Figure 4

hNAP-1 cooperates with Tat in LTR transactivation. A. hNAP-1 synergizes with Tat in transcriptional activation. HeLa cells were cotransfected with a reporter construct containing the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. B. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. C. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. D. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.