Figure 1
From: SMARCA2-regulated host cell factors are required for MxA restriction of influenza A viruses
RNAi and proteomic screens identify SMARCA2 as potential MxA co-factor. (a) For the RNAi screen A549-MxA cells were transfected in triplicate with siRNA library pools in 384-well plates for 72 hours. Cells were then infected with H5N1-RL virus (MOI of 8) and luciferase activity determined 24 h later. (b) For the proteomic screen A549-Strep-MxA and A549-Strep-GFP cells were treated with 1 μg/mL of doxycycline for 24 hours to induce the expression of streptavidin-tagged MxA (Strep-MxA) or GFP (Strep-GFP). Then, cells were infected with H5N1-RL (MOI of 8) for 24 hours and Strep-tagged proteins were affinity purified and analyzed by mass spectrometry. (c) Results from (a) and (b) were analyzed using Ingenuity® Pathway Analysis (IPA®, Qiagen) and a core set of 13 enriched factors was identified. (d) The protein-protein interaction network of the 13 factors identified through bioinformatic analysis. (e) The effects of SMARCA2, ZC3HAV1 and CASP9 depletion on virus replication was assessed in A549-MxA cells, A549 wild type cells (A549-wt) or A549-shMxA cells. Cells were transfected with a non-targeting control siRNA (NT) or with one of four siRNAs targeting MxA (MX1), CASP9, SMARCA2 or ZC3HAV1. 72 h post transfection cells were infected with H5N1-RL or H7N7-RL (MOI of 0.3) and 24 h later virus reporter activity was measured. The heatmap indicates increased (green) or decreased (red) viral replication as compared to the NT control siRNA (white) and demonstrates the data of 3 independent experiments (4 technical replicates per experiment). (f) siRNA depletion in A549-MxA cells was performed as described above for MX1, SMARCA2 (the most efficient siRNA: GE Dharmacon D-017253-01) or a combination of MX1 and SMARCA2. 72 h post transfection cells were infected with A/Thailand/1(KAN-1)/2004 (H5N1) (MOI of 0.001). At 36 h post infection virus titers were determined by plaque assay. Error bars indicate the standard error of the mean of three independent experiments. Student’s t-test was performed to determine the P value. *P < 0.05, **P < 0.01. Expression of SMARCA2, MxA, NP and Actin was determined by western blot. Full-length blots are presented in Supplementary Figure S8a and S8b. Log2FC = Log2 Fold Change.
