The E2 genes of the papillomaviruses encode polypeptides important for viral DNA replication and transcriptional regulation. The E2 open reading frame of the bovine papillomavirus type 1 encodes at least three polypeptides. The full-length gene product functions as a transcriptional transactivator and is required for episomal DNA replication. The 3' half of the open reading frame encodes two smaller polypeptides that repress E2-mediated transactivation. All three E2 proteins share a common carboxyl-terminal domain that encodes a specific DNA binding function and which contains sequences which promote E2 dimer formation. An amino terminal domain, which is unique to the full-length transactivator polypeptide, encodes the transactivation domain. Less is known about the regions of the E2 protein important for DNA replication. We have previously demonstrated that the BPV-1 E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the DNA binding domain. We have mutated these residues and have shown that viruses containing substitutions at serine residue 301 in the E2 polypeptide replicate to a copy number that is much greater than that of the wildtype genome. It has recently been demonstrated that both the full-length E2 polypeptide and the El polypeptide are both necessary and sufficient for BPV-1 replication and that these polypeptides interact to form a complex. An attractive model is that phosphorylation could regulate the formation of this complex and we are currently investigating this hypothesis and analyzing which regions of the E2 polypeptide are important for replication and complex formation. In addition, we are examining which protein kinase is responsible for the phosphorylation of the serine residue at position 301. We have demonstrated that DNA binding can be regulated in vitro by oxidation reduction (redox) of a single highly conserved cysteine residue in each subunit of the DNA binding domain. E2 polypeptides containing mutations in this highly conserved residue are still able to bind DNA but are defective in transactivation. We are currently investigating the defect in these mutated polypeptides.
