Bunyavirus SFTSV nucleoprotein exploits TUFM-mediated mitophagy to impair antiviral innate immunity

Abstract

Severe fever with thrombocytopenia syndrome is an emerging viral hemorrhagic fever caused by a tick-borne bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), with a high case fatality. We previously found that SFTSV nucleoprotein (NP) induces macroautophagy/autophagy to facilitate virus replication. However, the role of NP in antagonizing host innate immunity remains unclear. Mitophagy, a selected form of autophagy, eliminates damaged mitochondria to maintain mitochondrial homeostasis. Here, we demonstrate that SFTSV NP triggers mitophagy to degrade MAVS (mitochondrial antiviral signaling protein), thereby blocking MAVS-mediated antiviral signaling to escape the host immune response. Mechanistically, SFTSV NP translocates to mitochondria by interacting with TUFM (Tu translation elongation factor, mitochondrial), and mediates mitochondrial sequestration into phagophores through interacting with LC3, thus inducing mitophagy. Notably, the N-terminal LC3-interacting region (LIR) motif of NP is essential for mitophagy induction. Collectively, our results demonstrated that SFTSV NP serves as a novel virulence factor, inducing TUFM-mediated mitophagy to degrade MAVS and evade the host immune response.Abbreviation: 3-MA: 3-methyladenine; ACTB: actin beta; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole, dihydrochloride; DMSO: dimethyl sulfoxide; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GFP: green fluorescent protein; HTNV: Hantan virus; IAV: influenza A virus; IFN: interferon; LAMP1: lysosomal associated membraneprotein 1; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associatedprotein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; Mdivi-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MT-CO2/COXII: mitochondrially encoded cytochrome C oxidase II; NP: nucleoprotein; NSs: nonstructural proteins; poly(I:C): polyinosinic:polycytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SFTSV: severe fever withthrombocytopenia syndrome virus; TCID50: 50% tissue culture infectiousdose; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20:translocase of outer mitochondrial membrane 20; TUFM: Tu translation elongationfactor, mitochondrial.

Keywords: Bunyavirus; MAVS; SFTSV; interferon; mitophagy; nucleoprotein.

Figure 1.

SFTSV NP inhibits the activation of MAVS-mediated signaling pathway (A) 293T cells were transfected with IFNB promoter-luciferase reporter (IFNB-luc) (100 ng), Renilla luciferase reporter plasmid (pRL-TK; 10 ng), and either HA-NP plasmid (200 ng) or empty vector (pCAGGS) for 24 h. The cells were then transfected with poly(I:C) or mock-transfected for 12 h. The luciferase activity of IFNB was measured with Dual-luciferase reporter assay. (B) 293T cells were transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cells were then transfected with poly(I:C) or mock-transfected for 12 h, and the transcription levels of IFNB1 were measured with RT-qPCR. (C) 293T cells were transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cells were then infected with or without SFTSV (MOI = 5) for 24 h, and the transcription levels of IFNB1 were measured with RT-qPCR. (D) 293T cells were transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cells were then transfected with poly (I:C) for 0 h, 6 h, or 12 h. The cell lysates were analyzed with western blot. (E) 293T cells were co-transfected with Flag-MAVS, Flag-RIGI, Flag-TBK1, or Flag-IRF3 plasmids (2 μg) along with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The mRNA levels of IFNB1, IFIT1, IFIT2, and CCL5 were subsequently measured with RT-qPCR. (F) 293T cells were transfected with IFNB-luc (100 ng), pRL-TK (10 ng), Flag-RIGI or Flag-MAVS plasmids (200 ng) along with HA-NP plasmid (200 ng) or empty vector (pCAGGS) for 24 h. The luciferase activity of IFNB was measured with Dual-luciferase reporter assay. (G and H) 293T cells were co-transfected with IFNB-luc (100 ng), pRL-TK (10 ng), an increasing amount (0, 100, 200 ng) of HA-NP, and either Flag-RIGI (G) or Flag-MAVS (H) plasmids for 24 h. The luciferase activity of IFNB was measured with Dual-luciferase reporter assay. Graphs show the mean ± SD; n = 3; ns indicates not significant.

Figure 2.

