Fig. 5.

Tat activates gene expression via U3 elements. HIV-rtTA-Tat^wt (wt) and HIV-rtTA-Tat^stop (stop) constructs with different 5′-LTR-promoter configurations were cotransfected with 0 to 50 ng of pTat into 293T cells. CA-p24 levels in the culture supernatants were measured after the cells were cultured with dox for 48 h. Average values obtained in multiple experiments are shown, with error bars indicating standard deviations. (A) HIV-rtTA promoter configuration with two tetO sites placed between the NF-κB and Sp1 binding sites (n = 12 to 16). (B) ΔNF constructs in which the NF-κB sites and upstream U3 region were deleted (n = 3). (C) In ΔNF ΔTAR constructs, the TAR hairpin was replaced with the unrelated ER3 hairpin (n = 6) (22). (D) tetO-CMV constructs in which the 5′ U3 and TAR regions were replaced by a promoter sequence consisting of three tetO elements coupled to a 30-bp minimal TATA box region from the CMV IE promoter and a hairpin that is based on the +1/+16 region of the CMV promoter (n = 12 to 16). (E) In the tetO-CMV-Sp1 constructs, the HIV-1 Sp1 sites were cloned between the tetO and TATA box regions of tetO-CMV (n = 3). All constructs have the tetO-CMV configuration in the 3′ LTR. A two-sided unpaired sample t test was used to identify statistically significant differences (P < 0.01) between the wt and corresponding stop constructs at 0 ng of pTat (^a, asterisk indicates a statistically significant difference between the wt and stop constructs) and between the construct at 0 ng of pTat and the same construct at 0.5, 5, or 50 ng of pTat (^b, asterisk indicates a statistically significant difference between the constructs without and with pTat). When no asterisk is shown, the difference was not statistically significant (P > 0.01). CA-p24 production of all HIV-rtTA constructs was very low (>10-fold reduced) in the absence of dox (data not shown).