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. 2018 Dec 12;24(6):791-803.e6.
doi: 10.1016/j.chom.2018.11.001.

O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity

Tianliang Li  1 , Xinghui Li  1 , Kuldeep S Attri  2 , Changhong Liu  3 , Lupeng Li  1 , Laura E Herring  4 , John M Asara  5 , Yu L Lei  6 , Pankaj K Singh  7 , Chengjiang Gao  8 , Haitao Wen  9
Affiliations

O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity

Tianliang Li et al. Cell Host Microbe. .

Abstract

Increased glucose metabolism in immune cells not only serves as a hallmark feature of acute inflammation but also profoundly affects disease outcome following bacterial infection and tissue damage. However, the role of individual glucose metabolic pathways during viral infection remains largely unknown. Here we demonstrate an essential function of the hexosamine biosynthesis pathway (HBP)-associated O-linked β-N-acetylglucosamine (O-GlcNAc) signaling in promoting antiviral innate immunity. Challenge of macrophages with vesicular stomatitis viruses (VSVs) enhances HBP activity and downstream protein O-GlcNAcylation. Human and murine cells deficient of O-GlcNAc transferase, a key enzyme for protein O-GlcNAcylation, show defective antiviral immune responses upon VSV challenge. Mechanistically, O-GlcNAc transferase-mediated O-GlcNAcylation of the signaling adaptor MAVS on serine 366 is required for K63-linked ubiquitination of MAVS and subsequent downstream retinoic-acid inducible gene-like receptor -antiviral signaling activation. Thus, our study identifies a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates MAVS function and highlights the importance of glucose metabolism in antiviral innate immunity.

Keywords: MAVS; O-GlcNAc transferase; antiviral immunity; glucose metabolism; hexosamine biosynthesis pathway (HBP).

