Abstract
Metabolic pathways determine cellular fate and function; however, the exact roles of metabolites in host defence against influenza virus remain undefined. Here we employed pharmacological inhibition and metabolomics analysis to show that the metabolic pathways of oxaloacetate (OAA) are integrated with antiviral responses to influenza virus. Cytosolic malate dehydrogenase 1 senses intracellular OAA to undergo dimerization and functions as a scaffold to recruit the transcription factor ETS2 for phosphorylation by the kinase TAOK1 at serine 313. The phosphorylated ETS2 translocates into the nucleus and supports optimal expression of TBK1, an indispensable activator of type I interferon responses. OAA supplementation provides a broad-spectrum antiviral ability, and OAA deficiency caused by Acly genetic ablation decreases antiviral immunity and renders mice more susceptible to lethal H1N1 virus infection. Our results uncover a signalling pathway through cellular OAA sensing that links metabolism and innate immunity to coordinate defence against viral challenge.
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Data availability
The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD050149. RNA-seq and ATAC-seq analyses have been submitted to the Gene Expression Omnibus (GEO) under accession number GSE297098 and GSE259217, respectively. The scRNA-seq data used in this study can be found with GEO accession number GSE243629 and were previously deposited12. Source data are provided with this paper.
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Acknowledgements
We thank H. Zhang (Zhongshan Medical School, Sun Yat-sen University), C. Sun (School of Public Health, Shenzhen, Sun Yat-sen University), S. Chen (Sun Yat-sen University Cancer Center), X. Qin (Suzhou Institute of Systems Medicine), Y.-P. Li (Zhongshan Medical School, Sun Yat-sen University) and A. Yueh (National Health Research Institutes in Taiwan) for virus-related experiments. This work was supported by the National Natural Science Foundation of China (32170876 to S.J., 82341047 and 32270922 to J.C., 324B2020 to X.H., 32070918 to J.W., 32300724 to S.Y., 82471781 to Y.W.), the Fundamental Research Funds for the Central Universities, Sun Yat-sen University (23lgbj012 to S.J., 23yxqntd001 to J.C.), the Distinguished Young Scholars in Guangdong Province (2022B1515020109 to J.W.), start-up funding for the Pediatric Research Institute of GWCMC (3001082 to J.W.), the Guangdong Province Excellent Youth Team Project (2024B1515040009 to J.C.), the Science and Technology Planning Project of Guangdong Province (2023B1212060028 to J.C.) and Major Project of Guangzhou National Laboratory (GZNL2024A01016 to N.Q.).
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Contributions
J.C. and S.J. conceived of the project and designed the experiments. X.H. and S.J. performed the experiments. Z.W., T.Z., J.W., G.P., Y. Zhang, L.M., S.Y., L.W., Y.W., Y. Zou and N.Q. provided technical help. S.J. and X.H. analysed the data. J.C. provided resources and directed the research. S.J. and J.C. wrote the paper. All authors read and approved the final paper.
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Nature Microbiology thanks Steven Baker, Andreas Pichlmair, David Suter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Identification of OAA as a responsive metabolite upon viral infection.
a, The key metabolic pathways and their indicated inhibitory targets. b, Plaque titration of influenza virus in supernatants of A549 cells with H1N1 (PR8) virus (MOI = 1) infection for 24 h, together with 2-deoxyglucose (2-DG) (5 mM), fluasterone (10 μM), GOT1 inhibitor-1 (10 μM), MDH1-IN-2 (10 μM), dimethyl-malonate (DMM) (2 mM), MEDICA16 (200 μM), or TOFA (30 μM) treatment for 12 h. c, Content of cytosolic OAA in THP-1 cells cultured in the presence of DEOAA or OAA with indicated dosages for 4 h. d, A549 cells were pretreated with vehicle (alcohol), DEOAA (1 mM) or OAA (10 mM) for 12 h and challenged with H1N1 virus (MOI = 1) for 24 h. The supernatants were collected for plaque assay. e, Cell viability of THP-1 cells measured by the LDH assay when incubated with different concentrations of DEOAA or OAA for 24 h. f,g, Cell viability of A549 cells (f) or THP-1 cells (g) measured by the LDH assay with DEOAA (1 mM) treatment. h, The heatmap of metabolites determined by targeted LC-MS/MS metabolomics assay in THP-1 cells infected with H1N1 (PR8) virus (MOI = 1). i,j, Content of OAA in THP-1 cells (i) or A549 (j) cells infected H1N1 (PR8) virus (MOI = 1). k, Relative levels of OAA in the lung homogenates from C57BL/6 J mice (n = 6 per group) administered with or without sodium diethyl-oxaloacetate (20 mg/kg; i.p.) every day for 3 d. b–g,i,j, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). k, Data are represented as mean ± s.d. P values were determined using two-tailed unpaired Student’s t test.
