MAVS is energized by Mff which senses mitochondrial metabolism via AMPK for acute antiviral immunity

Fig. 1. Mff is required for MAVS-mediated antiviral signaling. a, b IFN-β (a) and IL-6 (b) secretion in WT and Mff KO MEFs. Cells were transfected with poly(I:C)-LMW (WT, KO: n = 6, 5 in a; n = 6 in b), poly(I:C)-HMW (n = 8), or poly(dI:dC) (WT, KO: n = 4, 5 in a; n = 8 in b), or cultured with LPS or palmitate (n = 6) for 8 h. P values; two-tailed unpaired t test. c Scheme of the innate immune cascades examined in this study. Defects of innate immune response were observed around MAVS under Mff deficiency. d–f qRT-PCR analyses of IFN-β (d), IL-6 (e), and IFN-α (f) mRNA in WT and Mff KO MEFs transfected with poly(I:C)-LMW (1 μg/mL) for the indicated times. P values; two-tailed unpaired t test (n = 3). The blue shaded parts of the graphs (0–8 h) represent the acute phase after poly(I:C), and the red shaded parts (8–24 h) represent the tolerant phase. g–j Immunoblot analyses of IRF3, TBK1 (g) and NF-κB (h) phosphorylation as in d–f. Phosphorylation levels of IRF3 and TBK1 were normalized to its total protein levels. P values; two-tailed unpaired t test (2–24 h; n = 3) or Mann–Whitney’s U test (0 h; n = 4). k, l qRT-PCR analysis of IFN-β mRNA in WT and Mff KO HeLa cells (k) or in HeLa cells…

Fig. 2. Mff regulates accumulated distribution of MAVS on mitochondria. a Confocal microscopic images of MAVS and Mff. WT, Mff KO, and Drp1 KO MEFs were immunostained with anti-MAVS (green) and anti-Mff (red) antibodies. b Confocal microscopic images of MAVS and mitochondria. WT and Mff KO MEFs were stained with MitoTracker Red (mitochondria; red) and immunostained with an anti-MAVS antibody (green). c Confocal microscopic images of MAVS and Drp1. WT and Mff KO MEFs were immunostained with anti-MAVS (green) and anti-Drp1 (red) antibodies. Nuclei were stained with Hoechst 33342 (blue) in a–c. Scale bars: 10 μm (whole) and 2 μm (magnified) in a–c. d Co-localization ratio between MAVS and mitochondrial tubules (mitoRFP) in WT and Mff KO MEFs. P value; two-tailed unpaired t test (n = 6). e Co-localization ratio between MAVS and Mff or Drp1 in WT MEFs. P value; two-tailed unpaired t test (n = 4). f Working hypothesis for the accumulated distribution of MAVS and the mitochondrial antiviral response regulated by Mff. Data represent means ± SEM examined over more than three biologically independent samples (d, e). All microscopic analyses were repeated independently more than three times with…

Fig. 3. Mff regulates MAVS-mediated antiviral response independent of mitochondrial fission. a Scheme of murine Mff deletion constructs. The amino acid numbering scheme used in the constructs corresponds to splice isoform 8 of Mff13. TM: transmembrane domain. b Confocal microscopic images of Mff and mitochondria. Mff KO MEFs were transfected with expression vectors for FLAG-tagged Mff (WT), Mff (ΔC20), or Mff (ΔN100), stained with MitoTracker Red (mitochondria; red), and immunostained with an anti-FLAG antibody (green). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. c qRT-PCR analysis of IFN-β mRNA in WT MEFs, Mff KO MEFs, and Mff KO MEFs transfected with an empty vector or expression vectors for FLAG-tagged Mff (WT), Mff (ΔC20), or Mff (ΔN100). Cells were transfected with or without poly(I:C)-LMW (1 μg/mL) for 8 h. Data represent means ± SEM from three independent experiments (vector, WT, ΔC20, ΔN100: n = 6, 4, 4, 5, P value; two-tailed unpaired t test). d Immunoblot analyses of endogenous and exogenous Mff and Mff phosphorylation in the cells listed in c. The indicated letters (a, b, c) correspond to the endogenous Mff bands observed in WT MEFs. e Confocal microscopic images…

