FIG. 2.

Effect of TAR mutations on gene expression and virus replication. (A) The LTR-tetO promoter region of HIV-rtTA with the original (TAR^m) or mutant (A to F) TAR sequence was placed upstream of the firefly luciferase gene. To determine dox responsiveness, C33A cells transfected with these plasmid reporter constructs and an rtTA-expression plasmid were cultured at different dox levels (0 to 1,000 ng/ml). A plasmid constitutively expressing Renilla luciferase was cotransfected to correct for differences in transfection efficiency, and the ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the promoter activity. A construct with the wild-type HIV-1 LTR promoter (TAR^wt) was included as a control. Average values obtained in three transfections are shown, with the error bars indicating standard deviations. (B) SupT1 T cells were transfected with the original HIV-rtTA (TAR^m) and TAR-mutated variants (A to F) and cultured with 1 μg/ml dox for several weeks. CA-p24 levels in the culture supernatant were measured by ELISA. No virus replication was observed in the absence of dox (not shown). This experiment was repeated three times with similar results.