FIG 3.

Construction of AcMNPV mutants. (A) Schematic diagram of the AcMNPV mutants used in this study. bpp78/83-pk1KO was constructed by replacing a 1,575-bp fragment of pp78/83 and pk1 in bMON14272 with a 1,038-bp CmR gene via Red/ET homologous recombination in Escherichia coli. The pp78/83-pk1-rescued virus (vPK1:FLAG) was constructed by transposing both gene cassettes of pp78/83 and pk1 tagged with a FLAG epitope as well as the polh and egfp genes into the mini-attTn7 locus of bpp78/83-pk1KO. vPK1KO was generated by inserting only the pp78/83, polh, and egfp genes into bpp78/83-pk1KO. To prevent the expression of a C-terminus-truncated pk1 mutant in insect cells, the initiation codon (ATG) of pk1 was mutated to ATT. vP6.9:HA contained a p6.9 ORF tagged with an HA epitope sequence as previously described (11). The 7 PK1-dependent Ser/Thr residues were replaced with Ala residues to construct vP6.9:7M. A series of P6.9-mutation viruses (vPK1:1M) were constructed as described for vP6.9:7M, in which each of the 7 PK1-dependent Ser/Thr phosphorylation sites were separately mutated to Ala. GenR, gentamicin resistance; CmR, chloramphenicol resistance. (B) Sf9 cells were transfected with vPK1KO bacmid. At 48 h p.t., the cells were immunoblotted with a rabbit polyclonal anti-Pp78/83 antibody. Cells transfected with vGP64KO bacmid were used as a positive control. (C) Sf9 cells were transfected with bacmids of vAcWT, vPK1KO, or vPK1:FLAG. At the indicated time points p.t., the cells were observed with a fluorescence microscope or a light microscope. (D) Sf9 cells were infected with vPK1:FLAG or vAcWT at an MOI of 5 TCID₅₀/cell or supernatants from vPK1KO bacmid-transfected cells at 96 h p.t. The supernatants were harvested at the indicated time points p.i., and BV titers were determined using TCID₅₀ assays. Each data point represents the average titer from three independent infections. The error bars represent the standard deviations lg TCID₅₀/ml, the logarithm of the TCID₅₀/ml value with base 10.