Figure 1.
Genomic HIV RNA induces innate immune responses dominated by ISGs.
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.
Figure 2.
HIV RNA oligos derived from the HIV genome induce an innate immune response in human PBMCs and primary macrophages.
(A) Schematic illustration of the size, position and secondary structure of the HIV-derived RNA oligos used in this study. (B-E) PBMCs were stimulated with different RNA oligos derived from the HIV genome (2 µg/ml) or with ssRNA40 (2 µg/ml). Total RNA and supernatants were harvested after 6 and 20 h, respectively, and CXCL10 (mRNA and protein) and IFN-β (mRNA) levels were measured. Data are shown as means of triplicates +/− st.dev. Similar results were obtained in three independent experiments. (F–K) Primary macrophages were differentiated from human PBMCs and stimulated for either 2 or 6 h with HIV Tar (oligo 2, 2 µg/ml). Transfected poly(I:C) (t-pIC) was included as a positive control (2 µg/ml). Total RNA was isolated at the indicated time points and analyzed by qPCR for the presence of IFN-β (E), IFN-λ1 (F), CXCL10 (G), RIG-I (H), IL-1β (I), and TNF-α (J). Data are presented as mean values from analysis of 2 independent donors +/− st.dev. Mock, Lipofectamine 2000 alone. RU, relative units. *, p<0.05.
Figure 3.
HIV RNA activates the NF-κB, p38, and IRF signaling pathways.
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.
Figure 4.
HIV Tar co-localizes with peroxisomes but not with mitochondria.
PBMCs stimulated with 1.2 µg/ml FAM-labeled HIV-Tar RNA (green) for 180 min were labeled for mitochondria (red) or peroxisomes (red). Nuclei were stained with DAPI (blue). Co-localization is shown in yellow, scale bar 20 µm. Representative images are shown in A, while panel B shows a quantification of percentage RNA-organelle co-localization as obtained by counting more than 100 cells per group. Similar results were obtained in two independent experiments.
Figure 5.
The innate immune response induced by HIV genomic RNA or RNA oligos is dependent on RIG-I and MAVS.
(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.