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Deactylation by SIRT1 enables liquid–liquid phase separation of IRF3/IRF7 in innate antiviral immunity

Nature Immunology volume 23, pages 1193–1207 (2022)Cite this article

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Abstract

Innate antiviral immunity deteriorates with aging but how this occurs is not entirely clear. Here we identified SIRT1-mediated DNA-binding domain (DBD) deacetylation as a critical step for IRF3/7 activation that is inhibited during aging. Viral-stimulated IRF3 underwent liquid–liquid phase separation (LLPS) with interferon (IFN)-stimulated response element DNA and compartmentalized IRF7 in the nucleus, thereby stimulating type I IFN (IFN-I) expression. SIRT1 deficiency resulted in IRF3/IRF7 hyperacetylation in the DBD, which inhibited LLPS and innate immunity, resulting in increased viral load and mortality in mice. By developing a genetic code expansion orthogonal system, we demonstrated the presence of an acetyl moiety at specific IRF3/IRF7 DBD site/s abolish IRF3/IRF7 LLPS and IFN-I induction. SIRT1 agonists rescued SIRT1 activity in aged mice, restored IFN signaling and thus antagonized viral replication. These findings not only identify a mechanism by which SIRT1 regulates IFN production by affecting IRF3/IRF7 LLPS, but also provide information on the drivers of innate immunosenescence.

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Fig. 1: IRF3 undergoes liquid–liquid phase separation.
Fig. 2: DBD, IAD and IDR are necessary for IRF3 LLPS.
Fig. 3: IRF3 compartmentalizes IRF7 in LLPS condensates.
Fig. 4: SIRT1 is required for IRF3 LLPS and innate antiviral immune responses.
Fig. 5: SIRT1 deficiency abolishes IRF3/IRF7 LLPS and leads to innate immune evasion.
Fig. 6: SIRT1 directly deacetylates IRF3/IRF7 in the DBD.
Fig. 7: Site-specific acetylation of IRF3/IRF7 DBD abrogates LLPS.
Fig. 8: Reinforcing SIRT1 activity rescues innate immunosenescence.

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Data availability

The de-identified datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The current work was supported by a special program from the Ministry of Science and Technology of China (2021YFA1101000), the Chinese National Natural Science Funds (U20A20393, U20A201376, 32125016, 31701234, 91753139, 31925013, 31671457, 31870902, 32070907, 32100699 and 31871405), the China National Postdoctoral Program for Innovative Talents (BX2021208), the China Postdoctoral Science Foundation (2021M692350), the Zhejiang Natural Science Fund (LD19C070001) and Jiangsu National Science Foundation (19KJA550003).

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Author notes

  1. These authors contributed equally: Ziran Qin, Xiuwu Fang.

Authors and Affiliations

  1. International Biomed-X Research Center, Second Affiliated Hospital of Zhejiang University School of Medicine, Zhejiang University, Hangzhou, China

    Ziran Qin & Long Zhang

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

    Ziran Qin, Xiuwu Fang, Wenhuan Sun, Zhenyu Ma, Tong Dai, Shuai Wang & Fangfang Zhou

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

    Zhi Zong, Heng Ru, Huasong Lu, Bing Yang, Shixian Lin & Long Zhang

  4. Faculty of Basic Medical Sciences, Chonqing Medical University, Chongqing, China

    Huizhe Huang

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

    Bing Yang

  6. Cancer Center, Zhejiang University, Hangzhou, China

    Long Zhang

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Contributions

Z.Q., X.F., T.D., W.S., Z.M. and S.W. designed the experiments and analyzed the data. Z.Q., F.C. and W.S. performed the experiments. Z.Z. designed the cartoon for the working model. B.Y. performed the mass spectrometry analysis. H.H., H.L., X.H. and L.Z. provided valuable discussion. L.Z. and F.Z. wrote the manuscript.

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Correspondence to Fangfang Zhou or Long Zhang.

