Abstract
The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs), whose role in antiviral immunity is elusive. Here, we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state, GLUT4 is trapped at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9, TUG). Following RNA virus infection, GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs, preventing them from activating IFN-β responses. UBXN9 deletion prompts constitutive GLUT4 translocation, sequestration of RLRs and attenuation of antiviral immunity, whereas GLUT4 deletion heightens RLR signaling. Notably, reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall, our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs.
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Data availability
Figure 6 and Extended Data Fig. 9 are derived from publicly available RNA-sequencing data at National Institutes of Health Gene Expression Omnibus under accession numbers GSE143323 and PRJNA491748. These raw data were reanalyzed to generate DEGs and are listed in source data titled ‘Supplementary Sequencing Dataset’. The source data for each bar graph and immunoblots are included in this manuscript, named ‘MainFig&EDFig_Sourcedata’. Raw uncropped gels are displayed in Supplementary Data as source data with the title ‘ Unprocessed blots for Main and Extended Figs_Source data file’. Source data are provided with this paper.
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Acknowledgements
We thank C. Yan at Tsinghua University for providing GLUT4 expression plasmids. We also thank A. Ménoret and R. Clark of UConn Health for their experimental advice and review of the manuscript. We acknowledge V. Graziano of UConn Health and M. Strine of Yale University for suggestions related to manuscript writing. We acknowledge J. Ruan, C. Wang, Y. Wang and S. Shivcharan of UConn Health for key reagents. This work was supported by National Institutes of Health grants R01AI132526, R21AI155820 and R21AI170981 and UConn Health startup fund to P.W. and R01DK129466 to J.S.B.
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A.G.H. performed most experimental procedures, acquired and analyzed the data. D.Y performed several experiments and analyzed the accompanying data. J.G.C., T.G., T.A.K., C.C.C. and X.L. assisted A.G.H. with some experimental procedures and/or provided technical support. Y.H. conducted the T-tubule isolations from mouse quadriceps. Z.C. carried out IPA analysis on RNA sequencing from DM and CIM studies. Z.F., A.T.V. and Y.Q. provided suggestions on writing. S.K.V., V.A.R., C.A.W. and J.S.B. offered critical comments and suggestions on data analysis, interpretation and writing. P.W. conceived the study. A.G.H. and P.W. designed the study and wrote the manuscript. All the authors reviewed and/or modified the manuscript.
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Extended data
Extended Data Fig. 1 UBXN9 is critical for RLR signaling in vitro and in vivo.
a, Immunoblots of UBXN9 protein in Ubxn9+/+ and Ubxn9−/− C2C12 myocytes. b, c, Cellular Ifnb1 mRNA levels (n = 4 biological replicates/group) (b) and secreted IFN-β protein (12 h, n = 2 biological replicates/group; 24 h, n = 4 biological replicates/group) (c) from C2C12 myocytes transfected with 3p-hpRNA. d, Secreted IFN-β protein from C2C12 myocytes (n = 2 biological replicates) transfected with interferon-stimulatory DNA (ISD). e-f, Secreted IFN-β protein (e) and viral RNA loads (f) in mouse C2C12 cells (n = 4 biological replicates/group) infected with ONNV (MOI: 1). g-h, Ubxn9+/+ and Ubxn9−/− littermates (n = 5 Ubxn9+/+ and 6 Ubxn9−/− mice) infected with ONNV as depicted in (g), and viral RNA in hindlimb muscles (h). Dpi: days post infection. The data are representative of 2-3 independent experiments (a-c, e, f) or 1 (d, g,h) independent experiment. Bar: mean ± SEM. P values were determined by by two-way ANOVA with Šídák multiple comparisons test (b-f) or two-tailed Mann-Whitney test (h). LOD: limit of detection, established with uninfected mice (h).
Extended Data Fig. 2 UBXN9 does not affect myotube gene expression or regulate RLR signaling in macrophages and human lung A549 cells.
