MAVI1, an endoplasmic reticulum–localized microprotein, suppresses antiviral innate immune response by targeting MAVS on mitochondrion

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  Fig. 1.. A microprotein MAVI1 is down-regulated in response to RNA virus infection. (A) HEK293 cells transfected with control siRNA (siCTL) or siRNA targeting genes as indicated were treated with or without VSV (2 × 106 p.f.u.) for 12 hours before examining IFNB1 expression. (B) Ribo-seq, RNA-seq, and PhyloCSF for SMIM30 are shown. (C and D) The expression of IFNB1 (C) and SMIM30 (D) were examined in HEK293 cells infected with VSV (mean ± SEM, * P < 0.05, ***P < 0.001). (E and F) The expression of IFNB1 (E) and SMIM30 (F) were examined in HEK293 cells transfected with Poly (I:C) (5 μg/ml) (mean ± SEM, ***P < 0.001). (G) HEK293 cells were subjected to polysome profiling, and the expression of SMIM30 was examined. (H) Schematic representation of Flag-tagged ORF with or without 5′ or 3′UTR. aa, amino acid. (I) HEK293 cells were transfected with constructs as shown in (H), followed by immunoblotting (IB) analysis. (J) Schematic representation of Flag-tagged ORF and its mutants with the first (M1), the second (M2), or both (M1 + M2) ATG mutated. (K) HEK293 cells were transfected with constructs as shown in (J), followed by IB analysis. (L) Schematic representation of Flag tag insertion at the…

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  Fig. 2.. MAVI1 negatively regulates RLR-mediated antiviral immune responses in vitro. (A) Hela cells were infected with lentivirus expressing control shRNA (shCTL) or two individual shRNAs specifically targeting MVAI1 (shMVAI1#1 and shMVAI1#2) and treated with or without poly (I:C) (5 μg/ml, 6 hours), followed by immunoblotting (IB). (B) Hela cells were infected with lentivirus expressing shCTL, shMVAI1#1, or shMVAI1#2 and treated with or without poly (I:C) (5 μg/ml, 6 hours), followed by examination of IFNB1(left) and MAVI1 (right) expression (mean ± SEM, ***P < 0.001). (C) HeLa cells infected with lentivirus expressing empty vector, WT MAVI1 (Flag-MAVI1), or mutant MAVI1 with the first ATG mutated [Flag-MAVI1 (M1)] were subjected to IB analysis. (D) Cells as described in (C) were subjected to examination of IFNB1 expression (mean ± SEM, *P < 0.05, ***P < 0.001). (E) Hela cells were infected with lentivirus expressing shCTL, shMVAI1#1, shMVAI1#2 and treated with or without VSV (2 × 106 p.f.u., 12 hours), followed by examination of IFNB1, MAVI1, and VSV expression (mean ± SEM, **P < 0.01, ***P < 0.001). (F) Hela cells transfected with siCTL or siMAVI1 were treated with VSV-GFP for 12…

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  Fig. 3.. MAVI1 negatively regulates RLR-mediated antiviral immune responses in vivo. (A) WT (Mavi1+/+) and Mavi1 knockout (Mavi1−/−) MEFs treated with or without poly (I:C) (5 μg/ml, 6 hours) were subjected to examination of Ifnb1 expression (mean ± SEM, ***P < 0.001). (B) Mavi1+/+ and Mavi1−/− MEFs treated with or without VSV (2 × 106 p.f.u., 12 hours) were subjected to examination of the expression of genes as indicated (mean ± SEM, **P < 0.01, ***P < 0.001). (C) Mavi1+/+ and Mavi1−/− MEFs were treated with VSV-GFP for 12 hours, followed by microscopy imaging analysis. (D) The number of GFP+ cells as shown in (J) were analyzed by ImageJ and normalized to Mavi1+/+ MEFs without VSV-GFP treatment. (E) The levels of Infβ and Il6 in serum from Mavi1 +/+ or Mavi1−/− mice (n = 5) treated with or without VSV (5 × 108 p.f.u. per mouse) for 12 hours were examined by enzyme-linked immunosorbent assay (mean ± SEM, **P < 0.01, ***P < 0.001). (F to H) The levels of Infb and VSV from the lung (F), spleen (G), and liver (H) of mice as described in (L) were examined (mean ± SEM, *P < 0.05, **P < 0.01). (I) Lung tissue sections from mice as described in (M) were subjected to hematoxylin and eosin…

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  Fig. 4.. MAVI1 is an ER membrane protein. (A and B) The SP (1 to 24 amino acids) (A) and TM domain (30 to 52 aa) (B) in MAVI1 predicted by SignaIP-4.1and TMHMM website, respectively, are shown. (C) HEK293 cells transfected with GFP-tagged MAVI1 were stained with Calnexin (ER maker), COXIV (mitochondria maker), EEA1 (endosome maker), LAMP2 (lysosome maker), RPL4 (ribosome maker), Vimentin (cytoskeleton maker), or Golgin-97 (Golgi apparatus marker) (Red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (D) Schematic representation of Flag-tagged, full length (FL) MAVI1, SP-deleted (△SP) MAVI1, and MAVI1 mutant with the SP cleavage site mutated (A > W) is shown. (E) HEK293 cells transfected with empty vector (CTL) or expression vectors as described in (D) were subjected to IB analysis. (F) HEK293 cells stably expressing Flag-tagged MAVI1 were subjected to cellular fraction, followed by IB analysis. W.C.L., whole cell lysate; Cyto., cytoplasmic fraction; Mem., membrane fraction. (G) HEK293 cells stably expressing Flag-tagged MAVI1 were subjected to membrane interaction assay, and the supernatant was treated with or without 0.1 M Na2CO3, 0.1 M NaOH, or 1 M NaCl. The…

