Mitochondria-localized β-actin is essential for priming innate antiviral immune signaling by regulating IRF3 protein stability

摘要

The mitochondrial pool of β-actin is required for priming innate antiviral immunity by maintaining IRF3 stability. a Gene Ontology (GO) enrichment analysis identified “defense to virus” as enriched in genes downregulated in β-actin KO MEFs. Statistics are in brackets (p value of each GO term). b Heatmap of the relative expression of ISGs (interferon-stimulated genes) in WT (n = 4 biological replicates) and KO (n = 3 biological replicates) MEFs; scale bar: Log2 (counts per million reads). c Scheme of the canonical cytosolic nucleic acid sensing pathway in innate antiviral immunity. Genes labeled red were downregulated in KO cells in comparison to their expression in WT cells. d Heatmap of significantly downregulated (FDR < 0.05) sensors or signal transducers in KO cells. e Heatmap of the relative expression of all Irf genes in WT and KO cells. f qPCR quantification of the Ifna4, Ifnb1, and Cxcl10 (IP10) genes in WT and KO cells with or without poly(dA-dT) stimulation. g qPCR quantification of the Ifna4, Ifnb1, and Cxcl10 (IP10) genes in WT and KO cells with or without poly(I:C) stimulation. Statistics in f and g: mean ± SEM, n = 3 biological replicates, representative of three independent experiments). **P < 0.01, ***P < 0.001 by unpaired two-tailed Student’s t-test. h Heatmap of the expression levels of ISG genes in WT and KO cells with or without poly(dA-dT) stimulation by RNA-seq analysis. Three biological replicates of each condition were analyzed. The violin plot shows the distribution of the net increase in the expression of ISGs in poly(dA-dT)-treated and mock-treated KO and WT cells, respectively. Statistics: The Wilcoxon signed-rank test was used to show difference in the net increase of ISGs between WT and KO cells, ***P < 0.001. i Western blot showing the reduced IRF3 protein level in KO cells in mock- and poly(I:C)-stimulated conditions detected by two different antibodies from Abcam and Cell Signaling Technology (CST). j IRF3 protein decay assay: The same number of WT or KO cells were treated with cycloheximide (150 µg/ml) for 0, 2, 4 and 6 h. IRF3 and β-tubulin protein levels in total cell lysates at each time point were determined by western blotting. The band intensity was normalized to that at the 0-h time point. n = 3 independent experiments. Statistics: mean ± SEM, two-way ANOVA with Bonferroni post-hoc test: **P < 0.01, ***P < 0.001. k Control cells (DMSO-treated) or cells treated with 20 mM β-NAD for 18 h were stained with 0.5 µM TMRE. The mitochondrial membrane potential (MMP) shown by TMRE staining was measured by FACS. Data are the summary of three biological replicates representative of two independent experiments. Statistics: mean ± SEM, one-way ANOVA with Bonferroni post-hoc test: **P < 0.01, ***P < 0.001. l The relative IRF3 protein levels in control or β-NAD-treated cells were determined by western blotting. Data are the summary of three independent experiments. Statistics: mean ± SEM, one-way ANOVA with Bonferroni post-hoc test: *P < 0.05, **P < 0.01. m Schematics of retroviral vector containing GFP (negative control), Actb (β-actin gene), Actb with SV 40 nuclear localization signal (NLS) and Actb with COX4 mitochondrial targeting sequence (MTS). The GFP, Actb, ActbNLS, and ActbMTS constructs were introduced into KO cells by retroviral transduction, and cells expressing the rat CD8a surface transduction marker were sorted by FACS. IRES: internal ribosome entry site. n Relative IRF3 protein levels in KO cells stably expressing GFP (KO::GFP), Actb (KO::Actb), ActbNLS (KO::ActbNLS) and ActbMTS (KO::ActbMTS). Data are the summary of three independent experiments. Statistics: mean ± SEM, one-way ANOVA with Bonferroni post-hoc test: **P < 0.01. o qPCR quantification of the Ifna4, Ifnb1, and Cxcl10 (IP10) genes in mock cells or cells stimulated by poly(I:C). Data following poly(I:C) stimulation are the summary of four independent experiments, and mock data are the summary of two experiments. Statistics: mean ± SEM, one-way ANOVA with Bonferroni post-hoc test: *P < 0.05, **P < 0.01