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MAVS deSUMOylation by SENP1 inhibits its aggregation and antagonizes IRF3 activation

Nature Structural & Molecular Biology volume 30, pages 785–799 (2023)Cite this article

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Abstract

Mitochondrial antiviral signaling protein (MAVS) is an adapter that recruits and activates IRF3. However, the mechanisms underpinning the interplay between MAVS and IRF3 are largely unknown. Here we show that small ubiquitin-like modifier (SUMO)-specific protease 1 negatively regulates antiviral immunity by deSUMOylating MAVS. Upon virus infection, PIAS3-induced poly-SUMOylation promotes lysine 63-linked poly-ubiquitination and aggregation of MAVS. Notably, we observe that SUMO conjugation is required for MAVS to efficiently produce phase-separated droplets through association with a newly identified SUMO-interacting motif (SIM) in MAVS. We further identify a yet-unknown SIM in IRF3 that mediates its enrichment to the multivalent MAVS droplets. Conversely, IRF3 phosphorylation at crucial residues close to SIM rapidly disables SUMO-SIM interactions and releases activated IRF3 from MAVS. Our findings implicate SUMOylation in MAVS phase separation and suggest a thus far unknown regulatory process by which IRF3 can be efficiently recruited and released to facilitate timely activation of antiviral responses.

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Fig. 1: Senp1 deficiency potentiates antiviral responses and restricts virus replication.
Fig. 2: SENP1 antagonizes RLR signaling via deSUMOylating MAVS.
Fig. 3: SENP1 inhibits Lys63-linked poly-ubiquitination of MAVS and represses MAVS aggregation.
Fig. 4: MAVS is poly-SUMOylated by PIAS3 mainly at Lys461 and Lys500.
Fig. 5: Lys461 and Lys500 poly-SUMOylation on MAVS contributes to its Lys63-linked poly-ubiquitination and aggregation and thus is required for MAVS activation.
Fig. 6: SUMOylation promotes LLPS of MAVS.
Fig. 7: IRF3 contains an SIM that mediates its recruitment to the MAVS droplet.
Fig. 8: IRF3-SIM-destructive KI abrogates IRF3 function and results in innate immune evasion.

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Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD037467. Source data are provided with this paper.

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Acknowledgements

We thank J. Cheng from Shanghai Jiao Tong University School of Medicine for Senp1f/f mice and F. Hou from the University of the Chinese Academy of Sciences for technical support. The current work was supported by the National Key R&D Program of China (grant no. 2022YFA1105200 to F.Z., grant no. 2021YFA1101000 to Long Zhang), the Chinese National Natural Science Funds (grant no. 32125016 to F.Z., grant nos. 31925013 and U20A20393 to Long Zhang, grant no. 32100699 to T.D., grant nos. 92253302 and 22222705 to S.L.), the China National Postdoctoral Program for Innovative Talents (grant no. BX2021208 to T.D.), the China Postdoctoral Science Foundation (grant no. 2021M692350 to T.D.) and the Suzhou Innovation and Entrepreneurship Leading Talent Program (grant no. ZXL2022505 to F.Z.). We thank Y. Li, G. Xiao, W. Yin and Z. Lin from the Core Facilities of Zhejiang University School of Medicine for their technical support.

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  1. These authors contributed equally: Tong Dai, Lei Zhang, Yu Ran.

Authors and Affiliations

  1. Center for Infection & Immunity of International Institutes of Medicine, The Fourth Affiliated Hospital, ZheJiang University School of Medicine, Yiwu, China

    Tong Dai & Fangfang Zhou

  2. Institutes of Biology and Medical Science, Soochow University, Suzhou, China

    Tong Dai, Meirong Zhang & Fangfang Zhou

  3. MOE Laboratory of Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China

    Tong Dai, Yu Ran, Bing Yang, Huasong Lu, Shixian Lin & Long Zhang

  4. Department of Orthopaedic Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China

    Lei Zhang

  5. Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco, CA, USA

    Bing Yang

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Contributions

T.D., Lei Zhang and F.Z. designed the experiments. T.D., Y.R., M.Z. and B.Y. performed the experiments and analyzed the data. Lei Zhang, S.L. and H.L. provided valuable discussion. T.D., Long Zhang and F.Z. wrote the manuscript.