a, Diagram illustrating the design of Ubxn9flox/flox mice. b, Immunoblots of UBXN9 and housekeeping tubulin proteins in various tissues of Ubxn9−/− mice ( + TMX) compared to Ubxn9+/+ littermates ( + corn oil). c, Protocol for isolation of primary mouse myoblast from Ubxn9+/+ and Ubxn9−/− mice. d, Representative light microscopic images demonstrating pre-plating steps described in (c). Scale bar, 10μm. Cells were isolated from Ubxn9+/+ mice as an example of the purification process. DM, differentiation medium. e, Immunoblots of UBXN9 and GAPDH in purified myoblasts isolated from Ubxn9+/+ and Ubxn9−/− mice. f, Myf5 and MyoG mRNA expression in Ubxn9+/+ and Ubxn9−/− myoblasts and myotubes, respectively (n = 3 Ubxn9+/+ and 4 Ubxn9−/− mice). g, Immunoblots of UBXN9 and β-actin in bone marrow-derived macrophages (BMDMs) isolated and differentiated from Ubxn9+/+ and Ubxn9−/− mice (n = 2 mice/group as a representative of UBXN9 knockout efficiency). h, Immunoblots of UBXN9 and β-actin in UBXN9+/+ and UBXN9−/− A549 lung cells. i, Cellular Ifnb1 transcript levels in Ubxn9+/+ and Ubxn9−/− BMDMs (n = 3 mice/group) before and after transfection with poly(I:C) (left) or 3p-hpRNA (right). j, IFN-β protein levels from UBXN9+/+ and UBXN9−/− A549 cells (n = 2 biological replicates from two independent experiments) stimulated with poly(I:C) for 12 h. The data are representative of one (f) and two independent experiments (g-j). ns, not significant based on two-way ANOVA with Šidák’s multiple comparisons test (i). Bar: mean ± SEM.
Extended Data Fig. 3 GLUT4 inhibits RLR signaling in C2C12 myocytes.
a, Mean fluorescence of VSV-GFP+ in Slc2a4+/+ and Slc2a4−/− C2C12 cells (N = 4 biological replicates/group) at 24 h post VSV infection (MOI: 1). b, VSV-G protein in Slc2a4+/+ and Slc2a4−/− C2C12 cells before and after VSV infection (MOI: 1). c, d, EMCV titers (c) and IFN-β protein levels (d) from Slc2a4+/+ and Slc2a4−/− C2C12 cells (c, n = 4 biological replicates/group; d, n = 2-4 biological replicates/group) infected with EMCV (MOI: 0.5). e, f, Phosphorylated (p) and total IRF3 before and after ONNV (e) or EMCV (f) infection in C2C12 cells. EMCV MOI: 0.5; ONNV MOI: 1. The data are representative of 2 independent experiments. Bar: mean ± SEM. P values determined by two-tailed Student’s t-test (a) or two-way ANOVA with Šidák’s multiple comparisons test (c, d).
Extended Data Fig. 4 Immunofluorescence confirmation of cardiac troponin T expression in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM).
a, Timeline and experimental protocol for the generation of induced pluripotent stem cells (iPSCs), differentiation into iPSC-derived cardiomyocytes (iPSC-CMs) and subsequent treatment conditions for assessing RLR responses. CHIR99021, GSK3 inhibitor/Wnt activator; IWP-4, inhibitor of Wnt production-4. b, Confocal microscopic images of cardiac troponin T (cTnT) counterstained with DAPI (nuclear DNA) in iPSC-CM on day 35 of differentiation. Top panel, 20x magnification; bottom panel, 100x magnification. Scale bar, 100μm (top) and 25μm (bottom).
Extended Data Fig. 5 RLR signaling is regulated by a lactate and glucose-independent mechanism despite increased glucose tolerance after Ubxn9 deletion.
a, b, Immunoblots of LDHA and β-actin in Ldha+/+ and Ldha−/− C2C12 myocytes (a) and extracellular lactate levels after 12 h of culture (n = 5 biological replicates, two independent experiments (b). c, d, Cellular Ifnb1 transcripts in (c) and secreted IFN-β protein levels (d) from C2C12 cells (n = 2 biological replicates/group) stimulated with 3p-hpRNA. e, Immunoblot loading controls for the T-tubule and muscle homogenate fractions from Ubxn9+/+ and Ubxn9−/− mice (n = 3 mice/group for example purposes) shown in Fig. 3c. f, Plasma lactate concentrations (n = 6 mice/group) before and after infection with EMCV (100 PFU/mouse). g, Schematic for intraperitoneal (i.p.) glucose tolerance test (IPGTT) with Ubxn9+/+ and Ubxn9−/− littermates. h, Glycemia (left) and area under curve (AUC) (right) during IPGTT (n = 13 Ubxn9+/+ and 12 Ubxn9−/− mice pooled from 3 independent experiments). i, Intracellular lactate levels in C2C12 cells (n = 4 biological replicates/group) before and after EMCV (MOI: 0.5). j, IFN-β protein release from Ldha+/+ and Ldha−/− C2C12 cells (n = 2 biological replicates/group for untreated; n = 4 biological replicates/group for 3p-hpRNA) that were transfected with an empty vector or GLUT4 expression plasmid (pGLUT4) before 3p-hpRNA treatment for 12 h. k, 2-DG uptake in DMSO or Fasentin (25 μM) pretreated WT C2C12 cells (n = 3 biological replicates/group). l, Ifnb1 transcripts in C2C12 cells (n = 4 biological replicates/group) treated simultaneously with Fasentin (25 μM) and EMCV (MOI: 0.5) for 6 h. Bar: mean ± SEM. P values determined by unpaired two-tailed Student’s t-test (b, h, i-k) and two-way ANOVA with Šidák’s multiple comparisons test (f, h, l).
