Fig. 2

Construction of recombinant viruses and their confirmation. (a) Strategy for bAcvp39KO construction. AcMNPV vp39 ORF has 1044 nt. The first nucleotide of the initiation codon ATG of vp39 is indicated as + 1. The 5′ late transcription start site of cg30 is located 759 nt downstream of the initiation codon ATG of vp39, and the 5′ transcription start site of lef4 is located 37 nt downstream of the initiation codon ATG of vp39. The 344-bp fragment between 300 and 645 nt downstream of vp39 ATG in the bMON14272 genome was replaced with a 1038-bp Cm cassette via ET homologous recombination to generate the vp39 deletion bacmid, bAcvp39KO. (b) PCR confirmation of bacmids. Primer pairs used in this study are shown below. Bacmid DNA templates are shown above each lane, and the migration of DNA markers is shown to the left in base pairs (bp). (c) Schematic diagram of wild-type control virus vAcWT, which was generated by inserting polh and gfp genes into the polh locus of bMON14272 via Tn7-mediated transposition. (d) Schematic diagram of vAcvp39KO, vAcvp39:FLAG, and vAcSpltvp39:FLAG. The vp39 deletion virus, vAcvp39KO, was constructed by inserting polh and gfp genes into the polh locus of bAcvp39KO. The AcMNPV vp39 (Acvp39) gene under the control of its native promoter with a FLAG tag (indicated as a grey triangle) prior to the vp39 stop codon together with the polh and gfp genes were inserted into the polh locus of bAcvp39KO to construct the rescue virus, vAcvp39:FLAG. The SpltMNPV vp39 (Spltvp39) gene under the AcMNPV vp39 promoter control with a FLAG tag (indicated as a grey triangle) prior to the SpltMNPV vp39 stop codon together with the polh and gfp genes were inserted into the polh locus of bAcvp39KO to construct the vp39-pseudotyped virus, vAcSpltvp39:FLAG. (e) Sf9 cells were transfected with vAcvp39KO or vAcSpltvp39:FLAG and harvested at 72 h p.t. FLAG-tagged SpltMNPV VP39 or actin was detected by immunoblotting with anti-FLAG or anti-actin (endogenous control) monoclonal antibodies, respectively