U3 and TAR mutations do not affect dox and Tat responsiveness of the SIV-rtTA promoter. (A) To assay dox responsiveness, C33A cells were transfected with LTR-promoter/luciferase reporter constructs corresponding to the original and evolved SIV-rtTA variants and an rtTA-expressing plasmid. After two days of culturing with 0 to 1000 ng/ml dox, the intracellular luciferase level, which reflects promoter activity, was measured. The error bar represents the standard deviation (SD) for 3 to 8 experiments (B) To assay Tat responsiveness, C33A cells were transfected with the promoter/luciferase plasmids and 0 to 50 ng SIV Tat-expressing plasmid. Two days after transfection, the promoter activity was analyzed by measuring the intracellular luciferase activity. The error bar represents the SD for 2 to 4 experiments. (C) 293T cells were transfected with the SIV-rtTA proviral constructs and cultured for two days with or without dox. Virus production was quantified by measuring the CA-p27 level in the culture supernatant. The error bar represents the standard deviation for 2 experiments.