The 68-kDa bovine papillonavirus (BPV) type 1 replication protein El and the 48-kDa transactivator protein E2 form a complex that specifically binds DNA [Mohr, I. J., Clark, R., Sun, S., Androphy, E. J., MacPherson, P. & Botchan, M. R. (1990) Science 250, 1694Science 250, -1699. We have confirmed this observation and shown that the E1-E2 complex binds to DNA fragments that contain the BPV plasmid maintenance sequence 1 and a site for the initiation of bidirectional BPV DNA synthesis. The El protein was found to bind preferentially to non-or underphosphorylated species of E2, suggesting that the phosphorylation state of E2 modulates the association of the two proteins. Replication-deficient El mutants with single amino acid substitutions and deletions in the carboxyl terminus failed to interact with E2, indicating that a region in the El carboxyl terminus is required for El to interact with E2. Our results suggest that the replication deficiency of some El mutants reflects their inability to associate with E2.Bovine papillomavirus (BPV) type 1 (BPV-l) provides an attractive system to study regulatory control circuits of eukaryotic DNA replication. The viral DNA replicates and maintains itself stably as a multicopy nuclear plasmid in transformed rodent cells (1, 2). Several viral early genes have been implicated in plasmid maintenance (3, 4), and recent studies indicated a direct requirement for the El and E2 gene products in the replication of BPV plasmids (5).Genetic analysis suggested that the BPV El open reading frame (ORF) encodes at least two trans-acting functions, a negative modulator M and a positive replication function R (6). Two El-encoded phosphoproteins were identified: a 23-kDa protein that contains the El amino terminus and corresponds to El M and a full-length 68-kDa El protein that contains both the El M and the El R domain (7-9). The 68-kDa El protein binds ATP, and BPV genomes with a mutation in the ATP-binding domain are replication deficient (8,9). This suggested a direct involvement of the 68-kDa El protein in the initiation of DNA synthesis. In addition, a repressor function for viral gene expression has been associated with El (10, 11).Mutations in the BPV E2 ORF have multiple effects on viral transcription, DNA replication, or both (12). Three site-specific DNA-binding proteins are encoded by E2: a 48-kDIa full-length transactivator protein and two transcriptional repressor proteins (E2C and E8-E2), which lack the amino-terminal transactivation domain (13-18). Frameshift mutations throughout the E2 ORF eliminated BPV plasmid replication (4,(19)(20)(21). In addition, a deletion removing most of the E2 ORF (3, 22) led to replicationdeficient BPV genomes (M.L. and M. Botchan, unpublished observation). Mutant BPV genomes lacking the E2C repressor function displayed an increased plasmid copy number (23).Furthermore, BPV genomes containing a temperature-sensitive E2 transactivator function were replication deficient at nonpermissive temperatures (24). Taken together, the genetic data ...

No abstract

No abstract

No abstract

No abstract