Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters

Vaccinia virus contains ~200 genes classified temporally as early, intermediate or late. We analyzed 53 intermediate promoters to determine whether any have dual late promoter activity. Our strategy involved (i) construction of a cell line that stably expressed the three late transcription factors, (ii) infection with a vaccinia virus mutant that expresses RNA polymerase but neither intermediate nor late transcription factors, and (iii) transfection with plasmids containing a luciferase reporter regulated by an intermediate promoter. After confirming the specificity of the system for late promoters, we found that many intermediate promoters had late promoter activity, the strength of which correlated with a TAAAT at the initiator site and T-content from positions −12 to −8 of the coding strand. In contrast, intermediate promoter activity correlated with the A-content from positions −22 to −14. The sequence correlations were confirmed by altering the specificities of strict intermediate and late promoters.