Abstract
The herpes simplex virus (HSV)-1 f 0 promoter contains a putative cAMP response element (CRE) located at positions m 68 to m 60 with respect to the initiation of transcription. In this report, the authors examined the functionality of this element using (1) luciferase reporter gene assays in nerve growth factor-differentiated (ND)-PC12 cells and (2) virus-induced activation from quiescently infected (QIF)-PC12 cells. The putative f 0 CRE was completely eliminated by digestion with the restriction enzyme Tsp45I followed by mung bean nuclease treatment. The mutated region was verified by DNA sequencing and was inserted into the f 0-luciferase reporter plasmid (pRD f 0-LUC) creating (pRD f 0 j CRE-LUC), and into the HSV-1 genome of strain 17 + ( f 0 j CRE). Insertion into both copies of the f 0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRD f 0-LUC and pRD f 0 j CRE-LUC plasmids responded similarly to forskolin (50 w M), with approximately 250% increases in luciferase activity compared to mock-treated cultures as measured 3 days following treatment. When QIF-PC12 cultures established with HSV-1 strain 17 + and f 0 j CRE were treated with forskolin (50 w M) 17 days post infection, virus was detected in 9/24 (37.5%) and 13/24 (54.2%) of induced cultures by day 8 post treatment, respectively. In contrast, virus was detected in 0/23 and 1/24 (4.2%) of mock-treated cultures by day 8 post treatment for wild-type and mutant viruses, respectively. These findings indicate that the f 0 promoter is forskolin responsive, the purported CRE of the f 0 promoter does not confer forskolin responsiveness in ND-PC12 cells, and this element is not required for reactivation of HSV-1 from QIF-PC12 cells.