Figure 6.

HspBP1 inhibits NF-κB mediated activation of HIV-1 transcription by obstructing p65-recruitment on LTR promoter. (A) HspBP1 competes with p65 for regulation of LTR driven gene-expression. HEK293T cells were transfected with indicated plasmids. Luciferase assays were performed 36 h post-transfection. (B) HspBP1 inhibits p50/p65-mediated activation of LTR promoter in a dose-dependent manner. HEK293T cells were co-transfected with the indicated plasmids. HspBP1-construct was transfected in three doses (0.25–0.75 ug). Luciferase assays were performed 36 h post-transfection. Expression of various constructs used in the assay was confirmed by immunoblotting. (C) HspBP1 over-expression diminishes the occupancy of p65 at LTR promoter. HEK293T cells were co-transfected with either control DNA and pNL4-3 or HspBP1 and pNL4-3. 48 h post-transfection, cells were fixed and chromatin was prepared followed by immunoprecipitation with p65 antibody or IgG control antibody. qRT-PCR analysis was performed with indicated primers spanning three different regions of LTR promoter. (D) HspBP1 knockdown causes an increase in p65-mediated induction of LTR promoter. HEK293T cells were transfected with either control siRNA or HspBP1 siRNA. 24 h post transfection, LTR promoter, Tat and p65 were over-expressed as indicated and luciferase assay was performed after 24 h of second transfection. Results represent data from three experiments and presented as the mean ± SD (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001).