Extended Data Fig. 6 Insulin, 3p-hpRNA and virus infection promotes glucose uptake and RIG-I translocation to the plasma membrane in a GLUT4-dependent manner.
a-c, Endogenous exofacial expression of GLUT4 in myocytes stimulated with or without insulin (200 nM) for 20 min. Scale bar, 10 μm. d, Mean fluorescence intensity (MFI) of plasma membrane GLUT4 (pmGLUT4) from cells (n = 5-7 individual images/group for vehicle; n = 11 WT, n = 11 Ubxn9−/− and n = 10 individual images for Slc2a4−/− during insulin) stimulated as in (a-c). Dotted line indicates background fluorescence. e, Colocalization of plasma membrane GLUT4 with ZO-1 (white arrows) in wild-type C2C12 myocytes before or after EMCV infection (MOI: 0.5, 24 h). Scale bar, 5 μm. f, 2-DG uptake in C2C12 cells treated with insulin (200 nM) for 20 min (n = 4 replicates/group), transfected with 3p-hpRNA for 12 h (n = 4 replicates/group), treated with IFN-β for 12 h (n = 6 replicates/group), or infected with EMCV (MOI: 0.5) for 24 h (n = 6 replicates/group). The results are normalized to untreated Slc2a4+/+ cells. g, Confocal images of PM staining, RIG-I and nuclear DNA in C2C12 cells mock treated. Scale bar, 5 μm. h, Cytoplasmic fraction from C2C12 myocytes (left) and 3T3-L1 myc-GLUT4-GFP adipocytes (right) before and after various treatments. Insulin for 20 min (200 nM); 3p-hpRNA for 6 h. i, Endogenous MAVS-RIG-I interaction from C2C12 cells stimulated with 3p-hpRNA for 6 h. j, 2-DG uptake by C2C12 cells (n = 12-13 WT, n = 9 Ubxn9−/− and n = 10-11 Slc2a4−/− biological replicates) before or after insulin for 20 min (200 nM). k, Proteins in the PM and TCL of C2C12 cells treated with insulin as in (j). l, Protein band ratios of GLUT4 to caveolin in the PM compartment of WT C2C12 cells (n = 3 biological replicates/group from 3 experiments) infected with VSV and EMCV as in (Fig. 4l). m, GLUT4-RIG-I interaction in 3T3-L1 myc-GLUT4-GFP adipocytes before or after insulin (200 nM) for various times. The data are representative of 2-3 independent reproducible experiments. Bar: mean ± SEM. P values determined by one-way ANOVA with Dunnett multiple comparisons test (d, f, j,) or two-way ANOVA with Šidák’s multiple comparisons test (l).
Extended Data Fig. 7 The CARD domain of RIG-I competes with GLUT4-UBXN9 binding in a glucose-uptake independent manner.
a-b, GLUT4-RIG-I colocalization in Ldha+/+ and Ldha−/− C2C12 cells after 3p-hpRNA treatment. a, representative images of overexpressed GLUT4 with endogenous RIG-I in Ldha+/+ and Ldha−/− C2C12 cells before or after 3p-hpRNA treatment for 12 h. Scale bar, 10 μm. b, GLUT4-RIG-I colocalization before (n = 16 for Ldha+/+ and n = 23 for Ldha−/−) and after 3p-hpRNA treatment (n = 25 for Ldha+/+ and n = 21 for Ldha−/−) from cells in (a). c, Co-IP of WT, CARD domain and helicase domain of RIG-I (FLAG) with GLUT4 (Myc). Experiment is a repeat of Fig. 5l to equalize the FLAG expression of FL and the helicase domain of RIG-I. d, Co-IP of various doses of WT-FL or R169A GLUT4 with endogenous RIG-I. e, 2-DG uptake in Slc2a4−/− cells reconstituted with WT or R169A GLUT4 plasmids. Cells were transfected with either vector (Vec) (n = 4 biological replicates), FLAG-FL (n = 3 biological replicates) or FLAG-R169A (n = 3 biological replicates) and then treated with insulin for 20 min (200 nM). f, GLUT4 pulldown with endogenous RIG-I in the presence of increasing concentrations of UBXN9 plasmid in HEK293T cells. All results are representative of 2 independent experiments. Bar: mean ± SEM. P values determined by one-way ANOVA with Tukey’s multiple comparisons test (b) or unpaired two-tailed Student’s t-test (e).
