FIG 1.
Treatment with purified Toll-like receptor (TLR) ligands alters HIV-1 replication at the level of transcription. (A) Monocyte-derived macrophages (MDMs) were infected with a single-round, replication-defective HIV-luciferase reporter virus and, 48 h after infection, were treated with the TLR2 ligand PAM3CSK4 (100 ng/ml), the TLR3 ligand poly(I·C) (25 μg/ml), the TLR4 ligand lipopolysaccharide (LPS) (100 ng/ml), or the TLR5 ligand FLA-ST (100 ng/ml) for 18 h. The cells were then lysed and assayed for luciferase activity. Bars represent the mean (± standard deviation [SD]) of 11 donors, each donor tested in triplicate. (B to D) MDMs were infected as described above. At 48 h after infection, cells were treated with PAM3CSK4 (100 ng/ml) or LPS (100 ng/ml) for 6 h (B and C) or 24 h (D). Cells were then lysed and assayed for viral RNA accumulation by reverse transcription-PCR (RT-PCR). Shown are data from one representative donor at 6 h (B) and composite data from eight donors at 6 h (C) and four donors at 24 h (D). (E) MDMs were infected as in panel A. At 48 h after infection, cells were treated with the TLR4 ligand LPS (100 ng/ml) for 4 h. Cells were then treated with actinomycin D (10 μg/ml) to inhibit transcription. Total cytoplasmic RNA was prepared from the treated cultures at the indicated time points following actinomycin D treatment and analyzed by reverse transcription-quantitative PCR (RT-qPCR) for the expression of HIV-1 RNA. The data are the means (± SD) from four donors. (F) MDMs were infected and treated with TLR ligands as in (A). The cells were then lysed and assayed for luciferase activity. Bars represent the mean (± SD) of five male donors and five female donors; each donor was tested in triplicate. Although virus replication was decreased overall in MDMs from female donors compared to MDMs from male donors, it was activated by treatment with PAM3CSK4 and FLA-ST and repressed by poly(I·C) and LPS, in a manner similar to that seen in MDMs from male donors. (G to H) MDMs were infected as in panel A, and, 48 h after infection, cells were treated with PAM3CSK4 (100 ng/ml) in the presence or absence of 10 μM BAY 11-7082 (G) or 10 μM celastrol (H) for 18 h. The cells were then lysed and assayed for luciferase activity. The data are the mean (± SD) of three donors (BAY 11-7082) or six donors (celastrol); each donor was tested in triplicate. (I) HEK293-TLR2CFPTLR1YFP cells were transfected with HIV-1 long terminal repeat (LTR)-luciferase reporter constructs with intact NF-κB, mutated NF-κB, or deleted NF-κB binding sites. Following transfection, cells were treated with PAM3CSK4 (100 ng/ml) for 18 h and then harvested and assayed for luciferase activity. Data represent the mean (± SD) of three independent experiments, each performed in triplicate. (J) MDMs were infected as in (A), and 48 h after infection, cells were treated with PAM3CSK4 (100 ng/ml) in the presence or absence of U0126 (10 μM), PD98059 (50 μM), or SB203580 (10 μM) for 18 h. Cells were then lysed and assayed for luciferase activity. The data are the mean (± SD) of six donors; each donor was tested in triplicate. (K) HEK293-TLR2CFPTLR1YFP cells were transfected with HIV-1 LTR-luciferase reporter constructs with intact AP-1 sites (wild-type [WT] LTR) or deleted AP-1 binding sites (−158 LTR). Following transfection, cells were treated with PAM3CSK4 (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. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.