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Correspondence to Shixian Lin, Long Zhang or Fangfang Zhou.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Florian Ullrich, Carolina Perdigoto and Dimitris Typas were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 SENP1 negatively regulates RLR signaling.

(a) IB of SENP1 knockdown efficiency by Senp1 shRNAs (sh-Senp1 no. 1 to no. 4). (b) IFN-β-Luc (left) and PRD I-III Luc (right) activity analysis of RAW264.7 cells transfected with Co.sh or sh-Senp1, followed by infection with or without SeV for 12 h. (c) qPCR of Ifnb1 mRNA in RAW264.7 cells transfected with Co.sh or sh-Senp1 and treated with SeV for indicated time points. Senp1 knockdown efficiency is shown on the left panel. (d) qPCR of IFNB1 mRNA in HEK293T cells transfected with Co.sh or SENP1 shRNA (sh-SENP1) and treated with SeV for indicated time points. SENP1 knockdown efficiency is shown on the left panel. (e) IB of HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt. (f) IFN-β-Luc (left) and PRD I-III Luc (right) activity analysis of HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by infection with or without SeV for 12 h. (g) qPCR of IFNB1 mRNA in HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by SeV infection (left) or poly (I:C) LMW transfection (right) for various time. (h) qPCR of Ifnb1 mRNA in RAW264.7 cells transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by SeV infection (left) or poly (I:C) LMW transfection (right) for various time. b-d, f-h, n = 3 biological independent samples; data are presented as mean ± s.d.; statistical analyses were performed using a two-tailed Student’s t-test. IB=immunoblot, Co.sh=control shRNA, Co.vec=control empty vector, wt=wild-type, mt=mutant.

Source data

Extended Data Fig. 2 SENP1 inhibits the innate antiviral response.

(a) qPCR of Ifnb1 mRNA (left) and VSV RNA (middle) and plaque assay of VSV (right) in RAW264.7 cells transfected with Co.sh or sh-Senp1 for 24 h, followed by infection with or without VSV for 12 h. (b) qPCR of IFNB1 mRNA (left) and VSV RNA (middle) and plaque assay of VSV (right) in HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt for 24 h, followed by infection with or without VSV for 12 h. (c) IB of VSV-G in HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by infection with or without VSV for 12 h. (d, e) Brightfield microscopy (top) and fluorescence microscopy (bottom) of VSV-GFP in HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt (d), or with Co.sh or two sh-SENP1 (e), followed by infection for 12 h with GFP-expressing VSV (MOI, 0.1). Scale bars, 100 μm. GFP intensity is analyzed by Image J software and shown right. a-b, d-e, n = 3 biological independent samples; data are presented as mean ± s.d.; statistical analyses were performed using a two-tailed Student’s t-test. IB=immunoblot, Co.sh=control shRNA, Co.vec=control empty vector, wt=wild-type, mt=mutant.

Source data

Extended Data Fig. 3 Senp1 deficiency potentiates innate antiviral immunity.

(a) Schematic diagram of Senp1 knockout strategy. Deletion of exon 14 and exon 15 results in frame shift and disrupts its open reading frame (ORF), leading to the loss of Senp1 expression. (b) qPCR of Ifnb1, Cxcl10, and Ccl5 mRNA in Lyz2-Cre-Senp1f/f and Lyz2-Cre+Senp1f/f BMDMs infected with SeV (top) or transfected with 5'-ppp RNA (bottom) for indicated time. (c) qPCR of Ifnb1 mRNA in Lyz2-Cre-Senp1f/f and Lyz2-Cre+Senp1f/f BMDMs transfected with poly (I:C) LMW (left) or infected with VSV (right) for indicated time. b-c, n = 3 biological independent samples; data are presented as mean ± s.d.; statistical analyses were performed using a two-tailed Student’s t-test.

Source data

Extended Data Fig. 4 SENP1 antagonizes RLR signaling by suppressing MAVS.

