Herpesvirus quiescence in neuronal cells. V: Forskolin-responsiveness of the herpes simplex virus type 1 α0 promoter and contribution of the putative cAMP response element

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8 Scopus citations

Abstract

The herpes simplex virus (HSV)-i α0 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 α0 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 α0-luciferase reporter plasmid (pRDα0-LUC) creating (pRDα0ΔCRE-LUC), and into the HSV-1 genome of strain 17+(α0ΔCRE). Insertion into both copies of the α0 promoter was verified by Southern blot analysis. ND-PC12 cells transfected with pRDα0-LUC and pRDα0ΔCRE-LUC plasmids responded similarly to forskolin (50 μM), 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 α0ΔCRE were treated with forskolin (50 μM) 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 α0 promoter is forskolin responsive, the purported CRE of the α0 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.

Original languageEnglish
Pages (from-to)489-497
Number of pages9
JournalJournal of NeuroVirology
Volume9
Issue number4
DOIs
StatePublished - Aug 2003

Keywords

  • Cyclic AMP
  • Forskolin
  • Herpes simplex virus
  • Neuronal
  • Reactivation
  • Viral latency

ASJC Scopus subject areas

  • Neurology
  • Clinical Neurology
  • Cellular and Molecular Neuroscience
  • Virology

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