Article Figures & Data
Figures
- FIGURE 1.
PACT is essential for EMCV-induced activation of MDA5. (A–C) WT and PACT−/− MEFs were infected with EMCV at MOI = 0.01. Expression levels of Ifnb (A) and Ifna4 (B) mRNAs, as well as EMCV genome levels (C), were analyzed by quantitative real-time RT-PCR. The relative expression level was calculated with respect to GAPDH mRNA using the 2−ΔCT method. Results are expressed as mean ± SD (n = 3). *p < 0.05, PACT−/− versus WT at the same time point, Student t test. (D) WT and PACT−/− MEFs were infected with EMCV (MOI = 0.01) or SeV (80 HAU/ml) for 18 h. Expression levels of Ifnb mRNA were analyzed by quantitative real-time RT-PCR. (E) PACT was reintroduced into PACT−/− MEFs by ectopic expression 24 h prior to EMCV infection at MOI = 0.01. The fold induction of the Ifnb gene was calculated with respect to the GAPDH transcript in mock-treated cells at time zero. *p < 0.05, PACT versus vector at the same time point, Student t test. ND, not detected.
- FIGURE 2.
Knockdown of PACT attenuates EMCV-induced activation of MDA5. WT MEFs (A) and L929 cells (B and C) were transfected with siPACT-1, siPACT-2, or siGFP for 48 h. Endogenous PACT mRNA levels (left panels) and protein levels (middle panels), as well as relative expression levels of Ifnb mRNA (right panels), in EMCV-infected cells (MOI = 0.01). *p < 0.05, siPACT versus siGFP at the same time point, Student t test. ND, not detected.
- FIGURE 3.
PACT augments dsRNA-induced activation of MDA5. (A) HEK293 cells carrying a stably expressed IFN-β–luc plasmid were transfected with PACT and MDA5 expression plasmids for 24 h before poly(I:C) induction (1 μg/ml). A luciferase reporter assay was carried out; relative luciferase activity is shown in arbitrary units. Data are the mean and SD of three replicates. *p < 0.05, PACT + MDA5 versus PACT at the same time point, Student t test. WT and PACT−/− MEFs were induced with poly(I:C) as above and analyzed for the expression of Ifnb (B) and Ifna4 (C) mRNAs. *p < 0.05, PACT−/− versus WT at the same time point, Student t test. (D) PACT was reintroduced into PACT−/− MEFs by ectopic expression 24 h prior to poly(I:C) induction at 1 μg/ml. The fold induction of the Ifnb gene was calculated with respect to GAPDH transcripts in mock-treated cells at time zero. *p < 0.05, PACT versus vector at the same time point, Student t test. (E) HEK293 cells were rendered deficient in PACT expression by double-nicking via CRISPR/Cas9-mediated genome editing. Two positive clones (clones 1 and 2) were validated for the expression of PACT protein by Western blotting using parental untreated WT cells as control (left panel). Luciferase reporter assay of IFN-β promoter activity was performed in these cells with overexpression of MDA5 and induction by poly(I:C) (right panel). Results are representative of three independent experiments. Error bars indicate SD. *p < 0.05, clones 1/2 versus WT, Student t test. ND, not detected.
- FIGURE 4.
PACT facilitates IRF3 activation. (A) HEK293 cells were transfected with PACT and MDA5 expression plasmids, together with a luciferase construct driven by tandem copies of IRF3 (IRF3-luc; upper panel) or κB (κB-luc; lower panel) elements, for 40 h. Results are representative of three independent experiments. Error bars indicate SD. (B) HEK293 cells were transfected with IRF3 expression plasmid and increasing doses of PACT expression plasmid for 24 h followed by poly(I:C) induction (1 μg/ml). Total lysate was resolved on nondenaturing native PAGE (upper panel) to visualize IRF3 dimerization or on denaturing SDS-PAGE (lower panel) to verify protein expression. *p < 0.01, PACT + MDA5 versus MDA5, Student t test.
- FIGURE 5.
PACT interacts with MDA5, and its activation of MDA5 requires dsRNA binding. (A) HEK293 cells were transfected with MDA5 and PACT expression plasmids for 48 h. Total lysate was subjected to pull-down with poly(I:C)- or polyC-conjugated agarose beads. Bound fraction and input were analyzed by Western blotting. (B) A poly(I:C) pull-down experiment was carried out with endogenous MDA5 and PACT. HEK293 cells were mock treated or treated with rIFN-β (1000 U/ml) for 24 h before harvest. (C and D) HEK293 cells were transfected with MDA5 and PACT expression plasmids, alone or in combination, for 30 h before poly(I:C) stimulation (1 μg/ml). Coimmunoprecipitation was carried out with anti-V5 (C) or anti-FLAG (D) Ab. (E) HEK293 cells carrying a stably expressed IFN-β–luc were transfected with expression plasmids for MDA5 and either PACT-WT or its dsRNA-binding defective mutant (PACT-M1, PACT-M2, or PACT-DM) for 24 h, followed by poly(I:C) induction (1 μg/ml) for 6 h. Protein expression was validated by Western blotting (inset). *p < 0.01 versus PACT-WT, Student t test.
- FIGURE 6.
PACT facilitates MDA5 oligomerization. (A) HEK293 cells were transfected with PACT expression plasmid for 24 h followed by poly(I:C) induction (1 μg/ml). Total lysate was resolved on nondenaturing native PAGE (upper panel) to visualize endogenous MDA5 oligomerization or denaturing SDS-PAGE (lower panel) to confirm protein expression. HEK293 cells carrying a stably expressed IFN-β–luc were transfected with expression plasmids for PACT and MDA5-WT, its monomer-interface mutant (MDA5-570/572 or MDA5-841/842) (B), or its constitutively active mutant (MDA5-S88A or MDA5-R337G) (C). Error bars indicate SD (n = 3). *p < 0.01, MDA5 mutant versus MDA5-WT, Student t test.
- FIGURE 7.
PACT potentiates MDA5 recruitment to dsRNA ligand. (A) HEK293 cells were transfected with MDA5 expression plasmid, alone or together with PACT expression plasmid, and harvested for a poly(I:C) pull-down experiment. The bound fraction of poly(I:C)-conjugated beads was evaluated for MDA5 recruitment at 15 and 30 min after lysate incubation. (B) HEK293 cells were transfected with MDA5 and PACT expression plasmids, alone or in combination for 30 h, and then subjected to mock treatment or poly(I:C) induction. Coimmunoprecipitation was carried out with anti-FLAG or anti-V5 Ab. (C) JEG-3 cells were transfected with expression plasmids of MDA5 and PACT and subjected to mock treatment or poly(I:C) induction. Subcellular localization of PACT (red) and MDA5 (green) was visualized by confocal microscopy. Nuclear morphology (blue) was revealed by DAPI staining. Relative colocalization (yellow) is shown in the merged image. Condensation of the merged signal is indicated by the arrowhead. Scale bars, 20 μm.
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