ISOLATION OF AN AMINO-TERMINAL REGION OF BOVINE PAPILLOMAVIRUS TYPE-1E1 PROTEIN THAT RETAINS ORIGIN-BINDING AND E2 INTERACTION CAPACITY

In vitro DNA binding results from a series of E1 proteins containing a mino-terminal or carboxy-terminal truncations indicated that sequences between amino acids 121 and 284 were critical for origin binding. Add itional binding experiments with E1 proteins containing internal, in-f rame insertions or deletions confirmed the importance of the region de fined by truncated E1 proteins and also demonstrated that downstream s equences were not required for binding activity in the contest of the full-length E1 protein. On the basis of mapping results from the E1 mu tants, a clone (pE1(121-311)) was constructed that expressed E1 amino acids within the approximate boundaries of the critical sequences for DNA binding. The E1(121-311) protein retained origin-specific DNA bind ing, confirming that this region was not only necessary but was also s ufficient for origin recognition. In addition to origin binding, E1(12 1-311) bound E2 protein in a cold-sensitive manner. Therefore, DNA bin ding and E2 binding activities colocalize to a 191-amino-acid function al domain derived from the amino-terminal half of the E1 protein. Fina lly, three E1 proteins, with mutations in this region all lacked DNA b inding activity and were all defective for in vivo replication. Two of these E1 mutants retained E2 binding capability, demonstrating that o rigin recognition by E1 is critical for replication and cannot necessa rily be rescued by an interaction with E2 protein.