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Nature Immunology thanks Andrew Bowie and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 IRF3 undergoes LLPS.

a, Domain structure (upper) and the intrinsically disordered tendency (lower) of IRF3. IUPred2, ANCHOR2 and VSL2 assigned scores of disordered tendencies between 0 and 1 to the sequences were shown. b, Bacterial purified GFP-IRF3 wt, d_DBD, d_IDR, d_IAD and d_ID proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. c–e, 5 μM GFP-IRF3 were treated with 5% Hex (c), heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) (d), or treated with 100 μg/ml Proteinase K for 30 min at 40 °C (e) and then subjected to droplet formation assay in vitro (200 mM NaCl, pH 7.0, room temperature). Mean ± s.d., n = 6 independent experiments. f, 3D reconstruction of activated GFP-IRF3 puncta in HeLa cells followed by stimulation with SeV for 12 h. Series z-stack images of live cells were captured by confocal microscope and then 3D reconstruction was performed. Typical optical sections of z-stack were shown. g, Left: a schematic describing the generation of site-specific phosphorylated recombinant IRF3 protein with a Sep-accepting tRNA (tRNASep) and its cognate phosphoseryl-tRNA synthase (SepRS), which incorporates the phosphorylated serine on the amber codon. Right: IB of the purified protein with antibody specific to phospho-Ser386 and phospho-Ser396 of IRF3. The anti-Myc blot indicates loading of lanes. h, Immunofluorescence microscopy and DAPI staining of L929 cells showed nuclear puncta of endogenous IRF3 upon PBS or SeV stimulation for 12 h. i, Related to Fig. 2e: quantified percentages of cells harboring GFP puncta and GFP nuclear puncta upon SeV stimulation were shown. n = 3. j, qPCR analysis of IFNB1 mRNA in IRF3 KO cells transfected with indicated plasmid (s) followed by mock infected (PBS) or infection for 12 h with SeV. n = 3. Data are representative of three independent experiments (b, f, g, h–j). n = 6 biological independent samples (c–e). Scale bar, 5 μm (c–f, h). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–e, i, j).

Source data

Extended Data Fig. 2 IRF7 undergoes LLPS with IRF3.

a, Domain structure (upper) and the intrinsically disordered tendency (lower) of IRF7. IUPred2, ANCHOR2 and VSL2 assigned scores of disordered tendencies between 0 and 1 to the sequences were shown. b, Bacterial purified mCherry-IRF7 wt, d_DBD, d_IDR, d_IAD and d_ID proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. c–e 5 μM mCherry-IRF7 were treated with 5% Hex (c), 100 μg/ml Proteinase K for 30 min at 40 °C (d), or treated with heated-inactivated (5 min at 95 °C and immediately put on ice for 5 min) (e), and then subjected to droplet formation assay in vitro (200 mM NaCl, pH 7.0, room temperature). f, Representative fluorescence and DIC images of mCherry-IRF7 (5 μM) droplets formation at room temperature with indicated concentrations of NaCl at pH 7.0. g, Representative fluorescence and DIC images of mCherry-IRF7 (5 μM) droplets formation at indicated temperature with 200 mM NaCl at pH 7.0. Scale bar, 10 μm. h, Left: representative micrographs of mCherry-IRF7 (5 μM) droplets before and after photobleaching. Right: quantification of FRAP of mCherry-IRF7 droplet over a 200 s time course (mean ± s.d., n = 3 droplets). i, Time-lapse micrographs of the fusion of AF594 labeled IRF7 (10 μM) droplets at room temperature with 200 mM NaCl at pH 7.0. n = 3. j, Purified mCherry-IRF7 wt, d_DBD, d_IDR, d_IAD, and d_ID proteins (5 μM) was analyzed using droplet formation assays at room temperature with 200 mM NaCl at pH 7.0. k, Fusion upon contact of droplets formed by GFP-IRF3 and mCherry-IRF7 proteins. l, Related to Fig. 3k: the endogenous IRF7 puncta were quantified in control and IRF3 KO cells. Data are representative of three independent experiments (b, i, k). n = 6 biological independent samples (c–g, j, l). Scale bar, 5 μm (c–k). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c–g, j, l).

