FIG 5.
LPS- and GC-mediated repression of HIV-1 in MDMs is associated with changes in interferon regulatory factor (IRF) recruitment to the interferon-stimulated response element (ISRE). (A) MDMs were infected with a single-round, replication-defective HIV-luciferase reporter virus and, 48 h after infection, were treated with PAM3CSK4 (100 ng/ml) or LPS (100 ng/ml). At various time points after TLR stimulation, cells were harvested, lysed, and total cytoplasmic RNA was extracted. Viral RNA accumulation was assessed by RT-PCR. The data are the mean (± SD) of four donors. (B) HEK293-TLR4CFP/MD-2/CD14 cells were transfected with HIV-1 LTR/Gag-leader sequence (GLS)-luciferase reporter constructs with an intact ISRE or mutated ISRE binding site. Following transfection, cells were treated with LPS (100 ng/ml) for 18 h and then harvested and assayed for luciferase activity. Data are the mean (± SD) of three independent experiments, each performed in triplicate. (C and D) MDMs were infected with a single-round replication-defective HIV-GFP reporter virus and, 48 h after infection, cells were treated with LPS (100 ng/ml). At either 1 or 24 h after LPS treatment, cells were fixed with formaldehyde, lysed, sonicated, and subjected to immunoprecipitation with antibodies against IRF1, IRF2, IRF4, IRF8, or rabbit IgG (isotype control). Association with the HIV-1 ISRE was assessed by PCR using HIV-1 specific primers. Data from one representative donor (C). Composite data representing the mean (± SD) from five donors (D). (E) MDMs were infected as in panel C and, 48 h after infection, were treated with heat-killed GC (MOI = 10). At 24 hours after GC treatment, cells were fixed with formaldehyde, lysed, sonicated, and subjected to immunoprecipitation with antibodies against IRF1, IRF2, IRF4, IRF8, NF-κB p65, or rabbit IgG (isotype control). Association with the HIV-1 ISRE was assessed by PCR using HIV-1-specific primers. Composite data from five donors are shown. (F and G) MDMs transfected with a controlled scrambled shRNA (white bars) or with shRNA targeting IRF8 (black bars). Knockdown of protein expression was detected by Western blot (F). Transfected MDMs were infected with a single-round, replication-defective HIV-luciferase reporter virus and, 48 h after infection, were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), a combination of PAM3CSK4 and LPS (each at 100 ng/ml), or GC (MOI = 10) for 18 h. Cells were then lysed and assayed for luciferase activity (G). The experiment was performed using cells from five different donors. (H and I) MDMs transfected with an empty vector (white bars) or a vector encoding IRF8 (black bars). IRF8 protein expression was detected by Western blot analysis (H). Transfected MDMs were infected with a single-round, replication-defective HIV-luciferase reporter virus and, 48 h after infection, were treated with PAM3CSK4 (100 ng/ml), LPS (100 ng/ml), a combination of PAM3CSK4 and LPS (each at 100 ng/ml), or GC (MOI = 10) for 18 h. Cells were then lysed and assayed for luciferase activity (I). The experiment was performed using cells from four different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.