Extended Data Fig. 2 OAA is involved in antiviral responses.
a, THP-1-derived macrophages cultured within 13C4 OAA (5 mM) for 12 h with either PBS or H1N1 (PR8) virus (MOI = 1) treatment for 8 h. Schematic representation of 13C4 OAA-derived carbon tracing of metabolites. b–i, Isotopomer analysis of 13C4 OAA-derived carbon incorporation of N-acetyl-D-glucosamine 6-phosphate (GlcNAc6P) (b), D-fructose 1,6-bisphosphate (Fructose-1,6P2) (c), phosphoenolpyruvate (PEP) (d), pyruvate (e), acetyl-CoA (f), citrate (g), malate (h), and aspartate (i) in THP-1-derived macrophages treated with either PBS or H1N1 (PR8) virus (MOI = 1) for 8 h in addition to 13C4 OAA treatment for 12 h (n = 3). j, qPCR analysis of mRNA level of indicated genes in THP-1 cells expressing corresponding single-guide RNAs (sgRNAs). k, THP-1 cells expressing indicated sgRNA were challenged with H1N1 (PR8) virus (MOI = 1) for 24 h, and the supernatants were collected for plaque assay. b–i, Data are represented as mean ± s.d. j,k, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-tailed unpaired Student’s t test.
Extended Data Fig. 3 OAA potentiates antiviral innate immune signaling.
a, Plaque titration of influenza virus in supernatants of A549 cells with H1N1 (PR8) virus (MOI = 1) infection, along with DEOAA (1 mM) in the absence or presence of 2-DG (5 mM) or DMM (2 mM) for 12 h. b,c, THP-1 cells pre-treated with DEOAA (1 mM) for 4 h and infected with H1N1 (PR8) virus (MOI = 1) for 8 h were used for OCR (b) and ECAR (c) analysis. Cells were supplied with 1.5 μM oligomycin (O), 1 μM FCCP, and 0.5 μM rotenone/antimycin (R & A) for OCR analysis, and 10 mM glucose (G), 1 μM oligomycin (O), and 50 mM 2-DG for ECAR analysis by using a Seahorse XFe96 analyzer system. d,e, C57BL/6 J mice (n = 6 per group) were administered sodium diethyl-oxaloacetate (20 mg/kg; i.p.) every day for 3 d and given OT-82 (10 mg/kg; i.p.) or dicoumarol (10 mg/kg; i.p.) once daily for 2 d. Relative NADH/NAD+ levels (d) and virus titers of influenza virus (e) in the lung homogenates were analyzed after intranasal H1N1 (PR8) virus inoculation for 3 d. f,g, C57BL/6 J mice (n = 6 per group) were administered sodium diethyl-oxaloacetate (20 mg/kg; i.p.) every day for 3 d and given N-acetyl-L-cysteine (NAC) (10 mg/kg; i.p.) or Mito-TEMPO (MT) (10 mg/kg; i.p.) once daily for 2 d. Relative ROS levels (f) and virus titers of influenza virus (g) in the lung homogenates were analyzed after intranasal H1N1 (PR8) virus inoculation for 3 d. h, PBMCs infected with H1N1 (PR8) virus (MOI = 1) in the absence or presence of DEOAA (1 mM). Relative expression levels of genes were measured by qPCR. i, qPCR analysis of IFNB1 and ISGs in A549 cells infected with H1N1 (PR8) virus (MOI = 1) in the absence or presence of DEOAA (1 mM). j, Intracellular NP vRNA levels from samples as in (i). k, PBMCs were vehicle-treated or treated with intracellular (IC) poly (I:C) (10 μg/mL), poly (dA:dT) (1 μg/mL), ISD (1 μg/mL) or LPS (100 ng/mL), along with DEOAA (1 mM). The transcription levels of IFNB1 were analyzed by qPCR. a–c,h–k, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). d–g, Data are represented as mean ± s.d. P values were determined using two-tailed unpaired Student’s t test.
