title: Herpesvirus quiescence in neuronal cells. V: forskolin-responsiveness of the herpes simplex virus type 1 alpha0 promoter and contribution of the putative cAMP response element.
abstract: The herpes simplex virus (HSV)-1 alpha0 promoter contains a putative cAMP response element (CRE) located at positions -68 to -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 alpha0 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 alpha0-luciferase reporter plasmid (pRDalpha0-LUC) creating (pRDalpha0deltaCRE-LUC), and into the HSV-1 genome of strain 17(+)(alpha0deltaCRE). Insertion into both copies of the alpha0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRDalpha0-LUC and pRDalpha0deltaCRE-LUC plasmids responded similarly to forskolin (50 microM), 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 alpha0deltaCRE were treated with forskolin (50 microM) 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 alpha0 promoter is forskolin responsive, the purported CRE of the alpha0 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.
authors.affiliation: Oral Medicine Section, Department of Oral Health Practice, University of Kentucky College of Dentistry and College of Medicine, Lexington, Kentucky 40536, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Danaher
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Jacob
authors.forename: Craig S
authors.initials: CS
authors.lastname: Miller
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000502:physiology; D006561:Herpes Simplex / Q000821:virology*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000254:growth & development; D008156:Luciferases / Q000235:genetics; D009474:Neurons / Q000821:virology*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000502:physiology; D051381:Rats; D020218:Response Elements / Q000502:physiology; D014709:Vero Cells
chemical_list: D005576:Colforsin; D000242:Cyclic AMP; D008156:Luciferases