HHV-8/KSHV Rta activates the hIL-6 promoter in a reporter system.

(A) Activation of the hIL-6 promoter by Rta in 2 different cell lines. Reporter plasmid phIL6-1200/SEAP (20 ng), pRL-CMV (2 ng), filler DNA (720 ng; plasmid DNA that lacks a mammalian promoter/enhancer), and either pcDNA3/Rta or pcDNA3 (50 ng) were transfected into 293T or R1T cells. Supernatants and cells were harvested at 48 hours after transfection and were assayed for SEAP and Renillaluciferase activities, respectively. SEAP activities from the hIL6 promoter were normalized to the corresponding Renillaluciferase activities. Fold activation by Rta was calculated by comparing the normalized SEAP activity stimulated by Rta to that by pcDNA3. (B) Dose-dependent activation of the hIL-6 promoter by Rta in 293T cells. Cells were transfected with 20 ng phIL6-1200/SEAP, 2 ng pRL-CMV, 720 ng filler DNA, and an increasing amount of pcDNA3/Rta (0-50 ng) and a correspondingly decreasing amount of pcDNA3 (50-0 ng) so that the total amount of pcDNA3 vector backbone remained the same. Reporter activities were assayed at 48 hours after transfection; fold activation by different amounts of pcDNA3/Rta was calculated by comparing the normalized SEAP activities to that stimulated by 0 ng pcDNA3/Rta and 50 ng pcDNA3.