(a) qPCR of Ifnb1 mRNA in Lyz2-Cre-Senp1f/f and Lyz2-Cre+Senp1f/f peritoneal macrophages transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by infection with SeV for 12 h. (b) IFN-β-Luc activity in control and SENP1 ectopically expressed (left) or SENP1-depleted (right) HEK293T cells transfected with Co.vec or indicated expression plasmids. (c) IB of IRF3 dimerization (upper panel, native gel) and p-IRF3 (Ser396), p-TBK1, and total IRF3 and TBK1 in Lyz2-Cre-Senp1f/f and Lyz2-Cre+Senp1f/f BMDM infected with SeV for various times. (d) IB of IRF3 dimerization (upper panel, native gel) and p-IRF3 (Ser396), p-TBK1, and total IRF3 and TBK1 in HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt, followed by infection with SeV for various times. (e) IB of TCL and proteins immunoprecipitated with anti-Flag antibody from Ubc9-ectopically expressed HEK293T cells transfected with Flag-MAVS along with indicated SUMO expression plasmids. (f) IB of TCL and His-SUMO3-associated proteins pulled down by Ni-NTA agarose in control or SENP1-depleted HEK293T cells transfected with Myc-tagged SENP1 wt or mt plasmids, followed by SeV infection for 12 h. (g) qPCR of IFNB1 mRNA in control and MAVS ectopically expressed HEK293T cells transfected with Co.vec, SENP1 wt, or SENP1 mt (left), or stimulated with DMSO or Ubc9 inhibitor 2-D08 (right). a-b, g, n = 3 biological independent samples; data are presented as mean ± s.d.; statistical analyses were performed using a two-tailed Student’s t-test; ns, not significant. IB=immunoblot, TCL=total cell lysates, Co.vec=control empty vector, wt=wild-type, mt=mutant.

Source data

Extended Data Fig. 5 MAVS is mainly poly-SUMOylated at Lys461 and Lys500.

(a) IB of TCL and proteins immunoprecipitated with anti-Flag antibody from HA-SUMO3- and Ubc9-stably expressed HEK293T cells transfected with Flag-MAVS wt or its lysine residue mutation plasmids. (b) Left: bacterial purified SAE1, SAE2, Ubc9, and mature SUMO3 proteins were analyzed by SDS-PAGE and detected by Coomassie blue staining. Right: in vitro SUMOylation of MAVS. Negative controls lacking ATP were also shown. (c) IB of TCL and proteins immunoprecipitated with anti-Flag antibody from HA-SUMO3-stably expressed HEK293T cells transfected with Flag-MAVS wt, K0 (all K mutated to R), K0 (R461K) or K0 (R500K) mutant expression plasmids. (d) IB of TCL and proteins immunoprecipitated with anti-Flag antibody from HA-Ub K63-stably expressed HEK293T cells transfected with Flag-MAVS wt or its lysine residue mutation plasmids without or with Myc-PIAS3. (e) A schematic diagram of purified MAVS and MAVS-SUMO fusion proteins. (f) E. coli purified GFP-MAVS and GFP-MAVS-SUMO fusion proteins (461-SUMO or 500-SUMO) were digested with PK (mass ratio 50:1) for the indicated time at 22 °C and then analyzed by Coomassie blue staining. The PK resistant MAVS segments were indicated as PK-MAVS. (g) Left: purified K63-Ub4 and RIG-IN proteins were analyzed by Coomassie blue staining. Right: purified GST-MAVS and SUMOylated GST-MAVS were incubated with K63-Ub4 and RIG-IN proteins at 22 °C for 10 min. The proteins were then digested with PK (mass ratio 50:1) for the indicated time at 22 °C and then analyzed by Coomassie blue staining. (h) Full-length MAVS and MAVS ▲CARD were expressed in MAVS KO HEK293T cells by transfection. Mitochondria (P5) from these cells were incubated with or without MAVS-wt and MAVS-SUMO (500-SUMO) chimera, as shown in (e), for 30 min before mitochondrial proteins were separated by sucrose gradient ultracentrifugation. n = 3 biological independent samples; data are presented as mean ± s.d. (i) MAVS or MAVS-SUMO fusion (500-SUMO) proteins were incubated with mitochondria (P5) from HEK293T cells. The mitochondria were then solubilized in a buffer containing 1% DDM and then analyzed by SDD-AGE and SDS-PAGE with a MAVS antibody. IB=immunoblot, TCL=total cell lysates, wt=wild-type, PK=proteinase K, KO=knockout.

Source data

Extended Data Fig. 6 MAVS undergoes LLPS contributed by SUMO.

