CHARACTERIZATION OF THE INTERACTION BETWEEN THE BACULOVIRUS REPLICATION FACTORS LEF-1 AND LEF-2

The Autographa californica multinucleocapsid nuclear polyhedrosis viru s has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyce s cerevisiae and glutathione S-transferase fusion affinity assays. Usi ng yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyse s of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are in activated by these deletions. All clones expressing LEF-1 and LEF-2 th at were unable to interact also failed to support significant levels o f transient DNA replication, suggesting that this interaction is requi red for DNA replication. Sequence analysis of LEF-1 revealed a primase -like motif, WVVDAD. when this motif was mutated to WVVQAD, LEF-1 no l onger supported transient DNA replication.