Figure 2

ADAP and SLP-76 regulates HIV-1 LTR transcription in a CD28- and NF-κB-dependent manner. (A) The pLTR-gag3-flag-luc luciferase reporter plasmid was transfected together with GFP or SLP-76-GFP into Jurkat, SLP-76 deficient J14 cells (upper left panels, P = 0.0112 left; P = 0.002 right) or ADAP deficient JDAP cells (lower left panel, P = 0.0145). Alternatively, this reporter plasmid was transfected into Jurkat or JDAP cells (upper right panel, P = 0.0203). These cells were stimulated with or without anti-CD3/CD28 to measure HIV-1 LTR transcription. (B) EMSA assay and immunoblotting with anti-IκBα showed that the loss of ADAP markedly decreased anti-CD3/CD28-induced NF-κB activation. The absolute integrated OD values of each band were analyzed by Gel-Pro (left panel). (C) Plasmids expressing GFP or ADAP/GFP or M12/GFP were transfected together with the luciferase reporter plasmid into Jurkat cells to assess the luciferase activity after anti-CD3/CD28 (left panel, P < 0.0001) or anti-CD3/CD80-Fc (right panel, P < 0.05) stimulation. The NF-κB activation was examined by an EMSA assay (middle panel). (D) Human primary PBLs were transfected with GFP or ADAP/GFP or M12/GFP together with the luciferase report plasmid to measure HIV-1 LTR transcription (P < 0.01). (E) CD28 deficient Jurkat cells (upper panel) or wild type Jurkat cells (lower panel) were stimulated by anti-CD3/CD28 with or without anti-LFA-1 blocking to check HIV-1 transcription (P > 0.05). (F) Jurkat cells expressing GFP or ADAP/GFP were treated with indicated specific inhibitors, followed by a measurement of the HIV-1 mRNA levels. (G) Human primary PBLs were transfected with GFP or ADAP/GFP, stimulated by anti-CD3/CD28 with or without the src kinase inhibitor PP2 to measure HIV-1 LTR transcription (NS/not significant; P = 0.1465). Three independent experiments were performed and the representative data are shown with error bars (*p = <0.05, **p = <0.01).