Figure 4

Transcriptional activity of the HIV-1 3' LTR in the reverse orientation. (A) A map of HIV-1 3' LTR and reporter plasmids. Top panel shows results of promoter prediction of the 5'-flanking region of the ASP-L TSS by TFsearch and Genetyx. Green box indicates putative TATA box. The sequence of the putative TATA box is described in F. The bottom panels are the structures of pGL4-asLTR-1, pGL4-asLTR-2 and pGL4-asLTR-3 plasmid. (B) Promoter activities of the LTR in the antisense orientation (asLTR). pGL4-5' LTR and pGL4-asEnv plasmids were used as positive and negative controls, respectively. mock, pGL-4.10; asEnv, pGL4-asEnv; asLTRs, pGL4-asLTR-1, pGL4-asLTR-2 and pGL4-asLTR-3; 5′ LTR, pGL4-5' LTR. The luciferase activities relative to that of pGL4-5' LTR are shown. (C) Effects of Tat protein on the asLTR promoter activity. A Tat expression plasmid, pME18S-tat, was co-transfected with pGL4-asLTR-1 or pGL4-5' LTR. (D) Dose–response effects of TNF-α on the asLTR promoter activity. At 12 h post-transfection of the reporters, cells were treated with various amounts of TNF-α (0–10 ng/mL) for 12 h. (E-H) Investigation of transcriptional regulatory elements in the asLTR. (E) A schematic description of series of asLTR mutant reporters. asLTR-ΔκB lacks two NF-κB binding sites. asLTR-TATA mut1 and mut2 contain mutated TATA box. (F) Sequences of the potential TATA box mutants. (G) Promoter activities of the asLTR mutants. The luciferase activities relative to that of pGL4-5' LTR are shown. (H) Effects of TNF-α on the ΔκΒ mutant. The experimental condition is identical to D. ‘delta kB’ stands for pGL4-sLTR-ΔκΒ. ‘wild type’ stands for pGL4-asLTR-1. The mean ± S.D. of quadruplicate (B) or triplicate (C, D, G, and H) experiments are shown. The asterisks shown in C, D, G, and H indicate statistical significance.