FIG 4.
Reinstating A26 promoter into the A26 locus restores plaque size. (A) Genomic organization of the engineered recombinant viruses expressing FLAG-tagged A26 under the control of the A26 promoter (vA26FLAG) or the F13 promoter (vA26FLAGpF13). (B) The genetic identity of the promoters introduced in the recombinant viruses was verified by restriction enzyme digestion of PCR amplicons produced with oligonucleotides flanking the promoters (indicated by arrows). The sizes of the amplicons and the expected digested fragments are indicated. (C) Restriction enzyme digestion of viral genome PCR amplicons from a number of plaque-purified revertant viruses (labeled R) or viruses retaining the mutant genotype (labeled M) corresponding to vA26FLAGpF13. Amplicons from revertant viruses failed to digest (like the parental vA26FLAG) as a consequence of reinstating the natural A26 promoter. (D) Analysis of viral spread by plaque assay. Cells were infected with the original vA26FLAG, vA26FLAGpF13 (3 representative isolates), and vA26FLAGpF13-Rev (2 representative isolates) and incubated in semisolid overlay for 72 h. The area of viral plaques (n = 15) was measured and presented as a percentage of the size obtained for vA26FLAG. Plaque area for vA26FLAGpF13 viruses was greater than 50% smaller than original vA26FLAG, and this was restored in vA26FLAGpF13-Rev isolates.