Source data

Extended Data Fig. 3 SIRT1 inhibition abolishes IRF3 LLPS and IFN signaling.

a, Immunofluorescence microscopy and DAPI staining of IRF3–GFP in IRF3–GFP stable cells pre−treated with DMSO or EX527 (20 μM), followed by infection for 8 h with HSV-1 (left). Scale bar, 5 μm. Quantified average number of nuclear IRF3 puncta and the percentages of cells with nuclear IRF3 puncta were shown (right). b, HeLa cells infected for 12 h with HSV-1 were grown on collagen-coated microchamber slides. After fixation, in situ PLA for IRF3/IRF7 was performed with α-IRF3 and α-IRF7 antibodies. The PLA-detected proximity (PROX) complexes are represented by the fluorescent rolling circle products (red dots) (left). Scale bar, 5 μm. Quantification of the PROX score is shown as means ± SD (right). c, IFN-β-Luc, PRD I-III-Luc and IFN-α-Luc activity in HEK293T cells pre-treated with DMSO or EX527 (20 μM) and infected for 12 h with SeV. d, qPCR analysis of Ifnb1 and Ifna mRNA level in RAW264.7 macrophages pre-treated with DMSO or EX527 (20 μM) and infected for 12 h with VSV (MOI, 0.1) or HSV-1 (MOI, 10). e, Immunoblot analysis of SIRT1 knockdown efficiency with independent sh-SIRT1 (#1 to #4 independent constructs) in HEK293T cells. f, IFN-β-Luc, PRD I-III-Luc IFN-α-Luc activity in HEK293T cells depleted for SIRT1 with sh-SIRT1 #1 and stimulated for 12 h with SeV. g, qPCR analysis of sh-SIRT1 #1 & #2 efficiency (left panel), IFNB1 and IFNA mRNA level (middle and right) in control and SIRT1-depleted HEK293T cells followed by SeV infection at the indicated time points. h, qPCR analysis of Sirt1 mRNA (left), Ifnb1(middle) and Ifna (right) mRNA in RAW264.7 cells transfected with siRNA (Co.) or si-Sirt1, followed by infection for various times (horizontal axis) with SeV; results are represented relative to those of the control gene Gapdh. Data are representative of three independent experiments (a, b). n = 6 (a, b) or 3 (c, d, f–h) biological independent samples. Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a (right), b (right), c, d, f–h).

Source data

Extended Data Fig. 4 SIRT1 enhances innate antiviral response.

a, IFN-β-Luc, PRD I-III-Luc and IFN-α-Luc activity in HEK293T cells transfected with control empty vector (Co.vec), wild-type SIRT1 (wt), or the catalytically inactive SIRT1 mutant (H363Y), followed by infection for 12 h with SeV. b, qPCR analysis of Ifnb1 and Ifna mRNA in HEK293T cells transfected with control empty vector (Co.vec), SIRT1 wt or H363Y and treated with SeV or poly(I:C). c, qPCR analysis of IFNB1 and IFNA mRNA in RAW264.7 cells transfected with control empty vector (Co.vec), SIRT1 wt or H363Y and treated with SeV or poly(I:C) for various time points. d, qPCR analysis of IFNB1 and IFNA1 mRNA in HEK293T cells transfected with control empty vector (Co.vec) or expression plasmid(s) encoding SIRT1 wt or H363Y, IRF3-5D or IRF7 as indicated. e, Bright field microscopy (top) and fluorescence microscopy (bottom) of VSV–GFP in HEK293T cells transfected with indicated control empty vector (Co.vec), SIRT1 wt or H363Y, followed by infection for 12 h with GFP-expressing VSV (MOI, 0.1) (left). Scale bars, 100 μm. The fold change in VSV–GFP intensity was quantified using ImageJ (right). f, qPCR analysis of IFNB1 (far left) and IFNA mRNA (left), VSV RNA (right), and plaque assay of VSV (far right), in HEK293T cells transfected with expression plasmids for SIRT1 wt or H363Y and mock infected (PBS) or infected for 8 h with VSV (MOI, 0.1). Data are representative of three independent experiments (e). n = 3 biological independent samples (a–f). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a–d, e (right), f).

