FIG 6.

NKILA mutants fail to abolish HIV-1 transcription and LTR promoter activity. (A) Schematic representation of NKILA with mutations at each of the hairpins (hairpins A, B, and C). (B) NKILA WT, the indicated mutants, or negative-control vector were cotransfected with pNL4-3ΔEnv and pVSV-G plasmids into HEK293T cells. Cells and partial supernatants were harvested for measurement of HIV-1 gene expression by IB analysis. (C) Supernatants containing virus-like particles (VLPs) were collected and used to infect TZM-bl cells. Viral infectious yield was measured by a luciferase reporter assay. (D) NKILA mutation completely abolished the ability to inhibit Tat with or without p65-mediated LTR promoter activity. NKILA WT or the indicated mutants were cotransfected with Tat or with both Tat and p65 plus pHIV-1-luciferase plasmids. Forty-eight hours posttransfection, reporter gene expression was analyzed by a luciferase reporter assay, and the expression of proteins was assessed by IB analysis. The corresponding value in the absence of Tat and p65 was set as 1 or 100% as appropriate. All results are representative of three independent experiments. The data are presented as the means ± SDs. **, P < 0.01; ***, P < 0.001.