FIG. 3.

Enhancement of RTA transactivation of various KSHV promoters by MGC2663. (A) Transfection of CV-1 cells was carried out with 50 ng of viral promoter reporter construct and 250 ng of pcDNA-ORF50 (RTA) and/or 500 ng of pcDNA-MGC2663 (MGC2663) (+) as indicated. The total DNA amount used in each transfection was normalized by adding pcDNA3.1(−) vector. Luciferase activities were measured 48 h posttransfection. The error bars indicate standard deviations. (B and C) Transfections of 3T3 and 293 cells were carried out as described for panel A, using the same amounts of DNA for transfection. (D) Transfection of BJAB cells. In each transfection, 10⁷ BJAB cells were mixed with 2 μg of reporter plasmid, 5 μg of RTA expression plasmid, and/or 5 μg of MGC2663 expression plasmid. Transfection was carried out using electroporation as described in Materials and Methods. (E) MGC2663 stimulation of RTA activation is dose dependent and is specific for RTA and its target promoters. CV-1 cells were transfected with 50 ng of KSHV promoter reporter construct, pGL3-promoter, or pHIVLTR-luc, as well as the indicated amount of pcDNA-ORF50, pcDNA-Tat, and/or pcDNA-MGC2663. Each result represents an average of at least three independent experiments. The standard deviations are shown as error bars. Transfection efficiency for each experiment was normalized using a β-Gal expression plasmid as an internal control. Fold activation was calculated based on the transfection of the reporter plasmid and the vector control, which was normalized to 1. (F) Western blot analysis of RTA expression in transfected CV-1 cells in the presence or absence of MGC2663. Lysates of cells transfected with either pcDNA-ORF50 alone (lane 1) or pcDNA-ORF50 and pcDNA-MGC2663 (lane 2) were analyzed using RTA antiserum. The protein molecular mass markers are indicated.