FIGURE 7.

Generation of recombinant vaccinia viruses expressing mutant A27 proteins in the infected cells. A, schematic representation of recombinant vaccinia virus genomes containing various A27L mutant ORFs. The wild-type WR virus is shown above each viral ORF, and the arrows indicate the direction of transcription. J2R represents the nonessential thymidine kinase locus. In all of the recombinant viruses except the WR virus, the viral A27L ORF is substituted with a dual-expression cassette, Luc-Gpt, which contains a luciferase (Luc) gene driven by a viral early promoter and the Eco-gpt (Gpt) gene driven by the viral p7.5k promoter. WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A also contain another expression cassette inserted in the J2R locus in which the WT-A27, A27-E87A, A27-I94A, or A27-E87A,I94A mutants are driven by a synthetic late promoter, and a lacZ gene is driven by the viral p11k promoter. The asterisks mark the mutant residues. B, morphology of plaques produced on BSC40 and BSC40-A27 cells by WR, WR-ΔA27L, WR-A27-Rev, WR-A27-E87A, WR-A27-I94A, and WR-A27-E87A,I94A infections. BSC40 and BSC40-A27 cells were infected with each virus, fixed at 2 days p.i., stained with 1% crystal violet in 20% ethanol, and photographed. The tiny plaques are shown by triangles. C, immunoblot analysis of A27 protein in the infected cells. HeLa cells were infected with each virus at an m.o.i. of 5 pfu/cell, and the lysates were harvested at 24 h p.i. for immunoblot analyses with anti-A26 (1:1,000), anti-A27 (1:5,000), anti-A17 (1:1,000), and anti-H3 (1:1,000) antibodies.