FIG 2.

HIV-C mediates efficient type I IFN responses via a MAVS/IRF3/NF-κB signaling axis. (a) Representative IB analyses for one donor of full-length MAVS (FL) and mini-MAVS 9 h after infection of DCs with differentially opsonized HIV-1 (HIV and HIV-C) strains BaL and YU-2. (Top) Representative IB using ERK1/2 as a loading control for IB; (bottom) quantitative results from 3 donors. Statistical significance was analyzed using GraphPad Prism software with one-way ANOVA and Tukeýs posttest. (b) MAVS aggregation was shown under nonreducing conditions in cells from three independent donors using 8% acrylamide native gels in DCs left untreated (iDC) or exposed for 9 h to HIV or HIV-C. IB analyses of phosphorylated IRF3 and activated NF-κB, recognizing the NLS of human p65, are shown. Tubulin was used as a loading control. Quantitative analyses of activated NF-κB from 4 donors are also depicted, and statistical significance was analyzed using unpaired Student's t test. (c) (Left) Confocal microscopic analyses of MAVS (orange) and HIV (pink) in iDCs and DCs infected with HIV or HIV-C-mCherry for 9 h. Representative 3D overviews are presented, and the experiment was repeated three times independently. (Right) Spot analyses were performed using RMS spot analysis in the Harmony software (Perkin Elmer), and HIV/MAVS colocalizing spots are illustrated from 5 independent areas and 300 cells in total. Statistical analysis was performed using GraphPad Prism software and unpaired Student's t test. (d) RT-PCR analyses of type I IFN (IFN-β) levels after silencing (siRNA) of MAVS expression in moDCs (MAVS siRNA). A control siRNA and moDCs without siRNA served as controls. Data are means and SD for analyses with cells from 4 donors, done in duplicate. A highly significant reduction in IFN-β was observed in DCs treated with MAVS siRNA and infected with HIV-C (red) compared to controls (control siRNA and no siRNA). One-way ANOVA with Tukeýs posttest was performed (*, P < 0.05; ***, P < 0.001).