FIGURE 6.

Nef inhibits LPS-induced IL-12p40 promoter activity through NFκB in THP-1 cells. A, the nucleotide positions corresponding to the NFκB, AP-1 and other transcription factors in 5′-terminal truncated human IL-12p40 promoter sequence are indicated. The binding motifs of transcription factors are shown in capital letters, whereas the coordinated mutants are shown in lowercase letters. B, LPS-induced IL-12p40 expression is regulated by the NFκB and AP-1 transcription factors in THP-1 cells infected with the control retroviruses. 2 × 10⁶ THP-1 cells were infected twice with pSRα retroviruses for 24 h followed by transient transfection with 1 μg of various IL-12p40 promoter/luciferase constructs containing either wild-type, mutant NFκB or AP-1 binding sequences, or control reporter vector plus 0.5 μgof β-galactosidase, an internal control, for 24 h. Cells were harvested following LPS (1 μg/ml) stimulation for another 24 h and lysed for analysis of luciferase activity. C, loss of IL-12p40 promoter activity by pSRα-Nef through NFκB binding motifs. THP-1 cells (2 × 10⁶) were infected with pSRα-Nef twice for 24 h each following which cells were transiently transfected with 1 μg of IL-12p40 promoter/luciferase constructs containing either AP-1 or NFκB binding motif plus 0.5 μgof β-galactosidase for 24 h. Cells were harvested following LPS (1 μg/ml) stimulation for another 24 h and lysed for analysis of luciferase activity. Luciferase activities were normalized with the baseline activity of control vector and the β-galactosidase activity. Results shown in B and C are represented as a mean ± S.D. of three independent experiments.