FIG 8.

PU.1 inhibits the basal activity of the HIV-1 LTR in a Tat-independent manner. (A) HEK293T cells were seeded in 24-well plates and transfected with pGL3-basic (0.1 μg), pGL-LTR-Luc (0.1 μg), or pGL-LTR-Luc (0.1 μg) plus pTat101 (0.1 μg). Total amounts of transfected DNA were adjusted to 0.5 μg using empty vector DNA (pUC19) as appropriate. Luciferase production was measured 2 days posttransfection. Means and error bars representing SEM from triplicate transfections are shown. The P value for the test comparing promoter activity between pGL3-basic and pGL-LTR-Luc is 0.001. The P value for the test comparing log₁₀ LTR promoter activity between pGL-LTR-Luc and pGL-LTR-Luc plus Tat101 is <0.001. (B and C) HEK293T cells were transfected in 24-well plates with pGL-LTR-Luc (0.1 μg) in the absence or presence of Tat (0.1 μg) as well as increasing amounts of pcDNA-PU.1 (0 μg, 0.02 μg, 0.06 μg, and 0.2 μg). Luciferase production was measured 24 h later. Results are shown as absolute luciferase units (B) or as relative units (C) with the signal obtained in the absence of PU.1 being defined as 100%. Means and error bars representing SEM from 12 transfections are shown. Statistical analyses demonstrate that the effect of PU.1 on LTR promoter activity is significant in the control group (no Tat) only. For details on the statistical analyses, see Fig. S1 in the supplemental material.