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  Fig. 5.. MAVI1 interacts with MAVS. (A) HEK293 cells stably expressing Flag-tagged MAVI1 were subjected to immunoprecipitation and mass spectrometry (IP-MS) analysis. (B) MAVI1-associated proteins identified were analyzed in REPRINT database to exclude common contaminants. Proteins with SAINT ≥ 0.95 and FC-B ≥ 4 were considered as specific binding. (C and D) HEK293 cells were subjected to IP analysis with anti-MAVI1 (C) or MAVS (D) antibody followed by IB analysis. (E) HEK293 cells transfected with or without MAVS in the presence or absence of MAVI1 were subjected to examine the expression of IFNB1 (mean ± SEM, *P < 0.05, ***P < 0.001). (F) HEK293 cells transfected with or without Flag-tagged MAVS in the presence of GFP or GFP-tagged MAVI1 were subjected to IB analysis.

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  Fig. 6.. MAVI1 interacts with MAVS through the TM domain. (A) Schematic representation of GFP-tagged, full length (FL), SP-deleted (△SP), linker region 1–deleted (△L1), TM domain–deleted (△L2), and linker region 2–deleted (△L2) MAVI1 is shown. (B) In vitro pull-down assay was performed by mixing GFP-tagged proteins purified from HEK293 cells as shown in (A) and purified, bacterially expressed GST-tagged MAVS. (C) Schematic representation of GFP-tagged MAVI1 (FL) and the TM domain only (TM) is shown. (D) In vitro pull-down assay was performed by mixing GFP-tagged proteins purified from HEK293 cells as shown in (C) and purified, bacterially expressed GST-tagged MAVS. (E) Schematic representation of Flag-tagged, MAVS (FL), caspase recruitment domain-deleted (△CARD), proline rich domain-deleted (△PRO), and TM domain–deleted (△TM) MAVS is shown. (F) In vitro pull-down assay was performed by mixing Flag-tagged proteins purified from HEK293 cells as shown in (E) and purified, bacterially expressed GST-tagged MAVS (△SP). (G) Schematic representation of Flag-tagged, MAVS (FL), CARD only (CARD), PRO only (PRO), unknown region only (unknown), and TM domain only (TM) is shown. (H) In vitro pull-down…

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  Fig. 7.. MAVI1 interaction with MAVS is critical for MAVI1 function in regulating antiviral innate immune responses. (A) Schematic representation of Flag-tagged MAVI1 and Flag-tagged MAVI1 with the lysosome sorting signal from LAMP1 attached (MAVI1-Lyso) is shown. (B) HEK293 cells transfected with Flag-tagged MAVI1-lyso were subjected to immunofluorescence analysis using anti-Flag (red) and anti-LAMP1 antibody (green). (C) HEK293 cells transfected with vectors as indicated were subjected to IB analysis. (D) HEK293 cells as described in (C) were subjected to examination of IFNB1, CCL5, and CXCL10 expression (mean ± SEM, ***P < 0.001). (E) HEK293 cells transfected with vectors as indicated were subjected to IB analysis. (F) HEK293 cells as described in (E) were subjected to examination of IFNB1, CCL5, and CXCL10 expression (mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001).

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  Fig. 8.. Peptide inhibitor targeting the interaction between MAVI1 and MAVS (PiMAVI1) is potent in activating type I IFN signaling and antiviral immune responses. (A) HEK293 cells were pretreated with peptide inhibitor PiMAVI1 at concentrations as indicated for 8 hours and then infected with or without VSV treatment (2 × 106 p.f.u., 12 hours) were subjected to examination of VSV replication (mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001). (B) HEK293 cells transfected with hemagglutinin (HA)–tagged MAVS and Flag-tagged MAVI1 and treated with or without PiMAVI1 (10 μM, 8 hours) were subjected to immunoprecipitation (IP) with anti-Flag antibody followed by immunoblotting (IB) analysis. (C and D) HEK293 cells pretreated with PiMAVI1 (10 μM, 8 hours) and then infected with VSV (12 hours) were subjected to IB (C) or RT-qPCR analysis (D). (E) HEK293 cells were pretreated with or without PiMAVI1 (10 μM, 8 hours) and then infected with or without VSV-GFP (2 × 106 p.f.u., 12 hours), followed by microscopy imaging analysis. (F) The number of GFP-positive (GFP+) cells as shown in (E) was analyzed by ImageJ and normalized to control cells. (G) The proposed model of MAVI1 function in antiviral innate…