Figure 2

N-terminal mutant CycT1 proteins (CycT1-U7) exhibit a strong dominant negative effect on HIV transactivation. A. CycT1-U7 cannot support Tat transactivation in murine cells. NIH 3T3 cells were transfected with HIV-Luc reporter gene in the presence (lane 2–6) or absence (lane 1) of Tat (0.1 μg) with or without increasing amounts (0.2 and 0.5 μg) of wt human CycT 1–280 (lanes 3 and 4) or CycT1-U7 (lanes 5 and 6). Twenty-four hours after transfection, luciferase activities were measured as described before. B. CycT1-U7 shows strong dominant negative effects on Tat-transactivation. Increasing amounts of CycT1-U7 (0.2, 0.4 and 0.6 μg) were transfected in HeLa/pHR-Luc cells in the presence of Tat (0.02 μg). Luciferase activities were measured as described above. C. CycT1-U7 was unable to inhibit basal HIV transcription and CMV-driven transcription. The plasmid (0.6 μg) encoding CycT1-U7 (gray bars) or an empty vector (black bars) was co-transfected in HeLa cells with HIV-LTR-Luciferase or CMV-Luciferase reporter plasmid (0.05 μg) in the absence of Tat. Luciferase activity was measured as described above. D. Tat has lower activity on HIV-LTR in cells stably expressing CycT1-U7. Increasing amounts of Tat were transfected in Hela/pHR-Luc cells stably carrying a lentiviral vector encoding no protein (empty vector; gray diamonds) or CycT1-U7 proteins (black triangle). Luciferase activities were measured as described in the Materials and Methods section. Error bars represent the standard deviation of triplicate measurements. Data are representative of four independent assays.