NOD1 Promotes Antiviral Signaling by Binding Viral RNA and Regulating the Interaction of MDA5 and MAVS

This information is current as Xiao Man Wu, Jie Zhang, Peng Wei Li, Yi Wei Hu, Lu Cao, of September 28, 2021. Songying Ouyang, Yong Hong Bi, Pin Nie and Ming Xian Chang J Immunol published online 13 March 2020 http://www.jimmunol.org/content/early/2020/03/12/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 13, 2020, doi:10.4049/jimmunol.1900667 The Journal of Immunology

NOD1 Promotes Antiviral Signaling by Binding Viral RNA and Regulating the Interaction of MDA5 and MAVS

Xiao Man Wu,*,†,1 Jie Zhang,*,1 Peng Wei Li,*,† Yi Wei Hu,*,† Lu Cao,*,† Songying Ouyang,‡,x Yong Hong Bi,* Pin Nie,*,†,{ and Ming Xian Chang*,†,{,‖

Nucleotide oligomerization domain–like receptors (NLRs) and RIG-I–like receptors (RLRs) detect diverse pathogen-associated molecular patterns to activate the innate immune response. The role of mammalian NLR NOD1 in sensing bacteria is well established. Although several studies suggest NOD1 also plays a role in sensing viruses, the mechanisms behind this are still largely unknown. In this study, we report on the synergism and antagonism between NOD1 and MDA5 isoforms in teleost. In zebraﬁsh, the overexpression of NOD1 enhances the antiviral response and mRNA abundances of key antiviral genes involved in RLR-mediated signaling, whereas the loss of NOD1 has the opposite effect. Notably, spring viremia of carp virus–infected NOD12/2

zebraﬁsh exhibit reduced survival compared with wild-type counterparts. Mechanistically, NOD1 targets MDA5 isoforms and Downloaded from TRAF3 to modulate the formation of MDA5–MAVS and TRAF3–MAVS complexes. The cumulative effects of NOD1 and MDA5a (MDA5 normal form) were observed for the binding with poly(I:C) and the formation of the MDA5a–MAVS complex, which led to increased transcription of type I IFNs and ISGs. However, the antagonism between NOD1 and MDA5b (MDA5 truncated form) was clearly observed during proteasomal degradation of NOD1 by MDA5b. In humans, the interactions between NOD1–MDA5 and NOD1–TRAF3 were conﬁrmed. Furthermore, the roles that NOD1 plays in enhancing the binding of MDA5 to MAVS and poly(I:C)

are also evolutionarily conserved across species. Taken together, our ﬁndings suggest that mutual regulation between NOD1 and http://www.jimmunol.org/ MDA5 isoforms may play a crucial role in the innate immune response and that NOD1 acts as a positive regulator of MDA5/MAVS normal form–mediated immune signaling in vertebrates. The Journal of Immunology, 2020, 204: 000–000.

he innate immune response is the first line of defense Over the last decade, significant progress has been achieved in against invading pathogens and is mediated by several characterizing NLR family members. According to their known T families of pattern recognition receptors (PRRs). It has physiological functions, the NLR family can be grouped into been demonstrated that RIG-like receptors (RLRs) and NOD-like inflammasome-forming, reproductive, and regulatory NLRs (9, 10).

receptors (NLRs) are the main cytosolic PRRs and that cytosolic Among all NLRs, NOD1, and NOD2 are the best-characterized by guest on September 28, 2021 RNAs derived from the viral genome or its replication interme- members. Previous studies have shown that NOD1 and NOD2 are diates are mainly recognized by RLRs including RIG-I, MDA5, important bacterial sensors that recognize peptidoglycans (11–13). and LGP2 (1–3). Upon the recognition of viral RNAs through C- Upon ligand binding, NOD1 and NOD2 associate with receptor- terminal RNA helicase domains, RIG-I and MDA5 are recruited to interacting protein 2 (RICK/RIP2) through CARD–CARD oligo- the downstream mitochondria-associated adaptor protein MAVS merization, thereby activating the downstream NF-kB and MAPK through their respective CARDs, which ultimately leads to the pathways to induce the production of proinﬂammatory cytokines production of type I IFNs and antiviral immune responses (2, 4). and antimicrobial molecules (14, 15). In addition to their role as The LGP2 lacking the N-terminal CARDs acts as a regulator of bacterial sensors, recent studies have uncovered the key function RIG-I and MDA5 signaling (5, 6). Unlike RLRs, NLRs are a large of NOD1 and NOD2 in sensing both ssRNA and DNA viruses family of intracellular PRRs and were originally reported to in- (16, 17). Additionally, it has been reported that deletion or func- duce the NF-kB pathway in response to bacterial pathogens (7, 8). tional knockdown of NOD2 results in a reduced IFN response and

*State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hy- analysis, and interpretation of the data, or in the preparation, review, or approval of drobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei Province, China; the manuscript. †University of Chinese Academy of Sciences, Beijing 10049, China; ‡Key Laboratory Address correspondence and reprint requests to Prof. Ming Xian Chang, Institute of of Innate Immune Biology of Fujian Province, Biomedical Research Center of South Hydrobiology, Chinese Academy of Sciences, No. 7 Donghu South Road, Wuhan China, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China; x 430072, Hubei Province, China. E-mail address: [email protected] Key Laboratory of OptoElectronic Science and Technology for Medicine, Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou 350117, The online version of this article contains supplemental material. China; {Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, ‖ Abbreviations used in this article: ATCC, American Type Culture Collection; co-IP, Wuhan 430072, Hubei Province, China; and Innovation Academy for Seed Design, coimmunoprecipitation; DExDc, DEAD/DEAH box helicase domain; dpf, day post- Chinese Academy of Sciences, Wuhan 430072, China fertilization; dpi, day postinfection; EPC, epithelioma papulosum cyprini; h, human; 1X.M.W. and J.Z. contributed equally to this work. HEK293T, human embryonic kidney 293T; Hela, human cervical carcinoma; HEL- ICc, helicase C-terminal domain; hpi, hour postinfection; IP, immunoprecipitation; ORCIDs: 0000-0003-4536-6578 (P.W.L.); 0000-0002-1120-1524 (S.O.); 0000-0001- MO, morpholino; MOI, multiplicity of infection; NLR, nucleotide oligomerization 6238-2313 (Y.H.B.); 0000-0002-9492-2940 (P.N.); 0000-0002-2352-4830 (M.X.C.). domain–like receptor; ORF, open reading frame; PRR, pattern recognition receptor; Received for publication June 19, 2019. Accepted for publication February 7, 2020. qRT-PCR, quantitative real-time PCR; RD, regulatory domain; RLR, RIG-like receptor; RSV, respiratory syncytial virus; SVCV, spring viremia of carp virus; This work was supported by Strategic Priority Research Program of the Chinese Acad- WT, wild-type. emy of Sciences Grant XDA24010308, the National Natural Science Foundation of China (31672687 and 31873046), and the Science Fund for Creative Research Groups Ó of the Natural Science Foundation of Hubei Province of China (2018CFA011). The Copyright 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 sponsors played no role in the design and conduct of the study, in the collection,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900667 2 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE increased viral titer of respiratory syncytial virus (RSV), influenza Cell culture and transfection A virus, and human CMV (16, 18, 19). Although significantly less Zebrafish ZF4 (embryonic fibroblast cell line, CRL-2050; American Type investigated, NOD1 has been shown to regulate innate antiviral Culture Collection [ATCC]) was grown at 28˚C in DMEM/F12 (1:1) responses following infection with ssRNA viruses such as hep- medium supplemented with 10% FBS (Life Technologies), 100 U/ml atitis C and dsDNA viruses such as human CMV (17, 20). penicillin, and 100 mg/ml streptomycin. EPC (CRL-2872; ATCC) cells However, during vesicular stomatitis virus and RSV infection, were maintained in M199 supplemented with 10% FBS at 28˚C. Human embryonic kidney 293T (HEK293T) and human cervical carcinoma (Hela) mammalian NOD1 failed to sense ssRNA viruses and inhibit cells were obtained from the ATCC and cultured in DMEM supplemented virus proliferation (16). Thus, the functional contradictions of with 10% FBS at 37˚C. Transfection of plasmids was performed in ZF4 NOD1 in these reports need more independent studies to confirm cells using the Amaxa Nucleofector II transfection system (Lonza) under if NOD1 regulates antiviral signaling, especially in response to program T20 and in EPC, Hela, and HEK293T cells using Lipofect- amine 2000 (Invitrogen). For all procedures, standard protocols were anti-ssRNA viruses. used according to the manufacturers’ manuals. Alternative splicing plays an important regulatory role in the immune system (21). In teleosts and mammals, NLRs and RLRs Bacterial and viral infections were found to undergo alternative splicing (22, 23). In an analysis Edwardsiella piscicida PPD130/91 and Flavobacterium columnare G4 of human and mouse databases, multiple splicing variants of were used for bacterial infection. E. piscicida was grown in tryptic soy NOD1 and two splicing variants of RIG-I and MDA5 were found broth (BD Biosciences) liquid culture at 28˚C and F. columnare in Luria- (22). However, the reference sequences were derived from geno- Bertani liquid culture at 28˚C. SVCV was propagated in EPC cells at 25˚C. mic sequences. Except for RIG-I–splicing variants (23), the Morpholinos microinjection and yolk sac infection functions of mammalian NOD1 and MDA5 splicing variants are Downloaded from unreported. In teleosts, our previous studies showed that two Morpholinos (MO) were designed and synthesized by Gene Tools and resuspended in nuclease-free water to give a solution of 500 mM. MO splicing variants were identified for RIG-I and MDA5, and all sequences are given in Table I. variants could interact with MAVS (24, 25). The overexpression of MOs were microinjected into one- or two-cell stage embryos. SVCV MDA5a (normal form of MDA5) and MDA5b (shorter splice (1.2 3 107 PFU/ml) was microinjected into the yolk sac of 24-h-old em- variant) in fish cells could induce the promoter activities of type I bryos microinjected with NOD1-MO and control MO. The typical injected volume was 2 nl. The injected embryos were raised at 25˚C in fish water.

IFNs and enhance the protection of transfected cpithelioma pap- http://www.jimmunol.org/ ulosum cyprini (EPC) cells against spring viremia of carp virus Gene cloning and plasmids construction (SVCV) infection (24). But the overexpression of RIG-Ib (normal Based on the zebrafish sequence (GenBank accession no. XM_002665060; form of RIG-I), but not RIG-Ia (long insertion variant of RIG-I), https://www.ncbi.nlm.nih.gov/nuccore/XM_002665060), primers D1GSP1, resulted in a marked enhancement of protection against SVCV D1GSP2, and D1GSP3 were designed and used for 59 rapid amplification of infection (25). Different from piscine RIG-I and MDA5, only one cDNA ends according to the method described previously (27), and D1Fout form of NOD1 exists in zebrafish, which is essential for CD44a- was used for 39 rapid amplification of cDNA end according to the man- mediated activation of the PI3K–Akt pathway and zebrafish larval ufacturer’s instructions for the GeneRacer Kit (Invitrogen). Zebrafish pcDNA3.1-MDA5a, pTurbo-MDA5a-GFP, pTurbo-MDA5b- survival (26). GFP, pt1GFP-MDA5b, p3xFLAG-NOD1 (NOD1-FLAG), and p3xFLAG- To elucidate the possible effects and functional mechanisms of MAVS (MAVS-FLAG) were described previously (24, 26, 28, 29). The by guest on September 28, 2021 piscine NOD1 in sensing viruses, we first assessed the antiviral complete open reading frames (ORFs) of NOD1 were amplified using the primer effects of NOD1 and examined the expression regulations of pairs ptGFP1NOD1F/ptGFP1NOD1R or pcDNANOD1F/pcDNANOD1R and inserted into the ptGFP1 expression vector (30) or pcDNA3.1/myc-His(2)A NOD1 on many antiviral genes and proinflammatory cytokines in vector (Invitrogen). The truncated mutants of zebrafish MDA5, NOD1, and NOD1-overexpressing ZF4 cells and NOD1 knockout and NOD1 MAVS, including pTurbo-MDA5-CARD1-GFP, pTurbo-MDA5-CARD2-GFP, knockdown zebrafish. This was accompanied by studying NOD1 pTurbo-MDA5–DEAD/DEAH box helicase domain (DExDc)–GFP, pTurbo- localization and the binding of NOD1 and its domain(s) to SVCV MDA5–helicase C-terminal domain (HELICc)–GFP, pTurbo-MDA5–regulatory in piscine cells. Based on in vitro and in vivo studies demon- domain (RD)–GFP, pTurbo-MAVS-CARD-GFP, CARD-MAVS-FLAG, ΔCARD-NOD1-FLAG, CARD-NOD1-FLAG, and LRRs-NOD1-FLAG, strating a role of NOD1 in the regulation of the expression of were generated and used for coimmunoprecipitation (co-IP) and RNA- MDA5 isoforms and MAVS, we next determined whether ver- binding experiments. The ORF of the TRAF3 gene was amplified using tebrate NOD1 had a functional linkage with RLR-mediated an- the primer pairs pTurboGFP-TRAF3F/pTurboGFP-TRAF3R or FLAG- tiviral signaling both in piscine and in mammalian cell lines. Our TRAF3F/FLAG-TRAF3R and inserted into the pTurbo-GFP expression vector (Evrogen) or p3XFLAG-CMV-14 Expression Vector (Sigma- data demonstrate that piscine NOD1 plays a crucial role in Aldrich). All the tags were attached to the C terminus of target pro- responding to SVCV infection and that the cumulative effects teins. The primers used for plasmid constructs are listed in Table I. between NOD1 and MDA5(a) in binding with dsRNA and in the Based on human (h)NOD1 (GenBank accession no. NM_006092; https:// formation of the MDA5(a)–MAVS complex are evolutionarily www.ncbi.nlm.nih.gov/nuccore/NM_006092), MDA5 (GenBank accession conserved across vertebrate species. no. AF095844; https://www.ncbi.nlm.nih.gov/nuccore/AF095844), MAVS (GenBank accession no. BC044952; https://www.ncbi.nlm.nih.gov/nuccore/ BC044952), and TRAF3 (GenBank accession no. NM_003300; https://www. Materials and Methods ncbi.nlm.nih.gov/nuccore/NM_003300) sequences, primer pairs hNOD1- Ethics statement HAF/hNOD1-HAR, hMDA5-FLAGF/hMDA5-FLAGR, hMAVS-FLAGF/ hMAVS-FLAGR, hMAVS-HAF/hMAVS-HAR, and hTRAF3-HAF/hTRAF3- All animal experiments were conducted in accordance with the guiding HAR were used to clone the ORFs of human NOD1, MDA5, MAVS, and principles for the care and use of laboratory animals and were approved by TRAF3 and inserted into the p3xFLAG or pcDNA3.1-HA expression vectors. the Institute of Hydrobiology, Chinese Academy of Sciences (approval The primers used for plasmid constructs are listed in Table I. identifier IHB 2013724). Subcellular localization Zebrafish care and maintenance mAbs against zebrafish NOD1 were generated according to our previous NOD1-1IS2/2 (1-bp insertion) and NOD1-2IS2/2 (2-bp insertion) zebra- method (26). mAbs against mammalian NOD1 were purchased from Santa fish were obtained as described in our previous report (26). Wild-type Cruz Biotechnology (catalog number sc-398696). To determine the sub- (WT) AB/TU and mutant zebrafish (China Zebrafish Resource Center) cellular localization of zebrafish NOD1, ZF4 cells were cultured in T25 were raised and maintained at 28˚C on a 12/12 h light/dark cycle in a flow- culture bottles overnight and then infected with E. piscicida and SVCV at a through system. Zebrafish embryos were obtained by artificial insemina- multiplicity of infection (MOI) of 1 or left untreated. To determine tion and reared at 28˚C. the subcellular localization of human NOD1, Hela cells were cultured in The Journal of Immunology 3

Table I. PCR primers used in this study

Name Sequence Application ptGFP1-NOD1F 59-GAACTCGAGAATGAAATGAAATTAAATATGG-39 Ligated to ptGFP1 vector ptGFP1-NOD1R 59-GAACCGCGGACTCATTCAGGGAAACTGG-39 ptGFP1-MDA5bF 59-GACCTCGAGATGGATCCAAACATGAGCAG-39 ptGFP1-MDA5bR 59-GTTGTCGACTCAGTTAGTGTCCATATCTTCATC-39 FLAG-ΔCARD-NOD1F 59-GTGGAATTCGAAGATGGAGCTCCTGAACG-39 Ligated to p3xFLAG-CMV-14 vector FLAG-ΔCARD-NOD1R 59-GAAGGATCCGCTGACTCCCTCTCGTTG-39 FLAG-LRRs-NOD1F 59-CGGAATTCATGCGTAGGAAGCATTTGGG-39 FLAG-LRRs-NOD1R 59-CGGGATCCGTTTTTCAGAGCCTCTGCG-39 FLAG-CARD-NOD1F 59-CGGAATTCATGAAATTAAATATGGGC-39 FLAG-CARD-NOD1R 59-CGGGATCCGGCCTGCGGGTTGAAG-39 FLAG-CARD-MAVSF 59-CCAAGCTTATGTCACTGACACGTGAG-39 FLAG-CARD-MAVSR 59-CGGGATCCTTGAAGAACAGAATCAGG-39 FLAG-TRAF3F 59-GTGAAGCTTGTCATGTCCGCAGGGCGTAA-39 FLAG-TRAF3R 59-GAAGGTACCGAAGGGTCAGGGAGGTCT-39 pcDNANOD1F 59-GACCTCGAGAAATGGGCTCATACAAGACTG-39 Ligated to pcDNA3.1 vector pcDNANOD1R 59-CGGGGTACCGCTGACTCCCTCTCGTTGCT-39 pTurboGFP-MAVSF 59-CCGCTCGAGATGGCTTCACTGACACGTGAGCA-39 Ligated to pTurboGFP vector pTurboGFP-MAVSR 59-TCCCCGCGGATGATTGAGCTTCCAGGC-39 pTurboGFP-MDA5-CARD1F 59-GACCTCGAGATGGATCCAAACATGAGCAG-39 Downloaded from pTurboGFP-MDA5-CARD1R 59-GTTGTCGACTGTTCGGCTTCTTCATCTGG-39 pTurboGFP-MDA5-CARD2F 59-GACCTCGAGATGGAGCCAGATGAAGAAGCC-39 pTurboGFP-MDA5-CARD2R 59-GTTGTCGACCCCTCGCACGGCTCTCCTC-39 pTurboGFP-MDA5-DExDcF 59-GTGCTCGAGATGGAGGTGGCCAGACCTGCT-39 pTurboGFP-MDA5-DExDcR 59-GTTGTCGACTGCTGGCTGACCGCTCCTCCC-39 pTurboGFP-MDA5-RDF 59-CTGCTCGAGATGGAGGAGAAAGTCAAGACC-39 pTurboGFP-MDA5-RDR 59-GTTGTCGACCAGTTAGTGTCCATATCTTCATC-39 http://www.jimmunol.org/ pTurboGFP-MAVS-CARDF 59-CCAAGCTTATGTCACTGACACGTGAG-39 pTurboGFP-MAVS-CARDR 59-CGGGATCCCGATAAATGTCGCTCATCTC-39 pTurboGFP-TRAF3F 59-GTGAAGCTTGTCATGTCCGCAGGGCGTAA-39 pTurboGFP-TRAF3R 59-GAAGGTACCGAAGGGTCAGGGAGGTCT-39 IRF3F 59-TGGCTGTCAATCACATTTCTCG-39 Quantitative real-time PCR IRF3R 59-AGTCGTTCTCCACCAACTGCTC-39 IL-1bF 59-GGCTGTGTGTTTGGGAATCT-39 IL-1bR 59-TGATAAACCAACCGGGACA-39 IL-8F 59-GTCGCTGCATTGAAACAGAA-39 IL-8R 59-CTTAACCCATGGAGCAGAGG-39

Actin F 59-CAGATCATGTTTGAGACC-39 by guest on September 28, 2021 Actin R 59-ATTGCCAATGGTGATGAC-39 SVCV-NF 59-GCCGATTATCCTTCCACCTT-39 Amplification of SVCV genes SVCV-NR 59-TCACTTGCCCTTCCCACTCT-39 SVCV-GF 59-CGACCTGGATTAGACTTG-39 SVCV-GR 59-AATGTTCCGTTTCTCACT-39 hNOD1-HAF 59-CGGAATTCATGGAAGAGCAGGGCCACAG-39 Construction of human NOD1 plasmid hNOD1-HAR 59-CGGGATCCGAAACAGATAATCCGCTTC-39 hMDA5-FLAGF 59-GGGGTACCATGTCGAATGGGTATTCCAC-39 Construction of human MDA5 plasmid hMDA5-FLAGR 59-CGGGATCCATCCTCATCACTAAATAAAC-39 hMAVS-HAF/hMAVS-FLAGF 59-CCCAAGCTTATGCCGTTTGCTGAAGACAAG-39 Construction of human MAVS plasmid hMAVS-HAR/hMAVS-FLAGR 59-GGGGTACCCCGTGCAGACGCCGCCGGTACAG-39 hTRAF3-HAF 59-CCGGAATTCATGGAGTCGAGTAAAAAG-39 Construction of human TRAF3 plasmid hTRAF3-HAR 59-CGGGATCCGGGATCGGGCAGATCCGAAG-39 Translation-blocking NOD1-MO 59-TCAGAGCCCGAATACCAGTCCAAAC-39 MOs of NOD1 Five-base mismatch control MO 59-TCAcAcCCCcAATACgAcTCCAAAC-39 six-well plates overnight and then stimulated with LPS (50 mg/ml) or expression of antiviral genes, the hatched larvae at 4 d postfertilization transfected with 5 mg poly I:C or left untreated. After 6 h postinfection (dpf) from WT zebrafish were exposed to 2 3 106 PFU/ml SVCV and (hpi) or stimulation, cytoplasm-bound, membrane-bound, nucleus-bound, collected at 1 and 2 d postinfection (dpi) for RNA extraction. To examine and chromatin-bound proteins were extracted using a Subcellular Pro- the effect of NOD1 deficiency on the expression of antiviral genes without tein Fractionation Kit (Thermo Fisher Scientific). The primary Abs used infection, the hatched larvae at 5 and 10 dpf from WT and NOD1-1IS2/2 were monoclonal anti-zfNOD1 Ab (1:2000), monoclonal anti-hNOD1 Ab zebrafish were used for RNA extraction. To examine the effect of NOD1 (1:2000), polyclonal anti–b-tubulin Ab (1:2000, ab6046; Abcam), poly- deficiency on the expression of antiviral genes in response to SVCV in- clonal anti-HDAC1 Ab (1:2000, ab41407; Abcam), and polyclonal anti- fection, the hatched larvae (4 dpf) from WT and NOD1-1IS2/2 zebrafish histone H3 Ab (1:2000, no. 9715; Cell Signaling Technology). The bands were exposed to 2 3 106 PFU/ml SVCV and collected at 1 and 2 dpi for were detected using MilliporeSigma Immobilon Western Chemilumines- RNA extraction. To examine the effect of NOD1 knockdown on the ex- cent HRP Substrate and ECL Western Blot System (LAS 4000 Mini, Fuji, pression of IRF3 and antiviral genes with or without SVCV infection, Japan) according to the manufacturer’s instructions. Western blotting re- zebrafish embryos were injected with 2 nl NOD1-MO and control MO sults were quantified using Quantity One Software. (500 mM) at the one-cell or two-cell stage and then injected with SVCV Quantitative real-time PCR (24 PFU) or culture medium at 24 h postfertilization. Fifteen to twenty embryos were lysed in TRIzol at 6 and 24 hpi to prepare total RNAs. To For understanding the expression change of NOD1 in response to different determine differences between the effects of MDA5 isoforms and MAVS pathogens, zebrafish ZF4 cells were infected with E. piscicida, F. cloum- and between NOD1 and MDA5 isoforms or domains on the expression nare G4, and SVCV at an MOI of 1 and collected at 6, 12, and 24 hpi for of antiviral genes, pcDNA3.1, ptGFP1, pcDNA3.1-NOD1, pcDNA3.1- RNA extraction. To examine the effect of SVCV infection on the MDA5a, ptGFP1-MDA5b, MDA5-RD-GFP, MDA5-HELICc-GFP, and 4 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE

MAVS-FLAG were diluted to the desired concentration of 100 ng/ml. Co-IP and Western blot Combinations of two kinds of plasmids were microinjected into fertilized zebrafish eggs at the one-cell stage, respectively. About 50 embryos per Polyclonal Abs against zebrafish MDA5 were generated according to our group were collected at 24 h and/or 48 h postmicroinjection. To deter- previous method (24). To investigate the endogenous interaction between mine NOD1-dependent or cytokine-mediated upregulation of antiviral MDA5 and NOD1, ZF4 cells were infected with SVCV at an MOI of 1 or genes, the naive ZF4 cells were incubated by the supernatant from left untreated. At 24 hpi, ZF4 cells were used for protein extraction using NOD1-overexpressing or ptGFP1-overexpressing cells. At 48 h, ZF4 cells lysis buffer containing 50 mM Tris HCl (pH 7.4) with 150 mM NaCl, overexpressed with NOD1 or ptGFP1, and the naive ZF4 cells incubated 1 mM EDTA, and 1% Triton X-100. Co-IP was carried out using the by the supernatant from NOD1-overexpressing or ptGFP1-overexpressing Thermo Scientific Pierce Co-IP Kit according to the manufacturer’s cells were collected and used for RNA extraction. PCR analysis was manual. The NOD1 Abs were first immobilized with AminoLink Plus performed using an ABI Prism 7000 system using primers specific to in- Coupling Resin using Coupling Buffer overnight. The resin was then dividual genes (Table I) or those described by others (31–33). The relative washed and incubated with ZF4 lysate overnight. After incubation, the expression of target genes was normalized to the expression of zebrafish resin was again washed and protein eluted using Elution Buffer (catalog GAPDH and expressed as fold changes relative to the corresponding number E6150; Sigma-Aldrich). Total lysate and eluted proteins were control group or arbitrary units relative to GAPDH. analyzed by Western blot analysis with anti-MDA5 (1:2000) and anti- NOD1 (1:2000) Abs. Luciferase activity assay To perform competitive binding interactions among NOD1, MDA5a/ MDA5b, and MAVS or among NOD1, MAVS, and TRAF3, EPC cells To examine the effect of NOD1 on the IFN induction mediated by MDA5a were transfected with the indicated plasmids. At 24 h posttransfection, or MDA5b and MAVS_tv1 (MAVS normal form), EPC cells seeded the cells were washed with ice-cold PBS buffer and then lysed in IP lysis overnight were transiently transfected with various plasmids at the indicated buffer containing Protease Inhibitor Cocktail. Co-IP was performed DNA concentration, together with 25 ng Renilla and 250 ng IFN1 and IFN3 using an FLAG-Tagged Protein Immunoprecipitation Kit (Sigma- reporter plasmids. Forty-eight hours posttransfection, the cells were lysed,

Aldrich) according to the manufacturer’s manual. The agarose was Downloaded from and luciferase activity was measured using the Dual-Luciferase Reporter washed four times with ice-cold wash solution, and protein was eluted Assay System (Promega). with Elution Buffer. Total lysates and eluted proteins were analyzed by Antiviral assay in vitro and in vivo Western blot analysis using anti-GAPDH (1:5000; Proteintech), anti- FLAG (1:5000, catalog no. F3165; Sigma-Aldrich), anti-pTurboGFP To explore the effect of NOD1 overexpression in SVCV infection in vitro, (1:5000, catalog no. AB513; Evrogen), anti-MDA5 (1:2000), or anti- ZF4 cells were transfected with ptGFP1 or ptGFP1-NOD1 and subjected to NOD1 (1:2000) Abs. G418 selection to enrich the GFP-positive cells. SVCV infection in the 24 To test for competitive binding interaction among hNOD1, hMDA5, and multiwell plates was performed as described previously (34, 35). Briefly, hMAVS or among hNOD1, hMAVS, and hTRAF3, HEK293T cells were http://www.jimmunol.org/ ZF4 cells stably transfected with ptGFP1 and ptGFP1-NOD1 were seeded transfected with the indicated plasmids. At 24 h, the cells were transfected overnight and then infected with SVCV with the MOI of 5, 0.5, and 0.05. again with poly(I:C) (2 mg/ml) or left untreated. Another 6 h later, the cells Postinfection for 5 d at 20˚C, the supernatants were collected for the de- were washed with PBS and then lysed in IP lysis buffer containing Pro- termination of virus titers by a standard plaque assay. The plates were fixed tease Inhibitor Cocktail. The cell lysates were incubated overnight at 4˚C in 10% paraformaldehyde for 1 h, then stained with 0.5% crystal violet and with the Anti-FLAG M2-Agarose Affinity Gel. Total lysates and eluted photographed. To quantify stained cells, the crystal violet was dissolved proteins were analyzed by Western blot analysis with anti-GAPDH, anti- in 100 ml 1% SDS solution for 10 min in an orbital shaker at 150 rpm. FLAG, anti-HA (1:5000), or anti-hNOD1 (1:2000, sc-398696; Santa Cruz The absorbance was read at a 562-nm wavelength using a BioTek Lab Biotechnology) Abs. Plate Reader. The relative OD value was calculated by dividing the av- To investigate the exact effects of MDA5 isoforms or domains on the erage OD value of the infected wells (n = 3) with that of the uninfected protein expression of NOD1, these proteins, including MDA5a and by guest on September 28, 2021 control (n =3). MDA5b, the different domains of MDA5 and NOD1 were expressed in To explore the effect of NOD1 deficiency in SVCV infection in vivo, the HEK293T cells, alone or in combination, because fish MDA5a or the 2 2 hatched larvae (4 dpf) from WT and NOD1-1IS / zebrafish were exposed different domains of MDA5 would be unlikely to induce the endogenous 2 2 to 2 3 106 PFU/ml SVCV or from WT and NOD1-2IS / zebrafish ex- expression of MDA5b in this human cell line. HEK293T cells seeded posed to 2 3 106 PFU/ml SVCV. After immersion in the SVCV suspension overnight were transiently transfected with various plasmids at the for 24 h, zebrafish larvae were maintained in 60-mm sterile disposable indicated DNA concentration. At 24 h, the cells were washed with PBS petri dishes with supplemental 25 ml fresh egg water. Exposures were and then lysed in Pierce RIPA Buffer containing Protease Inhibitor performed in triplicate, with 45 larvae per group or in quintuplicate with Cocktail. The cell lysates were analyzed by Western blot analysis with 53–55 larvae per group. The number of surviving larvae was counted daily anti-GAPDH, anti-FLAG, anti-pTurboGFP, anti-MDA5, or anti-actin for 5 or 7 d. GraphPad Prism 6 was used to generate survival curves, and (1:2000) Abs. the log-rank test was used to test differences in survival between the WT and NOD1-1IS2/2 or between the WT and NOD1-2IS2/2 zebrafish in- In vitro poly(I:C) pulldown assays fected with SVCV. 2 To explore the effect of NOD1 knockdown during SVCV infection EPC or HEK293T cells seeded in 25-cm cell culture flasks were in vivo, zebrafish embryos were injected with 8 ng MOs at one-cell stage and transfected with various plasmids. Transfected cells were harvested 24 h then SVCV (24 PFU) or medium injected at 24 h postfertilization. Mortality after transfection and lysed in IP lysis buffer containing Protease In- was monitored for 2 d. Experiments were performed in triplicate with 36–45 hibitor Cocktail. Cellular debris was removed by centrifugation at 3 embryos per group. 