Source data

Extended Data Fig. 5 Sirt1 deficiency potentiates innate antiviral immunity.

a, Schematic diagram of Sirt1 knockout strategy. Lyz2-Cre+Sirt1f/f mice in C57BL/6 N background was generated by targeting of exon 4 of Sirt1 using CRISPR/CAS9. Deletion of exon 4 results in frame shift and disrupts its open reading frame (ORF), leading to the loss of Sirt1 expression. b, Left: immunoblot analysis (IB) of Sirt1 in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages, assessed after immunoprecipitation (IP), was shown. Right: qPCR analysis of Ifnb1, Ifna, Cxcl10 and Ccl5 mRNA in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages infected with SeV for the indicated time points. c, qPCR analysis of Ifnb1, Cxcl10 and Ccl5 mRNA in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages stimulated for indicated time points with 5′-triphosphorylated RNA (5′-ppp RNA). d, qPCR analysis of Ifnb1 and Ifna mRNA in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages transfected with poly(I:C) for indicated time points. Data presented as mean ± s.d., n = 3 biological independent samples and the statistical analysis was performed using two-tailed Student’s t-test (b–d).

Source data

Extended Data Fig. 6 IFN signaling is down-regulated in Sirt1-deficient cells.

a, qPCR analysis of Ifnb1 and Ifna mRNA, VSV copy number, and plaque assay of VSV (right), in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages mock infected (PBS) or infected with VSV (MOI, 0.1) for various times (horizontal axis). b, qPCR analysis of Ifnb1 and Ifna mRNA, copy number of HSV-1 genomic DNA and plaque assay of HSV-1 in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f peritoneal macrophages mock infected (PBS) or infected with HSV-1 (MOI, 10) for various time courses (horizontal axis). c, qPCR analysis of Ifnb1 and Ifna mRNA in Lyz2-Cre−Sirt1f/f and Lyz2-Cre+Sirt1f/f Bone Marrow Derived Macrophages (BMDMs) treated with SeV (Upper), poly(I:C) (Middle) or HSV-1 (Lower) respectively for indicated time points. d, qPCR analysis of Ifnb1 and Ifna mRNA in wild-type and Sirt1−/−MEF cells treated with SeV (upper), poly(I:C) (middle) or HSV-1 (lower) (MOI, 10) respectively for indicated time points. Data are presented as mean ± s.d.; n = 3 biological independent samples and the statistical analysis was performed using two-tailed Student’s t-test (a–d).

Source data

Extended Data Fig. 7 Site-specific acetyl-mimicking mutations block IRF3/7 LLPS and their transcriptional activities.

a, IB of the TCL and IP with control IgG and α-acetyl-lysine (acetyl-K) derived from PBMCs (left) or MEFs (right) treated for 8 h with DMSO or EX527 (20 mM). b, Flag-tagged IRF3 and IRF7 were immunoprecipitated from HEK293T cells pre-treated for 8 h with DMSO or EX527 (20 mM) and stained with coomassie brilliant blue (left). The representative IRF3 peptide carrying acetylated Lys39 or Lys77 and IRF7 peptide carrying acetylated Lys45 or Lys92 were identified by Mass spectrometry (right). n = 3. c, Bacterially purified SIRT1 wt and HY (left), GFP-SIRT1 wt and HY (right) proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. n = 3. d, Bacterially purified IRF3 wt and 2KQ, IRF7 wt and 2KQ proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. n = 3. e, Related to Fig. 6l: representative micrographs of droplet formation by GFP-IRF3 wt and 2KQ (5 μM) mixed with ISRE DNA (500 nM) before and after photobleaching. n = 3. f, Related to Fig. 6m: representative micrographs of droplet formation by mCherry-IRF7 wt and 2KQ (5 μM) mixed with ISRE DNA (500 nM) before and after photobleaching. n = 3. g, qPCR analysis of ISG56 mRNA in IRF3 KO cells transfected with Co.vec and IRF3 wt/KQ mutations (left) or IRF7 wt/KQ mutations (right) as indicated, followed by mock infected (PBS) or infection for 12 h with SeV. n = 3. Data are representative of three independent experiments (a–g). Scale bar, 5 μm (e, f). Mean ± s.d.; statistical analysis was performed using two-tailed Student’s t-test (g).