Extended Data Fig. 4 OAA participates in TBK1-mediated immunity.
a, THP-1 cells were treated with DEOAA (1 mM) for 12 h, together with MG132 (10 μM), 3-methyladenine (10 mM), or bafilomycin A1 (0.2 μM) for 6 h. The protein extracts were collected for immunoblot analysis. b, qPCR analysis of Tbk1 mRNA in BMDMs from C57BL/6 J mice with increasing doses of DEOAA treatment for 12 h. c, TBK1 KO THP-1-derived macrophages expressing TBK1 under the control of synthetic hEF/HTLV strong promoter were pretreated with DEOAA (1 mM) for 12 h. The cells were challenged with H1N1 virus (MOI = 1) for 12 h and the protein extracts were harvested and analyzed by immunoblotting. d, The transcription levels of IFNB1 and ISGs in similar samples as (c) were analyzed by qPCR. e, Intracellular NP vRNA levels in the sample as (c). f, ATAC-seq volcano plots showing genes with significant chromatin accessibility across transcription start site (TSS) in PBMCs treated with DEOAA (1 mM) for 24 h as compared to those from vehicle-treated groups. g, Heatmap representation of chromatin accessibility (color) within 1.5 kb on either side of the TSS in PBMCs with DEOAA (1 mM) treatment for 24 h versus vehicle-treated group. b,d,e, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-tailed unpaired Student’s t test. a,c, Similar results were obtained in 3 independent biological experiments.
Extended Data Fig. 5 OAA positively regulates antiviral immune activation via ETS2.
a, Monocytes/macrophages and DCs identified by scRNA-seq from data deposited to the Gene Expression Omnibus (GEO) under accession number GSE243629. The UMAP projection from healthy controls and infected adults. b, Dot plots displaying marker gene expressions for pDCs, cDC2, CD14+ monocytes and CD16+ monocytes. c, The knockdown efficiency of the siRNAs in THP-1-derived macrophages was detected by qPCR. d, ELISA of IFNβ in PBMCs transfected with Scramble (Scr) or ETS2-specific siRNAs infected with H1N1 (PR8) virus (MOI = 1) for 24 h in the presence of diethyl-oxaloacetate (DEOAA) (1 mM). e, A549 cells transfected with Scr or ETS2-specific siRNAs were challenged with H1N1 (PR8) virus (MOI = 1) in the absence or presence of DEOAA (1 mM). The transcription levels of IFNB1 and ISGs were analyzed by qPCR. f, Intracellular NP vRNA levels in the sample as (e). g, The virus titers of A549 cells transfected with Scr or ETS2-specific siRNAs were infected with H1N1 (PR8) virus (MOI = 1) for 24 h in the presence of diethyl-oxaloacetate (DEOAA) (1 mM). h, 293 T cells transfected with Scr or ETS2-specific siRNAs, together with plasmid of TBK1-Luc. The cells were treated with DEOAA (1 mM) and the protein extracts were harvested for luciferase assay. i, Upper panel: violin plots comparing the antiviral response scores in different cell subpopulations between IAV-infected individuals and healthy controls. Single-cell antiviral response scores were determined using the AddModuleScore function in the R toolkit of Seurat to measure the average expression of a predefined set of antiviral genes in each cell. Lower panel: bar plots comparing the ETS2 regulon activities within myeloid subpopulations between IAV-infected individuals and healthy controls. The ETS2 regulon values were deduced using the runSCENIC_3_scoreCells function from the SCENIC toolkit to calculate the enrichment of the putative target gene set of ETS2 as an area under the recovery curve across the ranking of all genes in a particular cell, whereby genes were ranked by their expression values. c–h, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-tailed unpaired Student’s t test (c, d and f–i) or two-way ANOVA with Tukey’s multiple-comparison test (e).
