FIG. 6.

Construction and characterization of bKO-lef3-125. (A) Schematic diagram describing the construction of the LEF-3 amino acid 1 to 125 rescue bacmid, bKO-lef3-125. The location and orientation of primers used in donor plasmid constructions and the diagnostic PCR are shown as arrows with names. (B) Viral DNA replication in bacmid-transfected Sf21 cells. Total intracellular DNA was isolated from Sf21 cells transfected with bAcP, bKO-lef3-Ac, bKO-lef3-125, or bKO-lef3-Cf at various times posttransfection. The DNA was digested with DpnI for 16 h and analyzed by real-time PCR. The relative viral copy number per cell was calculated for each bacmid transfection. The results are expressed as the number of viral genome equivalents per cell and are displayed as averages of samples analyzed in triplicate from transfections performed in duplicate. Error bars indicate standard deviations. (C) Budded virus growth curves in Sf21 cells transfected with bAcP or bKO-lef3-125 DNA. Supernatants were harvested at various times posttransfection and titrated for the presence of infectious virus in TCID₅₀ endpoint dilution assays. (D) The virus supernatants harvested at 96 h from bacmid transfection mixtures were used to infect Sf21 cells (multiplicity of infection, 0.1). At various times after infection, cell culture supernatants were harvested and titer were determined in TCID₅₀ assays. Virus titers represent the averages derived from three independent assays. Error bars represent standard deviations.