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
RLR activation promotes glucose metabolism in macrophages. (A and B) Total metabolite profiling determined by LC-MS/MS metabolomics assay was assessed by principle component analysis (A) and pathway-enrichment analysis (B) in bone marrow-derived macrophages (BMMs) generated from C57BL/6 mice stimulated with vesicular stomatitis virus (VSV) (multiplicity of infection (MOI) = 1) for 4 h. (C) A summary of three glucose metabolic pathways, including the glycolysis (middle), PPP (left) and HBP (right). (D to G) Heatmap of metabolites (D) and fold changes in intermediate metabolites of the glycolysis (E), PPP (F), or HBP (G). (H) Fold change in 13C-labelled UDP-GlcNAc between non-treated and VSV-challenged BMMs in the presence of 13C6-glucose. (I) Immunoblotting (left) and densitometric analysis based on five independent experiments (right) to quantify ratio of total O-GlcNAc to actin in BMMs left untreated or treated with VSV or transfected with poly(I:C) (4 μg/ml) by lipofectamine 2000 for indicated periods. * P < 0.05, versus controls (two-tailed Student’s t-test (E to H)). Data are from one experiment representative of three experiments (A and B, D to G; mean ± s.d. of six biological replicates) or two experiments (H; mean ± s.d. of four biological replicates) or represent five independent experiments (I).
Figure 2.
Figure 2.
OGT deficiency impairs antiviral immune responses in vitro. (A to D) BMMs generated from Ogtfl/fl and OgtΔmye mice were left untreated or stimulated with VSV (MOI = 1) (A and B) or transfected with 4 μg/ml poly(I:C) by lipofectamine 2000 (C and D) for indicated periods. Gene transcripts in the cells (A and C), IFN-β, IL-6 and TNF-α proteins in the supernatants (B and D) were measured with RT-PCR and ELISA, respectively. (E and F) Gene transcripts in the cells (E) and cytokines in the supernatants (F) from Ogtfl/fl or OgtΔmye peritoneal macrophages left untreated or stimulated with VSV for indicated periods. * P < 0.05, versus controls (two-tailed Student’s t-test (A to F)). Data are from one experiment representative of five experiments (A to F; mean ± s.d. of four biological replicates).
Figure 3.
Figure 3.
OGT is critical for the activation of antiviral immune signaling and antiviral innate immune responses in vivo. (A to C) Phosphorylation of IFN-I signaling molecules including TBK1, IKKε, IRF3 and STAT1 (A), NF-κB (B), and MAPK (C) signaling molecules in Ogtfl/fl and OgtΔmye BMMs left untreated or stimulated with VSV (MOI = 1) for indicated periods. (D to F) Phosphorylated TBK1, IKKε, IRF3 and STAT1 (D), NF-κB (E), and MAPK (F) signaling molecules in Ogtfl/fl and OgtΔmye peritoneal macrophages treated with VSV for indicated periods. (G) Immunofluorescence staining of phosphorylated IRF3 (upper panel) or p65 (lower panel) in Ogtfl/fl and OgtΔmye BMMs left untreated or challenged with VSV for 4 h. Scale bar, 20 μm. (H) Survival of Ogtfl/fl (n = 18) and OgtΔmye (n = 20) mice after intraperitoneal injection with VSV (1 × 108 PFU per mouse). (I to L) Transcripts of Ifna4 and Ifnb1 in the spleen (I), liver (J), or lungs (K) and IFN-β protein in serum (L) from Ogtfl/fl and OgtΔmye mice challenged with either PBS or VSV (2 × 107 PFU) for 24 h (n = 6 per group). (M and N) VSV RNA in the spleen, liver and lungs (M) and histological analysis of the lung tissue (N) in Ogtfl/fl and OgtΔmye mice challenged with VSV (n = 5 per group). Scale bar: 20 μm. * P < 0.05, versus controls (Kaplan-Meier (H) or two-tailed Student’s t-test (A to F, I to M)). Data are from one experiment representative of three independent experiments and are expressed as mean ± s.d.
Figure 4.
Figure 4.
OGT promotes antiviral immune responses in human cells. (A to F) Immunoblotting of phosphorylated TBK1, IKKε, IRF3 and STAT1 (A and D), and NF-κB (B and E) signaling molecules in OGT-KO and WT control THP-1 (A to C) or HT29 (D to F) cells challenged with VSV (MOI = 1) for indicated periods. Transcripts of IFNB1, IL6 and TNFA in the cells were measured with RT-PCR (C and F). (G and H) Protein O-GlcNAcylation (G) and cytokine transcripts (H) in OGT-KO THP-1 cells virally transduced with either empty vector or expression vector for human OGT with synonymous mutation that was not recognized by OGT gRNA, followed by VSV challenge for 6 h. * P < 0.05, versus controls (two-tailed Student’s t-test (C, F and H)). Data are from one experiment representative of three experiments (A, B, D, E and G) or represent four independent experiments (C, F and H; mean ± s.d. of four biological replicates).