Extended Data Fig. 6 OAA prompts ETS2 activation in a MDH1-dependent manner.
a, Total, cytosolic, and nuclear fractions of THP-1 cells pre-treated with DEOAA (1 mM) for 12 h were immunoblotted with antibodies directed against ETS2, a cytosol marker (α-Tubulin), or a nucleus marker (PCNA). b, A549 cells transfected with Scr or MDH1-specific siRNAs were challenged with H1N1 (PR8) virus (MOI = 1) in the absence or presence of DEOAA (1 mM). The transcription levels of IFNB1 and ISGs were analyzed by qPCR. c, Intracellular NP vRNA levels in the sample as (b). d, Lysates of 293 T cells transfected with plasmids of Flag-MDH1, Flag-MDH2 and HA-ETS2 were immunoprecipitated with α-Flag M2 beads and immunoblotted with anti-HA. e, Luciferase activity of WT and MDH1 KO 293 T cells transfected with plasmid of TBK1-Luc in the absence or presence of DEOAA (1 mM). f, WT Flag-MDH1 or its indicated mutants purified from 293 T cells were incubated with biotin-labelled OAA in vitro, and subjected to pull-down assay using streptavidin-coated magnetic beads for further immunoblot analysis. g, Relative NADH/NAD+ levels of MDH1-deficent THP-1 cells reconstituted with plasmids of Flag-MDH1 and its indicated mutant. h, Extracts of THP-1 cells cultured in increasing doses of DEOAA were treated with 2 mM disuccinimidyl suberate (DSS) cross-linker and analyzed by immunoblotting. i, Extracts of 293 T cells expressing Flag-tagged WT MDH1 or its indicated mutants in the absence or presence of DEOAA (1 mM) were treated with 2 mM DSS cross-linker and analyzed by immunoblotting. j, Extracts of 293 T cells expressing Flag-tagged WT MDH1 or its indicated mutants in the absence or presence of DEOAA (1 mM) were treated with 2 mM DSS cross-linker and analyzed by immunoblotting. k, MDH1 KO 293 T cells were transfected with plasmid of TBK1-Luc, along with the vectors of encoding WT, or indicated mutant form Flag-MDH1. The cells were treated with DEOAA (1 mM) and the protein extracts were harvested for luciferase assay. l, MDH1 KO THP-1 cells expressing WT or indicated point mutated form of MDH1 were challenged with H1N1 (PR8) virus (MOI = 1) for 24 h and the samples were harvested and analyzed by ELISA. m, MDH1 KO THP-1 cells expressing WT or indicated point mutated form of MDH1 were infected with H1N1 (PR8) virus (MOI = 1) for 24 h, and the supernatants were collected for plaque assay. b,c,e,g,k–m, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-way ANOVA with Tukey’s multiple-comparison test (b) or two-tailed unpaired Student’s t test (c, e, g and k–m). a,d,f,h–j, Similar results were obtained in 3 independent biological experiments.