Fig. 4. The acute antiviral response is decreased in AICAR-treated cells with Mff phosphorylation. a WT MEFs were treated with the AMPK activator AICAR (2 mM) for the indicated times. Whole-cell lysates were analyzed by immunoblotting using antibodies against the indicated proteins. The indicated letters (a, b, c) correspond to the endogenous Mff bands observed in WT MEFs. b, c qRT-PCR analyses of IFN-β (b) and IL-6 (c) mRNA in WT MEFs transfected with poly(I:C)-LMW (1 μg/mL) in the absence (−) or presence (+) of AICAR (2 mM, pretreated for 2 h) for the indicated times. d WT MEFs were stained with MitoTracker Deep Red (mitochondria; red) and then treated with or without AICAR (2 mM) for 8 h. After fixation and permeabilization, cells were immunostained with an anti-MAVS antibody (green). Nuclei were stained with Hoechst 33342 (blue). Scale bars: 10 μm (whole) and 2 μm (inset). e, f qRT-PCR analyses of IFN-β (e) and IL-6 (f) mRNA in Mff KO MEFs transfected with poly(I:C)-LMW (1 μg/mL) in the absence (−) or presence (+) of AICAR (2 mM, pretreated for 2 h) for the indicated times. The blue shaded parts of the graphs (0–8 h) represent the acute phase after poly(I:C), and the red shaded parts…

Fig. 5. Mff phosphorylation impairs mitochondrial antiviral response. a Scheme of the murine phosphorylation-negative/mimic Mff constructs used in this study. A point mutation was introduced into FLAG-tagged Mff (WT) at S146 as indicated. b Mff KO MEFs stably expressing each Mff construct were analyzed by immunoblotting using anti-FLAG, anti-Mff, and anti-phospho-Mff antibodies. SD: S146D, SE: S146E, SA: S146A. WT MEFs treated with AICAR (AMPK activator: 2 mM) or compound C (AMPK inhibitor: 20 μM) for 2 h were used as controls. c, d qRT-PCR of IFN-β (c) and IL-6 (d) mRNA in Mff KO MEFs stably expressing each Mff construct with or without poly(I:C)-LMW (1 μg/mL) transfection for 8 h. Data represent means ± SEM of three independent experiments (n = 6, P values; two-tailed unpaired t test). e Representative live-cell images of MitoTracker Red (mitochondria, white) in Mff KO MEFs stably expressing each Mff construct. Scale bars: 10 μm (whole) and 2 μm (inset). f Magnified images of MAVS on mitochondria in Mff KO MEFs stably expressing each Mff construct. The cells were transiently transfected with a mitochondrial marker mitoRFP (red) and immunostained with an anti-MAVS antibody (green).…

Fig. 6. Mitochondrial dysfunction leads to Mff phosphorylation and impaired antiviral response. a WT MEFs were treated with the respiratory inhibitors rotenone (Rot, 0.5 μM) or antimycin A (AA, 1 μM) or the ATP synthase inhibitor oligomycin (Oligo, 1 μM) for 5 or 30 min. Whole-cell lysates were analyzed by immunoblotting using antibodies against the indicated proteins. AICAR (2 mM) or compound C (20 μM) was used as an AMPK activator or inhibitor, respectively. DMSO: dimethyl sulfoxide. b qRT-PCR analysis of IFN-β mRNA with or without poly(I:C)-LMW in WT MEFs and Mff KO MEFs stably expressing FLAG-Mff S146A. Cells were pretreated with or without oligomycin (Oligo, 1 μM) for 30 min and then transfected with poly(I:C)-LMW (1 μg/mL) in the absence (−) or presence (+) of oligomycin for 8 h. Data represent means ± SEM of two independent experiments (n = 4, P values; two-tailed unpaired t test). c Immunoblot analyses of Mff and Mff phosphorylation in WT MEFs and Mff KO MEFs stably expressing FLAG-Mff S146A with or without oligomycin (1 μM, 30 min). The indicated letters (a, b, c) correspond to the endogenous Mff bands observed in WT MEFs. d HeLa cells were transfected with control or Mfn1/2…

Fig. 7. Clearance of mitochondrial MAVS clusters by Mff. a Confocal microscopic images of MAVS and mitochondria. WT MEFs were transfected with poly(I:C)-LMW (1 μg/mL) for the indicated times. Cells were stained with MitoTracker Red (mitochondria; red) and immunostained with an anti-MAVS antibody (green). Nuclei were stained with Hoechst 33342 (blue). A magnified view of the boxed region is shown in the right panel. Scale bars: 10 μm (whole) and 2 μm (magnified). b WT, Mff KO, and Drp1 KO MEFs were stained with MitoTracker Red (mitochondria; red) and immunostained with an anti-MAVS antibody (green) with poly(I:C)-LMW (1 μg/mL) transfection for 8 h. Nuclei were stained with DAPI (blue). A magnified view of the boxed region is shown in the right panel. Scale bars: 10 μm. c The percentage of cells with cytoplasmic MAVS (MAVS signal without MitoTracker Red) was calculated. Data represent means ± SEM of three independent experiments (WT, Mff KO, Drp1 KO; n = 18, 18, 19, P values; two-tailed unpaired t test). d Working hypothesis for the mitochondrial membrane subdomains with MAVS clusters in antiviral innate immunity, dynamically regulated by the mitochondrial fission factor Mff and AMPK. All…