Figure 5.
Figure 5.
OGT promotes antiviral immune signaling via its enzymatic activity. (A to C) Transcripts of Ifna4, Ifnb1, Il6 and Tnfa in the cells (A), IFN-β, IL-6 and TNF-α proteins in the supernatants (B), and phosphorylated TBK1, IRF3, IκBα, IKKα/β and p65 (C) in OgtΔmye BMMs virally transduced with either empty vector or expression vector for OGT WT or K908A mutant, followed by the challenge with VSV (MOI = 1). (D to I) Transcripts of Ifna4, Ifnb1, Il6 and Tnfa in the cells (D and G), IFN-β, IL-6 and TNF-α proteins in the supernatants (E to H), and phosphorylated TBK1, IRF3, IκBα, IKKα/β and p65 (F and I) in Ogtfl/fl and OgtΔmye BMMs pretreated with or without either PUGNAc (50 μM) (D to F) or OSMI-1 (20 μM) (G to I) for 2 h, followed by VSV challenge. * P < 0.05, versus controls (two-tailed Student’s t-test (A, B, D, E, G and H). Data are from one experiment representative of three independent experiments (A to G). Data are from one experiment representative of three experiments (A, B, D, E, G and H; mean ± s.d. of three biological replicates) or represent two independent experiments (C, F and I).
Figure 6.
Figure 6.
O-GlcNAcylation of MAVS on S366 is critical for RLR signaling. (A) Luciferase activities in 293T cells transfected for 30 h with an IFNB1-luciferase reporter plasmid together with RIG-I N-terminus (RIG-I (N)), MDA5, MAVS, TBK1, IKKε, or IRF3, in the presence or absence of OGT. (B to D) 293T cells were transfected for 30 h with the indicated combination of Flag-tagged MAVS full-length (B and C) or N- and C-terminus (D), and Myc-tagged OGT WT (B and D) or K908A mutant (C). Immunoprecipitated MAVS was assessed for O-GlcNAcylation with specific anti-O-GlcNAc antibody. (E) Total MAVS was immunoprecipitated from Ogtfl/fl and OgtΔmye BMMs challenged with or without VSV (MOI = 1) for 4 h, followed by immunoblotting with anti-O-GlcNAc antibody. (F) LC-MS/MS analysis of Flag-tagged MAVS co-transfected with Myc-tagged OGT identified either T365 or S366 as MAVS O-GlcNAcylation site. MS/MS spectrum of the 2+ ion at m/z 924.99542 corresponding to O-GlcNAcylated MAVS peptide AGMVPSKVPTSMVLTK. (G and H) A series of point mutations of MAVS assayed for O-GlcNAcylation. (I to L) Total and phosphorylated IRF3 and NF-κB p65 (I and K) and IFN-β production (J and L) in Mavs−/− mouse embryonic fibroblasts (MEFs) transduced with either empty vector or GFP-tagged MAVS WT or S366A mutant (I and J), pretreated with or without thiamet G (10 μM) for 2 h (K and L), then followed by VSV challenge. (M) Cross-species sequence alignment of MAVS revealed conserved O-GlcNAcylation site S366. * P < 0.05, versus controls (two-tailed Student’s t-test (A) and ANOVA with Bonferroni post-tests (J)). Data are from one experiment representative of four experiments (A and J; mean ± s.d. of four biological replicates) or three experiments (B to L, G to I) or represent two independent experiments (F).
Figure 7.
Figure 7.
O-GlcNAcylation of MAVS on S366 promotes its K63-linked ubiquitination. (A to C) MAVS ubiquitination in 293T cells were transfected with Flag-tagged MAVS and Myc-tagged OGT, in the presence of HA-tagged ubiquitin WT (A), K63-only (B) or K48only (C) mutants. (D) Total MAVS was immunoprecipitated from Ogtfl/fl and OgtΔmye BMMs challenged with or without VSV (MOI = 1) for 4 h, followed by immunoblotting with specific anti-ubiquitin antibodies, as indicated. (E) Total or K63-only or K48-only ubiquitinated MAVS was assessed in 293T cells transfected with Flag-tagged MAVS together with either OGT WT or K908A mutant. (F) MAVS K63-linked ubiquitination assayed in Mavs−/− MEFs transduced with either empty vector or GFP-tagged MAVS WT or S366A mutant, followed by VSV challenge for 4 h. (G to I) Ubiquitination of MAVS in 293T cells co-transfected with Flag-tagged MAVS WT (G and H) or S366A mutant (I), GFP-tagged TRIM31 and HA-tagged ubiquitin, together with either OGT WT (G) or K908A mutant (H). (J and K) MAVS K63 ubiquitin or O-GlcNAcylation (J) and IFNB1 transcript in single TRIM31-KO or OGT/TRIM31-DKO THP-1 cells challenged with VSV. * P < 0.05, versus controls (two-tailed Student’s t-test (K)). Data are from one experiment representative of four experiments (A to K).
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