Extended Data Fig. 7 OAA promotes ETS2 phosphorylation at S313 by TAOK1.
a–c, Immunoprecipitation and immunoblot analysis of THP-1 cells treated DEOAA (1 mM) with indicated antibodies. d, Total, cytosolic, and nuclear fractions of THP-1 cells transfected with Scr or TAOK1-specific siRNA in the presence of DEOAA (1 mM) were immunoblotted with indicated antibodies. e, Representative image of HeLa cells transfected with Scr or TAOK1-specific siRNA in the presence of DEOAA (1 mM), followed by labelling of ETS2 (green) with specific antibody. Scale bar, 20 μm. f, Quantitative analysis of the similar samples as (e) from three biologically independent experiments (20 cells scored per condition per experiment). g, Total, cytosolic, and nuclear fractions of 293 T cells transfected with Flag-TAOK1 (WT or K57A) were immunoblotted with indicated antibodies. h, Representative image of HeLa cells transfected with plasmids encoding WT and the indicated mutant form of ETS2, together with TAOK1 (WT or K57A), followed by labelling of ETS2 (green) with specific antibody. Scale bar, 20 μm. i, Quantitative analysis of the similar samples as (h) from three biologically independent experiments (20 cells scored per condition per experiment). j, Total, cytosolic, and nuclear fractions of 293 T cells transfected with HA-tagged WT, K313A or K313D mutant form ETS2 were subjected to immunoblotting. k, 293 T cells transfected plasmids of HA-ETS2 and Flag-tagged WT MDH1 as well as its indicated mutants were treated with DEOAA (1 mM). The extracts of were immunoprecipitated using phosphoserine/theonine/tyrosine polyclonal antibody and immunoblotted with ETS2 antibody. l, Extracts of THP-1 cells transfected with Scr, MDH1- and/or TAOK1-specific siRNAs and overexpressed ETS2 S313D mutant were analyzed by immunoblotting. f,i, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-tailed unpaired Student’s t test. a–e,g,h,j–l, Similar results were obtained in 3 independent biological experiments.
Extended Data Fig. 8 Dynamic OAA production caused by ACLY modulation contributes to antiviral responses.
a, Schematic diagram depicting the production of cellular OAA. b, The knockdown efficiency of the siRNAs was detected by qPCR in THP-1 cells transfected with Scr, ACLY-, GOT1-, MDH2- or PC-specific siRNAs. c, The cellular OAA levels in THP-1 cells transfected with Scr, ACLY-, GOT1-, MDH2- or PC-specific siRNAs in the absence or presence of DEOAA (1 mM). d, THP-1 cells transfected with Scr, ACLY-, GOT1-, MDH2- or PC-specific siRNAs were infected with H1N1 (PR8) virus (MOI = 1) for 24 h in the absence or presence of DEOAA (1 mM). The total RNA was harvested and the transcription levels of IFNB1 were analyzed by qPCR. e, Intracellular NP vRNA levels in the sample as (d). f, Schematic diagram of Acly knockout strategy. Deletion of exon 3 and exon 6 results in frame shift and disrupts its open reading frame, leading to the loss of Acly expression. g, Activated macrophages enriched from lung tissues of Aclyfl/fl and Aclyfl/flLyz2Cre mice (n = 3 per group) given intranasal H1N1 (PR8) virus inoculation for 3 d, and the cell lysates were analyzed by immunoblotting with indicated antibodies. h, OAA levels in PBMCs cultured with MEDICA16 (200 μM) or BMS-303141 (200 μM) for 12 h with or without DEOAA (1 mM) treatment. i, ELISA of IFNβ in the supernatant of PBMCs infected with H1N1 (PR8) virus (MOI = 1) in the presence of DEOAA (1 mM), along with MEDICA16 (200 μM) or BMS-303141 (200 μM). j,k, OAA content (j) and qPCR analysis of Tbk1 (k) in BMDMs from Aclyfl/fl and Aclyfl/flLyz2Cre mice cultured with DEOAA (1 mM). b–e,h–k, Data are represented as mean ± s.e.m. (n = 3 independent biological experiments). P values were determined using two-tailed unpaired Student’s t test.
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Jin, S., He, X., Wang, Z. et al. Oxaloacetate sensing promotes innate immune antiviral defence against influenza virus infection. Nat Microbiol 10, 2521–2536 (2025). https://doi.org/10.1038/s41564-025-02107-3
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DOI: https://doi.org/10.1038/s41564-025-02107-3
