The innate immune response induced by HIV genomic RNA or RNA oligos is dependent on RIG-I and MAVS.

<p>(A) PBMCs were treated with bafilomycin A1 (0.5 µM) as indicated 15 min prior to stimulation with genomic HIV RNA, RNA oligos, or ssRNA40 (all 2 µg/ml). IFN-α was included as a positive control (10 ng/ml). Supernatants were harvested 18 h post stimulation for measurement of CXCL10. (B) BMMs from C57BL/6 wildtype and MAVS−/− mice were stimulated with genomic HIV RNA (2 µg/ml), Tar (2 µg/ml), Sendai virus (MOI 1), IFN-α (10 ng/ml), ssRNA40 (2 µg/ml), or R848 (2 µg/ml). Supernatants were harvested after 16 h and CXCL10 was measured by ELISA. UT, untreated cells. Data are shown as means of triplicates +/− st.dev. (C) Huh7, Huh7.5 (RIG-I mutant), Huh7.5 EV (empty vector), and Huh7.5 RIG-I cells were transfected with Tar RNA (2 µg/ml), or subjected to mock transfection with Lipofectamine 2000. Total RNA was harvested 6 h later and CXCL10 mRNA levels were analysed by qPCR. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone. RU, relative units *, p<0.05.</p

Genomic HIV RNA induces innate immune responses dominated by ISGs.

<p>PBMCs were stimulated for 16 h with HIV genomic RNA in increasing doses (from 0.3 to 3.0 µg/ml). Positive and negative controls included ssRNA40 (2 µg/ml) and ssRNA41 (2 µg/ml), respectively. Supernatants were harvested for measurement of (A) CXCL10, (B) IFN-β, (C) IFN-α, (D) TNF-α, and (E) IL-6. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. Mock, Lipofectamine 2000 alone.</p

HIV RNA activates the NF-κB, p38, and IRF signaling pathways.

<p>PBMCs were stimulated with HIV Tar (oligo 2, 2 µg/ml) and in panel A also with oligo 4 (2 µg/ml). (A–B) Whole-cell lysates were isolated 2 h post treatment, and (C–E) nuclear extracts were isolated at the indicated time points (IFN-γ, 10 ng/ml, 6 h; SeV, MOI 1, 6 h; R848, 500 ng/ml, 6 h). (A–B) P-IκBα and P-p38 were measured by Luminex technology and (C–E) DNA binding of IRF-1, 3, and 7 to an ISRE consensus sequence was measured by TransAM. Data are shown as means of duplicates or triplicates +/− st.dev. Similar results were obtained in two or three independent experiments. RU, relative units. Mock, Lipofectamine 2000 alone. *, p<0.05.</p