A PAPILLOMAVIRUS E2 PHOSPHORYLATION MUTANT EXHIBITS NORMAL TRANSIENT REPLICATION AND TRANSCRIPTION BUT IS DEFECTIVE IN TRANSFORMATION AND PLASMID RETENTION

Papillomavirus DNA persists in infected cells as a nuclear plasmid, ca using epithelial lesions in many hosts, including humans, The viral pr otein E2 is required for both replication and transcription to facilit ate this persistence, Bovine papillomavirus E2 protein is phosphorylat ed at two predominant sites, Phosphorylation of one of these sites (se rine 301) inhibits replication of the genome, Using mass spectrometry and Edman sequencing, we have mapped additional phosphorylation sites in tryptic peptides to positions which lie primarily in the putatively unstructured hinge region of E2, Mutation of the major sites facilita tes transformation in the absence of viral repressors and only has a m inor effect on transformation when the repressors are present. Mutatio n of the major phosphorylation sites combined with one additional chan ge at a newly discovered site (serine 235) blocks transformation, Tran sformation can be restored by mutating this residue to aspartic acid, mimicking a phosphorylated amino acid, suggesting that phosphorylation is key to the regulation, Transformation by the mutant genome can als o be rescued by ectopic expression of the E2 enhancer protein, demonst rating a loss of function by the mutant protein and not a toxic defect , In transient assays, phosphorylation site mutants of E2 protein were normal for all viral functions tested, including replication, transcr iptional activation and repression (by the overlapping mutant represso rs), protein accumulation, and surprisingly, viral oncogene E5 promote r activation, While the mutant genome transiently replicated to high l evels, stable replication was defective, suggesting that a function of E2 required for plasmid retention is regulated by phosphorylation.