Fig 1: Early recruitment of CD4+T cells and antiviral factors.CD4+ T cell recruitment to FGT was monitored by quantitative IHC image analysis. CD4 recruitment was correlated with vRNA (A) and vDNA (B) within the FGT. Representative images of CD4+ T cell recruitment (brown) are shown during local (C), transitional (D), and systemic (E) infection with macrophages (red). RNAscope was used to localize and identify the cell morphology of vRNA+ cells in the FGT from animals displaying local (F), transitional (G), and systemic (H) infection. The cell morphology is consistent with CD4+ T cells being the dominant target cell following vaginal transmission. Scale bars, 100 µm. Quantitative image analysis was used to identify the increase in Mx1 (I) and APOBEC3G (J) expression during progressive infection, which correlated to CD4+ T cell recruitment. The relative expression levels of Mx2, APOBEC3G, Tetherin, and TRIM5a were quantified together in SIV-negative or SIV-infected CD4+ cells using fluorescent confocal microscopy and expressed as the median fluorescence intensity (MFI) per T cell. Neither transitional nor systemic animals exhibited any difference in the MFI between SIV-positive and SIV-negative cells (K).
Fig 2: A whole-genome CRISPR/Cas9 screen to identify new HIV-1 inhibitors A Screen strategy. GeCKO cell populations (obtained by transduction of T98G/Cas9 cells with GeCKO v2 LV library) were IFN-treated, challenged with HIV-1 LVs coding for an antibiotic resistance gene and selected. Three rounds of IFN treatment, infection and selection were performed. Genomic DNAs of initial GeCKO and 3-time selected populations were extracted, the sgRNA-coding sequences amplified and sequenced.B Candidate gene identification. MAGeCK computational statistical tool (Li et al, 2014) was used to establish a Robust Rank Aggregation (RRA) score for each gene based on sgRNA enrichment and number of sgRNAs per gene. Genes belonging to the type 1 IFN response pathway (in blue) and DDX42 (in red) are shown (respective ranks into brackets) for 2 independent screens (the results of which were merged in the analysis). The dashed line indicates the significance threshold.C Candidate validation. T98G/Cas9/CD4/CXCR4/Firefly KO populations were generated for the 25 top hits of each screen. The control (CTRL) condition represents the mean of 4 negative CTRL populations, obtained with 4 non-targeting sgRNAs; IFNAR1 and MX2 KO populations were used as positive controls. KO cell populations were treated with IFN and infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and luciferase signals were measured 30 h later (Renilla signals were normalized to Firefly). IFN inhibition (i.e. ratio of untreated / IFN-treated conditions) was calculated and set at 100% inhibition for CTRL. Data from technical duplicates are shown.
Fig 3: DDX42 inhibits HIV-1 infection independently of the IFN response A Cell viability of siRNA-transfected U87-MG/CD4/CXCR4 cells was assessed 72 h post-transfection by measuring ATP levels. Data represent the mean ± S.E.M of 3 biological replicates.B U87-MG/CD4/CXR4 cells were transfected with siRNAs, pre-treated or not with IFN and infected with WT HIV-1 (NL4-3) in the presence or not of reverse transcription inhibitors (AZT/3TC). 48 h later, cells were lysed, RNA extracted and RT-qPCR analysis performed to measure HIV-1 RNAs. Data represent the mean ± S.E.M of 4 biological replicates. Two-way ANOVA on log-transformed data with Sidak's test.C Supernatants from (B) were harvested 48 h post-infection and AlphaLisa used to measure CA p24Gag production. Data represent the mean ± S.E.M of 4 biological replicates. Two-tailed, unpaired t test.D RT-qPCR analysis was performed RNA samples from (B) to measure relative expression of the ISG OAS1, ISG15, and MX1 (normalized to actin and GAPDH). Data represent the mean ± S.E.M of 4 biological replicates. Two-way ANOVA with Dunnett's test.E U87-MG/CD4/CXCR4 cells were transduced to express Cas9 and sgRNAs either targeting nothing (CTRL) or IRF9 and STAT1 (IRF9/STAT1). After 2 weeks, cells were treated or not with IFN, and, 48 h later, IFITM3 and MX2 induction was analyzed by immunoblotting, Actin served as a loading control. A representative immunoblot is shown.F siRNA-transfected MDMs were harvested 48 h post-transfection for RNA extraction and quantification of DDX42 mRNA levels by RT-qPCR. Actin and GAPDH were used as endogenous controls. Data represent the mean ± S.E.M of 3 biological replicates performed with cells from different blood donors (parallel samples from Fig 2E). One-way ANOVA with Dunnett's test.G U87-MG/CD4/CXCR4 cells were transduced with lentiviral vectors expressing either Firefly (negative control), WT DDX42 (WT) or a motif I point mutant, which has an impaired ATPase activity (K303E). Transduced cells were infected with HIV-1 Renilla (NL4-3/Nef-IRES-Renilla) and the infection efficiency was assessed 24 h later by measuring Renilla activity. Data represent the mean ± S.E.M of 3 biological replicates. Two-way ANOVA on log-transformed data with Dunnett's test. Data information: P values are denoted as follow: ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.
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