Journal: PLOS Biology
Article Title: Expression of intron-containing HIV-1 RNA induces NLRP1 inflammasome activation in myeloid cells
doi: 10.1371/journal.pbio.3003320
Figure Lengend Snippet: (A) Flow cytometry analysis (left panel) of MDMs infected with WT or mutant (M10 or M10-4xCTE) Lai∆envGFP/G viruses (MOI 1) in the presence of SIV3 + /Vpx VLPs, compared to mock (no virus infection). % infection (GFP + ) in MDMs was quantified at 3 dpi (right panel). (B) Immunoblotting analysis of whole cell lysates from MDMs infected with HIV-1 (WT, M10, or M10-4xCTE constructs) in the absence (untreated, DMSO) or presence of RT inhibitor (EFV, 1 μM) at 3 dpi. HIV-1 proteins (p55 Gag and p24 Gag ) were visualized using anti-HIV-1 IgG, β-actin was used as a loading control. (C) RT-qPCR quantification of unspliced (usRNA), and multiply-spliced (Tat/Rev) transcripts (msRNA) ( D ) in whole cell lysates from MDMs infected with WT, M10, or M10-4xCTE virus ± EFV, at 3 dpi compared to uninfected (mock) lysates. HIV-1 unspliced and spliced transcripts were quantified by RT-qPCR. Production of bioactive type-I IFN (E) , and secretion of IL-1β ( F ) in culture supernatants from MDMs infected with WT, M10, or M10-4xCTE virus ± EFV were harvested at 3 dpi and quantified using type I IFN bioassay or ELISA, respectively. (G, H) Transient knockdown of MAVS, STING and UNC93B1 by siRNA transfection in primary MDMs. Knockdown efficiency was quantified by RT-qPCR (G) at day 2 post-transfection and reported as relative expression normalized to that observed with scramble siRNA (siControl), and protein level analyzed by immunoblotting (H) . siRNA-transfected MDMs were infected with Lai∆envGFP/G (MOI 1) for 3 days (I–L) . Culture supernatants were harvested and analyzed at 3 dpi for p24 Gag (I) , IL-1β (J) , and IP-10 (K) secretion by ELISA. (L) Bioactive type I IFN in the culture supernatants from infected MDMs at 3 dpi quantified using interferon bioassay. The values were normalized to that of mock-infected control in each donor. The means ± SEM are shown, with each symbol representing an independent donor. The means ± SEM are shown, and each symbol represents an independent donor. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparisons (C, D) , or Dunnett’s post-test, comparing HIV-infected MDMs to uninfected (mock) control (E, F) , or with specific siRNAs to that from siControl (K, L) . P -values: **** < 0.0001; *** < 0.001; ** < 0.01; * < 0.05; ns: not significant ( p ≥ 0.05). The data underlying this figure can be found in .
Article Snippet: At day 2 post-transfection, cells were either infected with HIV-1 (as described above), or stimulated as follows to determine functional knockdown: AIM2 (cells were treated with ultra-pure LPS (100 ng/m, Invivogen) for 2 h, followed by transfection with linearized DNA (1 μg/mL) for 4 h); NLRP1 and CARD8 (cells were primed with Pam3CSK4 (0.5 μg/mL, InvivoGen) for 4 h, followed by stimulation with Val-boroPro (10 μM, InvivoGen) for 24 h; NLRP3 and caspase-1 (cells were primed with ATP (5 mM, Thermo Scientific) for 6 h, followed by activation with nigericin (10 μM) for 60 min; Caspase-4 (cells were transfected with ultra-pure LPS (5 μg/ml, InvivoGen) for 6 hrs; UNC93B1 (cells were treated with Resiquimod (5 μg/ml, Invivogen) for 24 h. At the indicated timepoints, culture supernatants were harvested for IL-1β and IP-10 ELISA, and cells were lysed for RNA or protein analysis.
Techniques: Flow Cytometry, Infection, Mutagenesis, Virus, Western Blot, Construct, Control, Quantitative RT-PCR, Bioassay, Enzyme-linked Immunosorbent Assay, Knockdown, Transfection, Expressing
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