Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2

Abstract

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 Stransferase 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. MATERIALS AND METHODS Insect cells. Spodoptera frugiperda (Sf-9) cells (42) were cultured in TNM-FH medium (18) supplemented with 10% fetal bovine serum (FBS). Cell culture maintenance was carried out according to published procedures (40). Bacterial and yeast cells. All bacterial plasmids were maintained in Escherichia coli DH5␣. S. cerevisiae Y166 (MATa ura3-52 leu2-3,-112 his3⌬200 ade2-101 trp1-901 gal4⌬ gal80⌬ RNR::GAL-URA3 LYS2::GAL-HIS3 GAL-lacZ) was used for the yeast-two-hybrid assays and was the gift of Steve Elledge. Plasmid constructs. All baculovirus constructs were originally derived from the AcMNPV E2 strain (39). Yeast-two-hybrid clones. lef-1 was originally cloned as an NruI-EcoRI fragment (map units [m.u.] 7.5 to 8.7 [21, 23]) into pUC19. lef-2 was cloned as an MluI fragment (m.u. 1.9 to 2.6) into pUC19. lef-1 and lef-2 were both subcloned into pBluescript (pKS Ϫ) (Stratagene, Inc.), and NcoI sites were generated at the ATG start codon of each gene by site-directed mutagenesis (38) to form pKSLEF1(NcoI) and pKSLEF2(NcoI), respectively. The primers used are shown in Table 1. Mutagenesis changed the second amino acid of LEF-1 from leucine to valine and left the LEF-2 amino acid sequence unchanged. NcoI-BamHI inserts from these constructs were subcloned into the yeast vectors pASI and pACTII (gifts from Steve Elledge) (8), creating fusions with the GAL4 DNA binding and GAL4 activation domains, respectively. C-terminal deletion constructs of LEF-1 (aa1-241 [containing amino acids {aa} 1 to 241], aa1-222, aa1-215, and aa1-197) were made by exonuclease III digestion (15). pASLEF1aa1-129 was created by digesting pASLEF1 with EcoRI and DraI (DraI cuts at codon 129) followed by subcloning the fragment into EcoRI-SmaI-digested pASI. C-terminal deletion aa1-175 and N-terminal deletion aa175-266 were created by digesting pKSLEF1(NcoI) with NdeI, which cuts at codon 175 of lef-1, filling in with T4 DNA polymerase, and then digesting with NcoI and BamHI. The resulting fragments were subcloned into pASI cut w...

The Baculoviruses Occlusion‐Derived Virus: Virion Structure and Function

“…ODV nucleocapsids contain all of these proteins but for LEF-2 (Braunagel et al, 2003). The absence of LEF-2 is surprising given its oligomerization with LEF-1 (Evans et al, 1997;Hefferon and Miller, 2002) and its stabilization of LEF-1 binding to DNA (Mikhailov and Rohrmann, 2002a). BV nucleocapsids do not contain IE-1 or P143 (Braunagel et al, 2003).…”

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution

“…(ii) Yeast two-hybrid transfections and liquid assays, GST fusion affinity assays, and transient DNA replication assays. These procedures were carried out as previously described (10).…”

Reconstructing the replication complex of AcMNPV

“…Our studies indicated that LEF-1 was able to interact with LEF-2. Interaction between LEF-1 and LEF-2 was previously demonstrated by Evans and Rohrmann, using both yeast two-hybrid and GST-fusion affinity experiments [29]. The authors showed that DNA replication could no longer be supported when the interaction domain of LEF-1 was destroyed, indicating that interaction between LEF-1 and LEF-2 is critical for AcMNPV infection to proceed.…”