Skip to main content
. 2005 Nov;79(21):13538–13547. doi: 10.1128/JVI.79.21.13538-13547.2005

FIG. 3.

FIG. 3.

Regulation of RTA promoter activity by PKA. (A) Activity of the RTA promoter was assessed by luciferase reporter assays in which pRpluc (firefly luciferase coding sequence controlled by ∼3 kb of KSHV genomic DNA upstream of ORF50) was electroporated into the Ramos or DG75 B cell lines, which are known to be free of KSHV and Epstein-Barr virus, or into BC3 PEL cells containing latent KSHV. Following electroporation, cells were incubated for 18 h in medium supplemented as indicated with the PKA activator db-cAMP (300 μM) or the PKC activator PMA (20 ng/ml). Firefly luciferase activity was normalized to Renilla luciferase activity generated by pRLCMV (Renilla luciferase under control of the CMV promoter), and the statistical significance of triplicate determinations was evaluated by t test. Results showed significant PKA-mediated induction of RTA promoter activity in both cell types, but db-cAMP up-regulated RTA promoter activity more strongly KSHV-containing BC3 cells (as indicated by a cell type × db-cAMP interaction term from a factorial analysis of variance, P < 0.0001). Similar effects were observed when PEL cell lines were treated with 10 μM norepinephrine (data not shown). (B) Bioinformatic analysis of the RTA promoter revealed six potential cAMP response elements (CREs) within 2 kb of the ORF50 transcription start site. To assess their role, we compared db-cAMP effects on the full-length promoter (pRpluc) with effects on a series of truncation mutants omitting the distal 1.05 kb but retaining all six putative CREs (pRp1), omitting the distal four CREs (pRp2), and omitting all predicted CREs (pRp8). Experiments were carried out KS-1 cells as described above (A), with data represented as the mean (+ standard error) fold enhancement in normalized luciferase activity for db-cAMP-treated cells relative to controls. Deletion of the distal four CREs led to a quantitative reduction in PKA-inducibility, but db-cAMP continued to activate the RTA promoter even in constructs lacking all predicted CRE sites (e.g., pRp8). Similar results emerged in BC3 cells (data not shown).