Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2.
The Autographa californica multinucleocapsid nuclear polyhedrosis virus 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 Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.
Evans
J T
JT
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301, USA.
Leisy
D J
DJ
Rohrmann
G F
GF
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019915:DNA Primase; D004261:DNA Replication; D017924:Nucleopolyhedroviruses / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D018411:Spodoptera / Q000166:cytology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C081258:late expression factor 1, baculovirus; C096108:late expression factor 2, Baculovirus; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases