ABSTRACT The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component

glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced

its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein

(MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (_Eprs_+/−) mice

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our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS THE E3 UBIQUITIN LIGASE ARIH1 PROMOTES ANTIVIRAL IMMUNITY AND AUTOIMMUNITY BY INDUCING MONO-ISGYLATION AND

OLIGOMERIZATION OF CGAS Article Open access 10 October 2022 CRYPTIC PHOSPHORIBOSYLASE ACTIVITY OF NAMPT RESTRICTS THE VIRION INCORPORATION OF VIRAL PROTEINS Article 21 November 2024 ALTERED

ISGYLATION DRIVES ABERRANT MACROPHAGE-DEPENDENT IMMUNE RESPONSES DURING SARS-COV-2 INFECTION Article 18 October 2021 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE75699

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Initiative Program (KGM4541622 to M.H.K.), the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning of Korea (NRF-2010-0029767 and

2014R1A2A1A01005971 to M.H.K.; NRF-M3A6A4-2010-0029785 to S.K.; and 2015020957 to J.-S.L.), the Korea Institute of Oriental Medicine (K12050 to J.-S.L.), the Ministry for Food, Agriculture,

Forestry and Fisheries (315044031SB010 to J.-S.L.) and the Korea Health Industry Development Institute (HI14C3484 to C.L.). AUTHOR INFORMATION Author notes * Eun-Young Lee and Hyun-Cheol

Lee: These authors equally contributed to this work. AUTHORS AND AFFILIATIONS * Infection and Immunity Research Laboratory, Microbiomics and Immunity Research Center, Korea Research

Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea Eun-Young Lee, Hyun-Kwan Kim, Song Yee Jang, Jungwon Hwang & Myung Hee Kim * College of Veterinary Medicine, Chungnam

National University, Daejeon, Korea Hyun-Cheol Lee, Hyun-Kwan Kim, Jae-Hoon Kim, Tae-Hwan Kim & Jong-Soo Lee * Center for Theragnosis, Biomedical Research Institute, Korea Institute of

Science and Technology, Seoul, Korea Seong-Jun Park & Cheolju Lee * Laboratory Animal Resource Center, KRIBB, University of Science and Technology (UST), Daejeon, Korea Yong-Hoon Kim 

& Chul-Ho Lee * Personalized Genomic Medicine Research Center, KRIBB, Daejeon, Korea Jong Hwan Kim & Seon-Young Kim * Department of Cellular and Molecular Medicine, Lerner Research

Institute, Cleveland Clinic, Cleveland, Ohio, USA Abul Arif & Paul L Fox * College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea Young-Ki Choi

* Department of Biological Chemistry, UST, Daejeon, Korea Cheolju Lee * Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los

Angeles, California, USA Jae U Jung * Department of Molecular Medicine and Biopharmaceutical Sciences, Medicinal Bioconvergence Research Center, Graduate School of Convergence Science and

Technology, Seoul National University, Seoul, Korea Sunghoon Kim * Biosystems and Bioengineering Program, UST, Daejeon, Korea Myung Hee Kim Authors * Eun-Young Lee View author publications

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View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS E.-Y.L. and H.-C.L. performed most of the experiments with help from H.-K.K., S.Y.J., J.H.,

J.-H.K. and T.-H.K. S.-J.P. and C.L. performed mass spectrometry. Y.-H.K. and C.-H.L. performed immunohistochemical analysis. J.H.K., S.-Y.K. and Y.-K.C. performed RNA-seq analysis. A.A.,

J.U.J., P.L.F. and S.K. contributed to the discussion and provided critical reagents. E.-Y.L., J.-S.L. and M.H.K. designed the study and wrote the manuscript. All of the authors helped with

data analysis. CORRESPONDING AUTHORS Correspondence to Jong-Soo Lee or Myung Hee Kim. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 EPRS EXPRESSION IN MULTIPLE CELL LINES UPON VIRAL INFECTION. (A,B) EPRS is slightly induced upon viral induction. qPCR of _Eprs_ mRNA (A)

and immunoblot analysis of corresponding endogenous EPRS expression (B) in multiple cell lines, including C57/B6 mouse-derived BMDM, U937, RAW264.7, A549, and 293T cells infected with

PR8-GFP or VSV-GFP. (C) qPCR analysis of representative _Isg_ mRNA expression under the same conditions as in (A). (D) qPCR analysis of _Eprs_ mRNA in U937 and RAW264.7 cells treated with

IFN-β (1000 units/ml). _Isg15_ mRNA was analyzed as a control. (E) qPCR analysis of _Eprs_ mRNA in RIG-I-sufficient (_Ddx58_+/+) or RIG-I-deficient (_Ddx58_-/-) MEF cells infected with

VSV-GFP. Data are representative of two (A-E) independent biological replicates with similar results (mean and s.d. of triplicate in A,C-E). SUPPLEMENTARY FIGURE 2 ANTIVIRAL EFFECTS OF EPRS

IN EPRS-DEFICIENT OR EPRS-OVEREXPRESSING IMMUNE CELLS. (A) Immunoblot analysis of EPRS expression. (B) Viral titer after infection with HSV-GFP (MOI = 1). RAW264.7 cells were transfected

with non-targeting control siRNA (siCtrl) or siEPRS (A,B). (C) Fluorescence microscopy images, (D) virus replication, and (E) secreted IFN-β or IL-6 levels in 293T cells transfected with

siCtrl or siEPRS for 36 h, followed by infection with VSV-GFP (MOI = 0.0001). (F) Immunoblot analysis of EPRS expression. (G) Fluorescence microscopy images, (H) PR8 titer, and (I) secreted

IFN-β or IL-6 level in cells infected with PR8-GFP (MOI = 1). (J) Fluorescence microscopy images, (K) VSV titer, and (L) secreted IFN-β or IL-6 level in stable EPRS-deficient cells infected

with VSV-GFP (MOI = 0.5). RAW264.7 cells were transduced with non-targeting control shRNA (shCtrl) or EPRS shRNA (shEPRS), followed by selection with puromycin (F–L). (M) Immunoblot analysis

of EPRS expression. (N) Fluorescence microscopy images, (O) VSV titers, and (P) secreted IFN-β or IL-6 level in EPRS-overexpressing cells infected with VSV-GFP (MOI = 0.5). RAW264.7 cells

were transfected with a FLAG-tagged empty vector (Ctrl) or with EPRS-FLAG (EPRS) plasmids, followed by selection with puromycin (M–P). Scale bars, 100 μm (C,G,J,N). *_P_ < 0.05, **_P_

< 0.01, and ***_P_ < 0.001 (Student’s _t_-test; D,E,H,I,K,L,O,P). Data are representative of three (A-P) independent biological replicates with similar results (mean and s.d. of

triplicate in B,D,E,H,I,K,L,O,P). SUPPLEMENTARY FIGURE 3 EPRS DEFICIENCY IN MOUSE BMDMS REDUCES ANTIVIRAL INNATE IMMUNE RESPONSES. (A) Immunoblot analysis of EPRS expression in BMDMs. BMDMs

were transfected with non-targeting control siRNA (siCtrl) or siEPRS for 36 h. (B) Plaque assay to determine virus titers and (C) ELISA to measure IFN-β and IL-6 levels at 12 and 24 h

post-infection. BMDMs were infected with PR8-GFP (MOI = 3) or VSV-GFP (MOI = 5) (B,C). (D) IFN-β and IL-6 levels measured in the culture supernatants from BMDMs treated with 40 μg of

Poly(I:C). (E) Induction of _Ifnb_ mRNA or IFN-related antiviral genes in virus-infected cells. RAW264.7 cells were transfected with siCtrl or siEPRS for 36 h, followed by infection with

PR8-GFP (MOI = 1) for 12 h. The graphs show the -fold induction of the indicated genes after normalization against _Gapdh_. (F) Viral titer (determined by plaque assay) and (G) secreted

IFN-β or IL-6 levels in cell culture supernatants after infection with HSV-GFP. BMDMs from _Eprs_+/+ and _Eprs_+/ – mice were infected with HSV-GFP (MOI = 2) (F,G). *_P_ < 0.05, **_P_

< 0.01, and ***_P_ < 0.001 (Student’s _t_-test; B–D). Data are representative of three (B-D) or two (E-G) independent biological replicates with similar results (mean and s.d. of

triplicate in B-G). SUPPLEMENTARY FIGURE 4 DISSOCIATION OF EPRS FROM THE MSC UPON VIRAL INFECTION. (A,B) Viral infection induces dissociation of EPRS from the MSC component proteins. Lysates

of RAW264.7 cells infected with PR8-GFP (MOI = 1) were subjected to immunoprecipitation with an anti-EPRS (A) or with an anti-KRS (B), followed by immunoblot analysis with anti-KRS,

anti-MRS, anti-AIMP3, and anti-GAPDH (A), or with anti-EPRS and anti-AIMP3 (B), respectively. (C) Confocal microscopy of endogenous EPRS (red) and KRS (green) in HeLa cells infected with PR8

(MOI = 5) for 6 or 12 h. IFN-γ (1000 units/ml) treatment for 12 h was used for comparison. Cells were permeabilized with a lower dose of digitonin (20 μg/ml, 5 min) than used in Fig. 4b (25

μg/ml, 10 min). Scale bar, 10 μm (2 μm in magnified images). (D) Confocal microscopy of endogenous EPRS (red) and NSAP1 (green) in HeLa cells infected with PR8 (MOI = 5) for 6 or 12 h, or

in cells treated with IFN-γ (1000 units/ml) for 12 h. Scale bar, 10 μm. Data are representative of three independent biological replicates with similar results (A-D). SUPPLEMENTARY FIGURE 5

IDENTIFICATION OF THE VIRAL-INFECTION-SPECIFIC PHOSPHORYLATION SITE IN EPRS. (A) Silver-stained Strep-EPRS (indicated by an asterisk) purified by Strep precipitation assay of 293T cells

infected or uninfected (–) with PR8-GFP (MOI = 5). EV, Strep-empty vector. (B) MS/MS spectra for a doubly charged EPRS peptide EYIPGQPPLSQSSDSpS*PTR (MH+ = 2125.93, z = 2+) obtained under

uninfected (upper) and PR8-infected (lower panel) conditions. The peptides contain the S886 phosphorylation site (marked by an asterisk). (C–E) Extracted ion chromatogram (XIC) of the

tryptic digests under uninfected (upper) and infected (lower panel) conditions, corresponding to doubly charged EYIPGQPPLSQSSDSSPTR (MH+ = 2044.96, z = 2+) (C), doubly charged

NQGGGLSSSGAGEGQGPK (MH+ = 1586.72, z = 2+) (D), and triply charged KDPSKNQGGGLSSSGAGEGQGPK (MH+ = 2142.02, z = 3+) (E) peptides from non-phosphorylated (left) and phosphorylated (right,

marked by an asterisk) EPRS. ND, not detected. (F) Immunoblot analysis of phosphomimetic (S990D) and phosphorylation-resistant (S990A) EPRS against with anti-phospho-EPRS(Ser990) in 293T

cells. (G–J) Immunoblot analysis of EPRS Ser990 phosphorylation in RAW264.7 cells infected with PR8-GFP (MOI =1) (G), 293T cells infected with PR8-GFP (MOI = 5) (H) or VSV-GFP (MOI = 0.001)

(I), or cells transfected with 2 μg of Poly(I:C) (J). (K,L) Secreted IFN-γ levels from U937 (K) or RAW264.7 (L) cells infected with PR8-GFP or VSV-GFP. Cells treated with IFN-γ (1000

units/ml) were used as a positive control. (M) Immunoblot analysis of Cp expression in PR8-GFP-infected RAW264.7 cells. Data are representative of three (G-M) independent biological

replicates with similar results (mean and s.d. of triplicate in K,L). SUPPLEMENTARY FIGURE 6 NON-TRANSLATIONAL ROLE OF EPRS IN REGULATING ANTIVIRAL IMMUNE RESPONSES. (A,B) Purified

His-tagged EPRS (aa 1–196) (A) or EPRS (aa 1–168) (B) was mixed with the GST-fused PCBP2 KH1 (aa 11-82). After His-tag precipitation, proteins were subjected to SDS-PAGE and stained with

Coomassie Brilliant Blue. (C) The purified His-tagged ERS (aa 1-732) and its mutant that is inactive for tRNA glutamylation (MT). Black arrows denote protein fragments derived from ERS

during purification (as in Fig. 6j). (D) Aminoacylation assay for ERS (WT) and its mutant (MT). CPM, counts per minute. Ctrl, buffer without protein. (E) _IFNB_ promoter activity in 293T

cells transfected with N-RIG-I plus empty vector (EV), Strep-EPRS (WT), or its mutants inactive for tRNA glutamylation only (E-MT), tRNA prolylation only (P-MT), or both (EP-MT). (F)

Immunoblot analysis of endogenous EPRS, MAVS, and RIG-I expression in sgEPRS 293T cells or non-targeting control (sgCtrl) cells. (G–K) Non-translational function of EPRS in antiviral immune

responses. Virus replication assay (examined by fluorescence microscopy) (G) and plaque assay (H) at 24 h post-infection with VSV-GFP (MOI = 0.0001). Immunoblot analysis of Strep-EPRS or

endogenous EPRS expression (I). IFN-β (J) and IL-6 (K) secreted by sgEPRS cells infected with VSV-GFP. sgCtrl or sgEPRS 293T cells were reconstituted with EV, Strep-tagged EPRS (WT), or its

catalytic mutant (EP-MT) (G–K). Scale bar, 100 μm (G). *_P_ < 0.01; NS, not significant (Student’s _t_-test; D,E,H,J,K). Data are representative of two (A-K) independent experiments (mean

and s.d. of triplicate in D,E,H,J,K). SUPPLEMENTARY FIGURE 7 INFECTION-SPECIFIC EPRS PHOSPHORYLATION IS ESSENTIAL FOR REGULATING MAVS. (A,B) _In vitro_ binding assay showing MAVS

interaction with PCBP2 KH1 (aa 11–82). (C,D) The precipitation (ppt) assays revealed no interaction between PCBP2 and LRS in 293T cells (C), whereas PCBP2 interacted with MAVS (D). (E)

Ubiquitination of exogenous MAVS in non-targeting control (siCtrl) or EPRS-deficient (siEPRS) 293T cells transfected with Ub, ITCH, MAVS, or PCBP2. (F) Ubiquitination of endogenous MAVS in

293T cells transfected with Ub, ITCH, PCBP2, and Strep-empty vector (EV), or with WT EPRS or its mutant (EP-MT, enzymatically inactive for both tRNA glutamylation and prolylation). (G)

Expression of endogenous MAVS in 293T cells transfected with PCBP2 and WT EPRS or EP-MT. The histogram shows the intensity of the MAVS band normalized against actin. (H–N) Ubiquitination of

endogenous MAVS (H) in non-targeting control (sgCtrl) or sgEPRS 293T cells transfected with HA-Ub and infected with VSV-GFP (MOI = 0.1). (I) Ubiquitination of endogenous MAVS in 293T cells

transfected with HA-Ub and infected with VSV-GFP. (J) IFN-β or (K) IL-6 levels in supernatants from cells infected with VSV-GFP. (L) Fluorescence microscopy images and (M) plaque assay at 24

h post-infection with VSV-GFP (MOI = 0.0001). (N) Immunoblot analysis of EPRS-FLAG or endogenous EPRS. sgEPRS 293T cells were reconstituted with a FLAG-EV, WT EPRS, S990A, or S990D (I–N).

Scale bar, 100 μm (L). *_P_ < 0.01; NS, not significant (Student’s _t_-test; J,K,M). Data are representative of two (A-N) independent biological replicates with similar results (mean and

s.d. of triplicate in J,K,M). SUPPLEMENTARY FIGURE 8 THE TAT-EPEP IS SPECIFIC TO INFECTION WITH RNA VIRUSES. (A–C) Tat-Epep has no significant effect on virus replication (A) or IFN-β (B)

and IL-6 (C) secretion in RAW264.7 infected with HSV-GFP (MOI = 1) for 12 h. HSV-GFP-infected RAW264.7 cells treated with PBS were used as negative controls. (D) Viability of RAW264.7 cells

as measured in an MTS assay after treatment with the indicated doses of Tat-Epep for 12 h. (E) Viability of 293T cells after treatment with Tat-Epep for 12 or 24 h. Ctrl, 293T cells treated

with a lytic detergent (digitonin, 30 μg/ml) for 15 min as a positive control. Results are expressed as the mean ± SD of two independent biological replicates incorporating triplicate

samples. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–8 and Supplementary Tables 1 and 2 (PDF 1905 kb) SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO

FIG. 2 SOURCE DATA TO FIG. 3 SOURCE DATA TO FIG. 4 SOURCE DATA TO FIG. 5 SOURCE DATA TO FIG. 6 SOURCE DATA TO FIG. 7 SOURCE DATA TO FIG. 8 RIGHTS AND PERMISSIONS Reprints and permissions

ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, EY., Lee, HC., Kim, HK. _et al._ Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity. _Nat Immunol_ 17,

1252–1262 (2016). https://doi.org/10.1038/ni.3542 Download citation * Received: 04 June 2016 * Accepted: 28 July 2016 * Published: 05 September 2016 * Issue Date: November 2016 * DOI:

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