THE BPV-1 REPLICATOR

Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA i s stably propagated in the transformed cells, we have constructed a ce ll line CHO4.15 expressing constitutively the viral proteins E1 and E2 , that are required for initiation of viral DNA replication. We show t hat these viral proteins are necessary and sufficient for stable extra chromosomal replication. Using the cell line CHO4.15, we have shown th at the bovine papillomavirus-1 (BPV-1) minimal origin of replication ( MO) is absolutely necessary, but is not sufficient for stable extrachr omosomal replication of viral plasmids. By deletion and insertion anal ysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV-1 which assures stable replication of the MO-containing plasmids. This element is com posed of multiple binding sites for the transcriptional activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity In contrast to, for example, Epstein-Barr virus oriP, stably maintained BPV-1 plasmids are not subject to once- per-cell cycle replication as determined by density labelling experime nts. These results indicate that papillomavirus episomal replicators r eplicate independently of the chromosomal DNA of their hosts.