SFTSV NP interacts with MAVS and promotes the degradation of MAVS. (A) 293T cells were infected with or without SFTSV (MOI = 5) for 24 h. Cells were stained with anti-NP (green) and anti-MAVS (red) antibodies and were analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B) 293T cells were transfected with HA-NP plasmid (2 μg) and Flag-MAVS plasmid (2 μg) for 24 h. Cells were stained with anti-HA (green) and anti-Flag (red) antibodies and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (C) 293T cells were transfected with Flag-MAVS plasmid (2 μg) along with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cell lysates were immunoprecipitated with either anti-HA or anti-Flag antibodies to detect the interaction between NP and MAVS. (D) 293T cells were infected with or without SFTSV (MOI = 5) for 24 h. The cell lysates were immunoprecipitated with either anti-NP or anti-MAVS antibodies and then immunoblotted with the indicated antibody. (E) The schematic diagram of MAVS truncation mutants. (F) 293T cells were transfected with Flag-NP plasmid (2 μg) and MYC-MAVS, MYC-ΔCARD, MYC-ΔTM, MYC-ΔN, MYC-N, or MYC-ΔN/ΔTM (2 μg) for 24 h. The cell lysates were immunoprecipitated with the anti-MYC antibody and then immunoblotted with the indicated antibody. (G) 293T cells were transfected with an increasing amount (0, 0.5, 1, or 2 μg) of HA-NP for 24 h. The mRNA levels of MAVS were detected with RT-qPCR. (H) 293T cells were co-transfected with Flag-MAVS plasmid (2 μg) and Flag-ACTB plasmid (2 μg), along with an increasing amount (0, 0.5, 1, or 2 μg) of HA-NP for 24 h. The cell lysates were analyzed with western blot. (I) 293T cells were transfected with an increasing amount (0, 0.5, 1, or 2 μg) of HA-NP plasmid for 24 h. The cell lysates were analyzed with western blot. (J) 293T cells were co-transfected with Flag-MAVS plasmid (2 μg) and Flag-ACTB plasmid (2 μg), along with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 20 h and then treated with MG132 (10 μM), chloroquine (CQ) (50 μM), or 3-methyladenine (3-MA) (10 mM) for 6 h. The cell lysates were analyzed with western blot. (K) 293T cells were co-transfected with Flag-MAVS (2 μg) along with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 20 h and then treated with Mdivi-1 (25 μM) or vehicle (DMSO) for 6 h. The cell lysates were analyzed with western blot. (L) 293T cells were co-transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 20 h and then treated with Mdivi-1 (25 μM) or vehicle (DMSO) for 6 h. The cell lysates were analyzed with western blot. Graphs show the mean ± SD; n = 3; ns indicates not significant.

Figure 3.

Mitophagy is triggered under SFTSV infection (A) HeLa cells were infected with SFTSV (MOI = 0, 1, 5) for 24 h or infected with SFTSV (MOI = 5) for 0, 12, or 24 h. The cell lysates were analyzed with western blot. (B) 293T cells were infected with or without SFTSV (MOI = 5) for 24 h. Cells were stained with anti-NP (purple), MitoTracker (red), and anti-LC3 (green) and subjected to confocal microscopy analysis. Nuclei were stained with DAPI (blue). The arrows indicate the presence of LC3 punctate aggregation. Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between mitochondria and LC3. (C) HeLa cells were infected with or without SFTSV (MOI = 5) for 24 h. The cell lysates were then analyzed with transmission electron microscopy to determine autophagosome and autolysosome formation. Red arrows indicate autophagic vacuoles and yellow arrows indicate autolysosomes containing mitochondria. Scale bar: 2 μm. (D) 293T cells were transfected with mCherry-GFP-mitochondria (mCherry-GFP-mito) for 12 h and then infected with or without SFTSV (MOI = 5) for 24 h. Cells were stained with anti-NP (purple) antibody and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (E) 293T cells infected with or without SFTSV (MOI = 5) for 24 h. Cells were stained with anti-NP (purple), MitoTracker (red), and anti-LAMP1 (green) and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between mitochondria and LAMP1. (F) HeLa cells infected with or without SFTSV (MOI = 5) for 20 h and then treated with CQ (50 μM) for 6 h. The cell lysates were analyzed with western blot. Data were obtained from three independent experiments (n = 3); ns indicates not significant.

Figure 4.

