Quantifying Promoter Activity and VP16 Function

Dual-Glo luciferase assays (Promega) were employed in co-transfection assays according to the manufacturers’ protocols. To assay for promoter strengths, the constructs in which firefly luciferase was driven by wt or mutant VP16 promoters as well as a cytomegalovirus immediate early gene promoter (CMVIE) driven luciferase construct (Promega) as a positive control, were co-transfected with the pRL-TK renilla expression plasmid (Promega) or no promoter vector. The HSV-2 regulatory sequences were slightly stronger in this assay (≤2-fold higher). To assay for VP16 function, the VP16 constructs employed to make the mutants described above were co-transfected with an ICP0 promoter (124,818 to 124,109 BP) firefly luciferase construct (Sawtell and Thompson, 2016a (link),b (link)). Transfection efficiency was determined and normalized by including the relevant renilla or firefly luciferase expression plasmids (Figure 3A).