Details

Autor(en) / Beteiligte

• Danaher, R J
• Jacob, R J
• Miller, C S

Titel

Herpesvirus quiescence in neuronal cells. V: Forskolin-responsiveness of the herpes simplex virus type 1 alpha 0 promoter and contribution of the putative cAMP response element

Ist Teil von

• Journal of neurovirology, 2003-08, Vol.9 (4), p.489-497

Erscheinungsjahr

2003

Quelle

SpringerLink

Beschreibungen/Notizen

• The herpes simplex virus (HSV)-1 alpha 0 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 alpha 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 alpha 0-luciferase reporter plasmid (pRD alpha 0-LUC) creating (pRD alpha 0 Delta CRE-LUC), and into the HSV-1 genome of strain 17 super(+) ( alpha 0 Delta CRE). Insertion into both copies of the alpha 0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRD alpha 0-LUC and pRD alpha 0 Delta CRE-LUC plasmids responded similarly to forskolin (50 mu 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 super(+) and alpha 0 Delta CRE were treated with forskolin (50 mu 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 alpha 0 promoter is forskolin responsive, the purported CRE of the alpha 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.