Extended Data Fig. 8 AKT2 is dispensable for GLUT4 translocation in C2C12 myocytes.
a, b, Immunoblot of indicated GLUT4 trafficking proteins from WT C2C12 cells before and after VSV (MOI: 0.5, top panels) or EMCV (MOI: 0.5, lower panels) infection. c, AKT2 expression in Akt2+/+ and Akt2−/− C2C12 cells. d, Confocal microscopic images of exofacial GLUT4 in Akt2+/+ and Akt2−/− myocytes before and after VSV (MOI: 0.5) or EMCV (MOI: 0.5) infection for 12 h. Scale bar, 10 μm. e, Immunoblot of proteins in the PM fraction and TCL from Akt2+/+ and Akt2−/− myocytes before and after VSV infection (MOI: 0.5) for 12 h. Mock, medium alone without virus. Results are representative of 2 independent experiments.
Extended Data Fig. 9 Myopathic diseases are associated with decreased expression of GLUT4 and its trafficking machinery.
a, Schematic of critical illness myopathy (CIM) and dermatomyositis (DM) studies. N = 7 CIM and 6 healthy controls; N = 39 DM and 20 healthy controls. b, Ingenuity Pathway Analysis (IPA) of the 6,257 differentially expressed genes (DEGs) (p < 0.05, fold change ≥ 1.5) in the CIM patients compared to healthy controls. Red circles highlight innate immune pathways activated in CIM patients compared to controls. c, Select DEGs in the canonical interferon pathways and SLC2A4 (GLUT4, red box) from (b). d, Expression of GLUT4 trafficking-related genes (FPKM) in dermatomyositis (DM) patients and healthy controls. e, Simple linear regression analysis of SLC2A4 or SLC2A1 expression with DDX58 and OAS1A in DM patients and healthy controls (IFITM1, IFIH1). For e, regression line + 95% confidence intervals (CI). Bar: mean ± SEM. P values determined by two-tailed Mann-Whitney test (d) or linear regression analysis (e).
Extended Data Fig. 10 GLUT4 compartmentalizes RLRs to the plasma membrane to attenuate RLR signaling.
Viral RNA is sensed by cytosolic RIG-I and MDA5 that leads to MAVS oligomerization at the mitochondria for IFN-β production. During viral infection, GLUT4 trafficking machinery are activated—including AKT, AMPK and c-Cbl—that promote UBXN9 cleavage, thus liberating GLUT4 storage vesicle (GSVs) for surface translocation. RLRs are then sequestered to the plasma membrane by mobilized GLUT4, leading to attenuated IFN-β responses and augmented virus replication. Of note, GLUT4 can effectively tether the steady state and IFN-β-induced pool of cytosolic RLRs. The loop 6 and C terminus of GLUT4 is responsible for binding to the RLR CARD domain. Colored ‘P’ circle denotes AKT, AMPK and c-Cbl are phosphorylated after virus infection. GLUT4 storage vesicles, GSVs. Mitochondrial antiviral-signaling protein, MAVS. Caspase activation and recruitment domains, CARDs. RIG-I-like receptor, RLR.
Supplementary information
Supplementary Data 1
Pathway analysis and differential gene expression analysis for the two human inflammatory myopathy studies. One excel file with tabs clearly labeled for each relevant main/extended data figure panel (Fig6a-b; EDFig9b). Each relevant panel has its own tab with the accompanying data.
Source data
Source Data
Unprocessed western blots/gels for all relevant main figure panels and extended data figure panels combined into one document. Each membrane is clearly marked with the panel number and the groups being compared.
Source Data
Statistical raw data for main and extended data figures combined into one Excel document. Each tab is one figure with every panel from that figure listed with statistical data (for those panels where graphs are used to present data). Immunoblots are found in Unprocessed blots for Main and Extended Figs_Source data file.pdf file. Larger sequencing datasets are found in the file ‘UBXN9-GLUT4 Supplementary Sequencing Dataset.xlsx’.
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Harrison, A.G., Yang, D., Cahoon, J.G. et al. UBXN9 governs GLUT4-mediated spatial confinement of RIG-I-like receptors and signaling. Nat Immunol 25, 2234–2246 (2024). https://doi.org/10.1038/s41590-024-02004-7
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DOI: https://doi.org/10.1038/s41590-024-02004-7