SFTSV NP overexpression induces mitophagy (A) HeLa cells were transfected with an increasing amount (0, 0.5, 1, or 2 μg) of HA-NP plasmid for 24 h. The cell lysates were analyzed with western blot. (B) HeLa cells were transfected with HA-NP plasmid (2 μg) for 0, 12, 24, or 48 h. The cell lysates were analyzed with western blot. (C) 293T cells were transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. Cells were stained with anti-NP (purple), MitoTracker (red), and anti-LC3 (green) and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). The arrows indicate the presence of LC3 punctate aggregation. Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between mitochondria and LC3. (D) 293T cells were co-transfected with mCherry-GFP-mito (2 μg) along with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. Cells were stained with anti-NP (purple) antibody and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (E) 293T cells were co-transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. Cells were stained with anti-NP (purple), MitoTracker (red), and anti-LAMP1 (green) and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between mitochondria and LAMP1. (F) HeLa cells were transfected with HA-NP plasmid (2 μg) or empty vector (pCAGGS) for 20 h and then treated with CQ (50 μM) for 6 h. The cell lysates were analyzed with western blot. Data were obtained from three independent experiments (n = 3); ns indicates not significant.

Figure 5.

SFTSV NP acts as a potential receptor of mitophagy to mediate the interaction of MAVS and LC3 (A and B) 293T cells were co-transfected with GFP-LC3 plasmid (2 μg) and Flag-SFTSV-NP plasmid (2 μg) or Flag-RVFV-NP plasmid (2 μg) for 24 h. The cell lysates were immunoprecipitated with either anti-Flag (A) or anti-GFP (B) antibodies to detect the interaction between NP and LC3. (C and D) 293T cells were transfected with Flag-SFTSV-NP plasmid (2 μg) or Flag-RVFV-NP plasmid (2 μg) for 24 h. The cell lysates were immunoprecipitated with either anti-Flag (C) or anti-LC3 (D) antibodies to detect the interaction between NP and LC3. (E) HeLa cells were transfected with Flag-NP plasmid (2 μg) or Flag-np-ΔWSRI plasmid (2 μg) for 24 h. The cell lysates were analyzed with western blot. (F and G) 293T cells were co-transfected with the indicated plasmid for 24 h. The cell lysates were immunoprecipitated with either anti-MYC (F) or anti-GFP (G) antibodies and then immunoblotted with the indicated antibody. (H) 293T cells were co-transfected with the indicated plasmid for 24 h. Cells were stained with anti-Flag (purple) and anti-MYC (red) antibodies and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between MAVS and LC3. Data were obtained from three independent experiments (n = 3); ns indicates not significant.

Figure 6.

SFTSV NP interacts with TUFM (A) HeLa cells were transfected with Flag-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. Cytoplasm and mitochondrial fractions were purified for western blot (fractions: cyto, purified cytosolic; mito, purified mitochondria. Organelle markers: TOMM20, mitochondria; ACTB, cytoplasm). (B) 293T cells were co-transfected with Flag-NP plasmid (2 μg) and GFP-TUFM plasmid (2 μg) for 24 h. Cells were stained with anti-Flag (purple) and MitoTracker (red) and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (C and D) 293T cells were co-transfected with Flag-NP plasmid (2 μg) and GFP-TUFM plasmid (2 μg) for 24 h. The cell lysates were immunoprecipitated with either anti-Flag (C) or anti-GFP (D) antibodies to detect the interaction between NP and TUFM. (E and F) 293T cells were transfected with Flag-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cell lysates were immunoprecipitated with either anti-Flag (E) or anti-TUFM (F) antibodies to detect the interaction between NP and TUFM. (G and H) 293T cells were infected with or without SFTSV (MOI = 5) for 24 h. The cell lysates were immunoprecipitated with either anti-NP (G) or anti-TUFM (H) antibodies and then immunoblotted with the indicated antibody. (I) 293T cells were transfected with MYC-TUFM plasmid (2 μg) and Flag-np, Flag-Δ2-32, Flag-Δ33-92, Flag-Δ93-127, Flag-Δ128-207, or Flag-Δ208-245 (2 μg) for 24 h. The cell lysates were immunoprecipitated with the anti-Flag antibody and then immunoblotted with the indicated antibody. Data were obtained from three independent experiments (n = 3); ns indicates not significant.

Figure 7.

