Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication.

The bovine papillomavirus protein E2 serves dual functions in viral transcription and in the initiation of viral replication. As a transcription factor, E2 can cooperatively interact with cellular proteins such as SP1 and stimulate transcription of distal promoters. In replication, E2 and the helicase El are the only viral proteins required for accurate replication of templates containing the viral origin. The amino terminus of E2 is a functionally separable domain critical for activation of both replication and transcription; its primary sequence is conserved between many strains of papillomavirus. We targeted conserved residues spanning the activation domain and constructed a series of 30 amino acid substitution mutants. These mutant E2 genes were analyzed for the ability to activate DNA replication and gene expression in cells. The majority of the substitutions affected the ability of E2 to support both viral replication and transcriptional activation, revealing substantial overlap of the functional determinants for these two processes. Replication and transcription activities are genetically separable, however, as mutations at amino acids 73 and 74 retained replication function but failed to activate transcription. Additionally, a mutation at position 39 substantially reduced replication activity but left transcriptional activation intact. Interestingly, over two-thirds of the mutations analyzed reduced function and protein accumulation, many in a temperature-dependent manner. The correspondence between the replication and transcription phenotypes of mutations spanning the activation domain may indicate that the entire region is folded into a single domain required for both functions.