Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143).

Abstract

LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

Characterization of the Interaction between P143 and LEF-3 from Two Different Baculovirus Species: Choristoneura fumiferana Nucleopolyhedrovirus LEF-3 Can Complement Autographa californica Nucleopolyhedrovirus LEF-3 in Supporting DNA Replication

“…We have also clearly demonstrated that with AcMNPV, LEF-3 is necessary for the transport of P143 from the cytoplasm to the nucleus (39). These results have been confirmed by a yeast two-hybrid analysis of P143 and LEF-3 that also revealed an interaction between these two proteins (12). P143 also binds to DNA in a non-sequence-specific manner (22), a characteristic of some replication proteins including DNA helicases, DNA polymerases, primases and their accessory factors, DNA ligases and DNA topoisomerases (4).…”

“…These changes permitted AcMNPV to replicate more efficiently in B. mori cell lines and to kill B. mori larvae (3, 18). In addition, several attempts have been made to complement AcMNPV P143 with homologous genes from other baculovirus species but all of these have failed (5,12,15), suggesting that there are important differences in P143 from different viral species that regulate P143 function during replication. …”

The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication

“…The nature of the interactions between these proteins is not yet clear, but studies have identified some specific interactions. Using two-hybrid data and glutathione transferase fusion experiments, Evans et al (14) have shown that LEF-1, a possible viral primase, interacts specifically with LEF-2, which has an unassigned function. The SSB protein LEF-3 forms homotrimers and has also been shown by twohybrid analysis to interact with P143 (15,16,20).…”

“…Using transient-replication assays, it has been shown that six genes, lef-1, lef-2, lef-3, dnapol, p143, and ie1, are essential for replication and that three genes, ie2, p34, and iap-1, are stimulatory (4). Based on studies with OpMNPV and the related viruses Autographa californica MNPV (AcMNPV) and Bombyx mori NPV, data suggest that lef-1 encodes a possible primase and interacts directly with lef-2, which has an unknown function (14). lef-3 encodes a single-stranded DNA binding (SSB) protein, forms homotrimers, and is essential for transport of P143 into the nucleus (1,15,56).…”

The Transcriptome of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus in Trichoplusia ni Cells

“…Represented among the 20 most abundant AcMNPV mRNAs detected at 6 h p.i. are transcripts from 5 lef genes (lef-2, lef-3, lef-6, pp31, and p35) (27), 4 genes that encode proteins known to bind viral DNA (lef-2, lef-3, dbp, and pp31) (72)(73)(74)(75)(76), and the gene encoding the major budded virus envelope glycoprotein (gp64) (see Fig. S3B).…”