Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication

Replication of the bovine papillomavirus (BPV-1) DNA requires both the viral E1 and E2 gene products. The minimal origin of replication, which resides in a 60-basepair fragment centered on the unique HpaI site of the BPV-1 genome, can be bound by the E1 protein and is flanked by E2 binding sites. The integrity of the region surrounding the unique HpaI restriction enzyme site is important for E1 binding and DNA replication, but the E2 binding sites are not required. The ability of E1 to complex with E2 and the requirement for both factors in DNA replication have led to the hypothesis that the E1/E2 complex may have a direct role in replication. Therefore we have studied mutations in the BPV-1 origin region for E1, E2, and E1/E2 complex binding and for their effect on transformation and replication in the context of the complete viral genome. Transformation and DNA replication were observed with some mutated viral genomes dramatically reduced for E1 binding. These mutated origins could bind significant amounts of E1 in the presence of E2, suggesting that binding of the E1/E2 complex could compensate for the loss of E1 binding in DNA replication. One mutation, with an eight-nucleotide insertion at the HpaI site, was still bound by E1 and the E1/E2 complex, yet did not replicate or transform efficiently. Thus although the binding of E1 or the E1/E2 complex to the origin appears to be necessary for replication, it is not sufficient.