(a) Domain structure and the intrinsically disordered tendency of MAVS. (b) Representative fluorescence and DIC images of GFP-MAVS and GFP-MAVS-SUMO (500-SUMO) droplets formation at room temperature with 250 mM NaCl. (c) Representative fluorescence and DIC images of 5 μM GFP-MAVS and GFP-MAVS-SUMO (500-SUMO) droplets formation at room temperature with the indicated concentrations of NaCl. (d) Phase separation diagram of different concentrations of GFP-MAVS (left) or GFP-MAVS-SUMO (500-SUMO) (right) at indicated concentrations of NaCl. The green dots and dot sizes indicate phase separation, and the black dots indicate no phase separation. (e) Representative fluorescence and DIC images of GFP-MAVS (5 μM) droplets formation at 4 °C or 37 °C with 250 mM NaCl. (f, g) 5 μM GFP-MAVS were treated with 5% Hex (f) or treated with 100 μg/ml Proteinase K for 30 min at 40 °C (g), and then subjected to droplet formation assay in vitro (250 mM NaCl, room temperature). (h) Time-lapse micrographs of the fusion of AF488 labeled MAVS and MAVS-SUMO (500-SUMO) droplets at room temperature with 250 mM NaCl. EqDiameter frequency distribution of AF488 labeled MAVS and MAVS-SUMO (500-SUMO) (20 μM) liquid droplets formed at the indicated timepoints is shown in the right panel. (i) Left: representative micrographs of MAVS and MAVS-SUMO droplets before and after photobleaching. Right: quantification of FRAP of MAVS and MAVS-SUMO droplets. Scale bar, 5 μm. (j) The in vitro interaction of purified MAVS-wt or MAVS-SIM3A mutant with MAVS-SUMO3. (k) Representative fluorescence images of GFP-MAVS wt or SIM3A mutant protein (5 µM) mixed with AF594 labeled MAVS-SUMO (500-SUMO) (1 µM). (l) Bleaching was performed after GFP-MAVS wt or SIM3A mutant protein (5 µM) and AF594 labeled MAVS-SUMO (500-SUMO) (1 µM) were mixed, and quantification of FRAP of GFP-MAVS liquid droplets. b-c, e-h, k, scale bar, 10 μm. b-c, e-g, n = 6 independent experiments, i, l, n = 3 condensates; data are presented as mean ± s.d. Statistical analyses were performed using a two-tailed Student’s t-test (b-c, e-g) or two-way ANOVA (i, l). DIC=differential interference contrast, wt=wild-type.

Source data

Extended Data Fig. 7 IRF3 contains a SIM that contributes to its association to the upstream adapter proteins and downstream transcriptional activity.

(a) In vitro interaction of purified IRF3 with MAVS-wt or MAVS-SUMO3 (500-SUMO) expressed and purified from bacteria (IRF3, MAVS-wt, and MAVS-SUMO3 fusion protein). After incubation, immunoprecipitations with anti-MAVS antibody were performed and followed by IB for MAVS and IRF3. (b) IB of indicated proteins in IRF3 KO HEK293T cells transfected with Flag-IRF3 along with MAVS-wt or MAVS-SUMO (500-SUMO) plasmids. (c) IB of TCL and proteins immunoprecipitated with anti-Flag antibody from IRF3 KO HEK293T cells transfected with Myc-STING together with Flag-IRF3 wt or SIM2A plasmids, followed by stimulation with cGAMP (100 nM). (d) IB of indicated proteins in IRF3 KO HEK293T cells transfected with Flag-IRF3 wt or SIM2A along with or without Myc-STING plasmids, followed by stimulation with cGAMP (100 nM). (e) qPCR of IFNB1, ISG15, and CXCL10 in wt and IRF3 KO HEK293T cells transfected with Myc-STING along with or without Flag-IRF3 wt or SIM2A plasmids, followed by stimulation with cGAMP (100 nM); n = 3 biological independent samples, results are presented as mean ± s.d., statistical analyses were performed using a two-tailed Student’s t-test. IB=immunoblot, TCL=total cell lysates, wt=wild-type, KO=knockout.

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Dai, T., Zhang, L., Ran, Y. et al. MAVS deSUMOylation by SENP1 inhibits its aggregation and antagonizes IRF3 activation. Nat Struct Mol Biol 30, 785–799 (2023). https://doi.org/10.1038/s41594-023-00988-8

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