SFTSV NP induces TUFM-dependent mitophagy (A) 293T cells were transfected with MYC-TUFM plasmid (2 μg), GFP-LC3 plasmid (2 μg) along with Flag-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. Cells were stained with anti-Flag (purple) and anti-MYC (red) antibodies and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between TUFM and LC3. (B and C) 293T cells were co-transfected with the indicated plasmid for 24 h. The cell lysates were immunoprecipitated with either anti-MYC (B) or anti-GFP (C) antibodies and then immunoblotted with the indicated antibody. (D) 293T cells were infected with or without SFTSV (MOI = 5) for 24 h. Cells were stained with anti-NP (purple), anti-TUFM (red), and anti-LC3 (green) antibodies and analyzed with confocal microscopy. Nuclei were stained with DAPI (blue). Scale bars: 20 μm. The right panel shows the quantification of Pearson’s colocalization coefficient between TUFM and LC3. (E) Sh-Vector or Sh-TUFM HeLa cells were transfected with Flag-NP plasmid (2 μg) for 24 h. Cytoplasm and mitochondrial fractions were purified for western blot (fractions: cyto, purified cytosolic; mito, purified mitochondria. Organelle markers: TOMM20, mitochondria; ACTB, cytoplasm). (F) Sh-Vector or Sh-TUFM HeLa cells were infected with or without SFTSV (MOI = 5) for 24 h. The cell lysates were analyzed with western blot. (G) Sh-Vector or Sh-TUFM HeLa cells were transfected with Flag-NP plasmid (2 μg) or empty vector (pCAGGS) for 24 h. The cell lysates were analyzed with western blot. Data were obtained from three independent experiments (n = 3); ns indicates not significant.

Figure 8.

Inhibiting mitophagy restricts SFTSV replication (A) HeLa cells were infected with SFTSV (MOI = 5) and treated with Mdivi-1 (0, 10, 25, 50 μM) for 24 h. The mRNA levels of the SFTSV S, M, and L segments were detected with RT-qPCR. (B) HeLa cells were infected with SFTSV (MOI = 5) and treated with Mdivi-1 (0, 10, 25 μM) for 24 h. The cell lysates were analyzed with western blot. (C) HeLa cells were infected with SFTSV (MOI = 5) and treated with Mdivi-1 (10 μM) for 0, 24 or 48 h. Endpoint 10-fold dilutions of an SFTSV stock were titrated. Values presented in the graph are calculated and expressed as the log10 of TCID₅₀/mL of supernatant. (D) HeLa cells were infected with SFTSV (MOI = 5) and then treated with FCCP (10 μM) or vehicle (DMSO) for 24 h. The mRNA levels of the SFTSV S, M, and L segments were determined with RT-qPCR. (E) HeLa cells were infected with or without SFTSV (MOI = 5) and then treated with FCCP (10 μM) or vehicle (DMSO) for 24 h. The cell lysates were analyzed with western blot. (F) HeLa cells were infected with SFTSV (MOI = 5) and treated with FCCP (10 μM) for 0, 24 or 48 h. Endpoint 10-fold dilutions of an SFTSV stock were titrated. Values presented in the graph are calculated and expressed as the log10 of TCID₅₀/mL of supernatant. (G) Sh-Vector or Sh-TUFM HeLa cells were infected with SFTSV (MOI = 5) for 24 h. The mRNA levels of the SFTSV S, M, and L segments were determined with RT-qPCR. (H) Sh-vector or Sh-TUFM HeLa cells were infected with SFTSV (MOI = 5) for 24 or 48 h. Endpoint 10-fold dilutions of an SFTSV stock were titrated. Values presented in the graph are calculated and expressed as the log10 of TCID₅₀/mL of supernatant. (I) Sh-Vector or Sh-TUFM HeLa cells were infected with SFTSV (MOI = 0, 1, 2, 5) for 24 h. Cells were stained with SFTSV NP (green) and the immunofluorescence data were semi-quantitatively analyzed. Graphs show the mean ± SD; n = 3; ns indicates not significant.

Figure 9.

A working model of the role of SFTSV NP in the regulation of MAVS-mediated signaling. Black arrows indicate the RIGI-MAVS-mediated type I IFN signaling pathway; red arrows indicate the process of SFTSV NP-induced mitophagy. SFTSV NP interacts with TUFM and translocates to mitochondria, where it induces mitophagy, leading to MAVS degradation, thus suppressing antiviral innate immunity. The model was created in BioRender.com.