Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein.

Bovine papillomavirus (BPV) replication requires only two viral proteins, E1 and E2 (40), with the rest of the replication machinery supplied by the host cell (27). Both the E1 and E2 proteins are site-specific DNA binding proteins which recognize sequences in the viral origin of replication (1, 41, 42). The E2 binding site (E2BS) is a 12-bp partial palindrome (1, 22), while E1 binds to an 18-bp imperfect inverted repeat sequence (18, 19, 34). In vitro, only the E1 protein is absolutely required, indicating that the E2 protein does not supply a requisite replication function (3, 5). Instead, E2 appears to act as an auxiliary factor that interacts directly with E1 (24, 35, 44) and increases binding site specificity (5, 32). Moreover, at low protein concentrations, E2-E1 complexes facilitate loading and assembly of additional E1 molecules on the origin to form the active initiation complex (30). Initial binding of E1 induces distortion in the origin DNA that is a likely prelude to origin unwinding, and this distortion of the origin DNA is also enhanced by E2 (13, 30). Ultimately, a hexameric E1 complex with ATP-dependent helicase activity is assembled (11, 33), though the precise steps in the formation of this hexamer are undefined. E1 also interacts with several host cell proteins, including DNA polymerase α and replication protein A (4, 14, 29), and presumably recruits these host replicative factors to the viral initiation complex. The origin binding function of E1 is central to its role in viral genomic replication. Truncation studies of the 605-amino-acid BPV E1 protein localized the DNA binding domain (DBD) of E1 (the E1DBD) to approximately amino acids 140 to 300 (21, 31). Isolated polypeptides expressing this DBD region in the absence of other E1 sequences retain origin-specific binding activity (7, 21, 31, 39). Since the ATP binding site and helicase activity map to the C-terminal portion of E1 and are not present in the DBD, neither of these activities is required for origin recognition (39). The E1DBD does retain the ability to interact with E2 protein (21), and this interaction cooperatively enhances origin binding (7). Clearly then, the E1DBD is a functional subdomain with binding properties similar to those of full-length E1 protein. In contrast to the E2DBD, which has been crystallized and studied in detail (15–17), little is known about the structure of the E1DBD or the sequences which mediate origin binding. A previous study identified a pair of conserved, heptad repeats of hydrophobic residues from amino acids 249 to 282 (39). A triple mutant with substitutions at two of the conserved hydrophobic residues failed to bind origin DNA, consistent with a role for these elements in origin binding. However, the third mutation in this triple mutant changed a conserved proline located between the heptads to an alanine. The proline-to-alanine substitution could likely have a significant effect on protein folding in this region and makes interpretation of the role of the heptad repeats uncertain. Two additional mutants with basic residue-to-alanine substitutions in the region adjacent to the N-terminal side of the first heptad (amino acids 241 to 247) were both severely defective for origin binding, demonstrating that this hydrophilic region was critical for E1-DNA interaction. One additional mutation at a distal site, an arginine 180-to-alanine substitution, reduced E1 DNA binding activity to less than 1% of the wild-type (WT) E1 level, though this region was not investigated further (39). In the present study, the hydrophilic region that includes E1 amino acid 180 was investigated in detail and was shown to be critical for site-specific DNA binding both in vitro and in vivo. Features of this region suggest that it may be part of a recognition sequence that mediates specific contact with nucleotides in the E1BS.