HIV-1 infection specifically induces viperin in MDMs without IFN induction. (A) HIV-1 infection of MDMs does not induce type 1 IFN. MDMs were treated with HIV-1 or AT2–HIV-1. IFN-α and IFN-β levels in the supernatants were determined by ELISA (R&D Systems). As a positive control for IFN induction, PBMCs and plasmacytoid DCs (IFN-α) or MDDCs (IFN-β) were treated with HSV-2₁₈₆. Data are representative of 3 experiments. (B) HIV-1 infection does not degrade IRF3. A representative Western blot of IRF-3 expression in MDMs on days 2 and 4 after treatment with HIV-1 at an MOI of 1 compared with mock treatment using an anti-IRF3 and GAPDH antibodies. (C) HIV-1 inhibits IRF3 translocation from cytoplasm to nucleus. IRF3 cellular localization was examined in mock- and HIV-1–treated MDMs at 48 hours. TZM-bl cells infected with Sendai virus were used as a positive control for the nuclear IRF3 translocation. Fluorescently labeled cells were visualized with an inverted Olympus IX-70 microscope (DeltaVision Image Restoration Microscope; Applied Precision/Olympus) using a numerical aperture oil immersion lens (1.4 or 1.43) and a photo-metrics CoolSnap QE camera. The representative z-series were deconvoluted, the pictures overlaid; colocalization and significance were performed using SoftWoRx software (Version 3.4.5; Universal Imaging Corporation) as described previously.²⁸  Images shown are representative of n = 20 cells. (D) HIV-1 does not lead to phosphorylation of IRF3. A representative Western blot of phospho-IRF-3 expression in MDMs after 3, 6, 9, 12, 18, and 24 hours after treatment with HIV-1 at an MOI of 1 compared with lipopolysaccharide stimulated (1 μg/mL) MDMs using antiphospho-IRF3 and GAPDH antibodies. (E) Kinetics of mRNA viperin induction by HIV-1 compared with IFN-α. MDMs were treated with either HIV-1 at an MOI of 0.25 or IFN-α/2β (500 U/mL). RNA was extracted at different time points, reverse transcribed, and viperin mRNA expression was assessed by quantitative PCR relative to GAPDH. The mean data from 3 experiments are shown with SE bars. (F) Inhibitors of HIV-1 binding, fusion, and entry inhibit viperin expression in MDMs. MDMs were either infected with HIV-1 only or treated with a combination of the fusion inhibitor T20 (1 mg/mL), soluble recombinant CD4 (NIH), and the anti-CCR5 (CD195, BD Biosciences PharMingen) antibodies at 20 μg/mL each, then infected. Total RNA was harvested, and viperin expression was measured by quantitative PCR relative to GAPDH. The mean data from 3 experiments are shown with SE bars.