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Extended Data Fig. 8 Aged mice show reduced SIRT1 activity and impaired innate antiviral immunity.

a, Cellular SIRT1 activity in PBMCs of non-aged (n = 12, 2–3 months old) and aged (n = 10, 20 months old) mice. b, ELISA of IFN-β in PBMCs from mice as in a infected for 12 h with VSV (MOI, 0.1) (left); Correlation of IFN-β and cellular sirt1 activity in PBMC cells from mice as in a after VSV stimulation (right). c, ELISA of IFN-β in PBMCs from mice as in a infected for 12 h with HSV-1 (MOI, 10) (left); Correlation of IFN-β and cellular sirt1 activity in PBMC cells from mice as in a after HSV-1 stimulation (right). d, IB of TCLs and proteins immunoprecipitated with antibodies to (anti-) acetyl-Lys39 or acetyl-Lys77 of IRF3 (upper) and (anti-) acetyl-Lys45 or acetyl-Lys92 of IRF7 (lower) from PBMCs of non-aged (n = 5) and aged (n = 5) mice, non-infected (−) or infected for 12 h with SeV. e, Immunofluorescence microscopy and DAPI staining of Mouse Pulmonary Fibroblasts (MPF) cells infected for 12 h with SeV. Intensity of intranuclear IRF3 or IRF7 puncta and the percentage of cells showing IRF3 or IRF7 puncta were quantified by ImageJ. Scale bar, 5 μm. f, qPCR analysis of Ifnb1 mRNA in the lungs, spleen and liver of non-aged and aged mice (n = 5 mice per group) given intraperitoneal injection of PBS or VSV (5 × 108 PFU per mouse) for 24 h. g, ELISA of IFN-β in serum from mice as in f. h, qPCR analysis of VSV mRNA in the lungs, spleen and liver of infected mice as in f (left); Plaque assay of VSV in the lungs, spleen and liver of infected mice as in f (right). i, Immunoblot analysis of VSV-G in the liver, lungs and spleen of infected mice as in f. j, Microscopy of hematoxylin-and-eosin (H&E)-stained lung sections from mice as in f. Scale bar, 100 µm. k, Survival rates of non-aged and aged mice (n = 5 mice per group) at various times (horizontal axes) after intraperitoneal infection with VSV (2 × 109 PFU per mouse). l, qPCR analysis of Ifnb1, Cxcl10 and Isg56 mRNA in the brain of non-aged and aged mice (n = 5 mice per group) given intraperitoneal injection of PBS or HSV-1 (5×108 PFU per mouse) for 24 h. m, ELISA of IFN-β in serum from mice as in l. n, qPCR analysis of HSV-1 genomic DNA in brain of mice as in l. o, Plaque assay of HSV-1 in brain of mice as in l. p, Survival rates of non-aged and aged mice (n = 5 mice per group) at various times (horizontal axes) after intraperitoneal infection with HSV-1 (2 × 109 PFU per mouse). Data are representative of at least three independent experiments (d, e, i, j). Mean ± s.d., n = 5 biologically independent animals (g, h, m–o); statistical analysis was performed using two-tailed Student’s t-test (a–c, e–h, l–o) or two-way ANOVA (k, p).

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Extended Data Fig. 9 STACs promote SIRT1-mediated deacetylation of IRF3/7 and elevate the innate antiviral response.

a, Immunoblot (IB) of the total cell lysate (TCL) and immunoprecipitates (IP) derived from Sirt1+/+ and Sirt1−/− MEFs treated for 12 h with control DMSO (−), SRT501 (50 μM) or SRT2183 (10 μM). b, ChIP in Sirt1+/+ and Sirt1−/− MEF cells pre-treated with control DMSO (−), SRT501 (50 μM) or SRT2183 (10 μM), followed by infection for 12 h with SeV. c–d, qPCR analysis of Ifnb1 (c, left), Ifna (c, right) and VSV mRNA (d) in spleen, liver and lung from mice as in Fig. 8g. e, Microscopy of hematoxylin-and-eosin (H&E)-stained lung sections from mice as in Fig. 8g. Scale bar, 100 µm. n = 3. f, Immunofluorescence microscopy of IRF3 (upper), IRF7 (lower) and DAPI staining in liver from mice as in Fig. 8g. n = 3. Data are representative of three independent experiments (a). n = 3 (b, e, f) or 4 (c, d) independent biological replicates. Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (b–d).

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Qin, Z., Fang, X., Sun, W. et al. Deactylation by SIRT1 enables liquid–liquid phase separation of IRF3/IRF7 in innate antiviral immunity. Nat Immunol 23, 1193–1207 (2022). https://doi.org/10.1038/s41590-022-01269-0

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