12,000 g for 10 min at 4˚C. Poly(I:C)-coated beads were generated as To explore the effect of NOD1 overexpression on the SVCV replication previously described (30). Briefly, poly(C)-conjugated agarose beads mediated by MDA5 isoforms, ZF4 cells stably transfected with ptGFP1 and and poly(I) were resuspended in buffer containing 50 mM Tris (pH 7) ptGFP1-NOD1 were seeded overnight and then transfected with empty and 150 mM NaCl to a final concentration of 2 mg/ml and mixed at a plasmid, MDA5a, or MDA5b. After 36-h transfection, these cells were ratio of 1:2 (v/v). The mixture was incubated at 4˚C overnight, centri- 5 3 infected with SVCV (5 3 10 PFU/ml) and finally collected at 24 hpi for fuged at 1000 g for1min,andwashedonce.Followingthis,the examining the expression of SVCV genes. poly(I:C)-coated beads were washed with binding buffer containing 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% NP-40. Interaction of NOD1 with viral RNA The beads were then incubated with cell lysates at 4˚C for 1 h, centrifuged at 1000 3 g, rinsed three times with binding buffer, and EPC cells were transfected with p3xFLAG empty vector, NOD1-FLAG, finallyresuspendedin80ml13 SDS sample buffer by boiling for Δ CARD-NOD1-FLAG, CARD-NOD1-FLAG, LRRs-NOD1-FLAG, 10 min at 95˚C. The input protein and the immunoprecipitates were or CARD-MAVS-FLAG and then infected with SVCV at an MOI of 1. analyzed by Western blot analysis with anti-GAPDH, anti-pTurboGFP, At 24 hpi, the cells were washed with ice-cold PBS buffer and then lysed anti-FLAG, or anti-hNOD1 Abs. in immunoprecipitation (IP) lysis buffer (Thermo Fisher Scientific) containing Protease Inhibitor Cocktail. Lysates were immunoprecipitated with Statistical analysis FLAG agarose (Sigma-Aldrich) for 4 h at 4˚C. After washing the beads with Wash Buffer (20 mM Tris [pH 7.5], 10 mM NaCl, and 0.1% Tween Expression data determined by quantitative real-time PCR (qRT-PCR) are 20 detergent) eight times, TRI Reagent was added to isolate bound RNA. presented as means and SEM. Significant differences were determined RT-PCR was performed using SVCV-N, SVCV-G, actin, and GAPDH- using a two-tailed Student t test or a one-way ANOVA, followed by a specific primers that are listed in Table I. Tukey test for multiple comparisons. The level of significance is shown as The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Expression and regulation of zebrafish NOD1. (A) The inducible expression of zebrafish NOD1 in ZF4 cells infected with F. columnare G4, E. piscicida, and SVCV (MO1 = 1). Zebrafish ZF4 cells seeded overnight in six-well plates were infected or left untreated. At 6, 12, and 24 hpi, the untreated cells and those cells infected with F. columnare G4, E. piscicida, or SVCV were collected and used for RNA extraction. The untreated cells were used as a control group. (B) The in vitro antiviral role of zebrafish NOD1 determined by plaque assay of viral titer. ZF4 cells (Figure legend continues) 6 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE follows: *p , 0.05 and **p , 0.01. Significance testing in the cumulative signaling. Although statistically significant, NOD1 knockout did survival analysis was carried out using GraphPad Prism software. not seem to have major effects on most antiviral genes (around 2- fold); however, it did have the most prominent effects on IL-1b and Results IL-8 genes (Fig. 1E). In infected zebrafish larvae, NOD1-1IS2/2 Expression and subcellular localization of NOD1 mutants demonstrated elevated expression of SVCV-N, SVCV-G, We first examined the inducible expression of NOD1 in ZF4 cells in and SVCV-P and decreased expression of IL-1b and IL-8, whereas response to different pathogens. NOD1 gene expression was in- the expression of most antiviral genes did not change at 1 dpi. At 2 2 duced by bacterial and viral infection at 6, 12, and 24 h following 2 dpi, NOD1-1IS / mutants demonstrated markedly reduced infection. The induction of expression was highest at 6 h and expression of most antiviral genes, including MDA5a, MDA5b, decreased thereafter (Fig. 1A). Then, we determined the subcel- IRF3, IFN1, IFN3, MXA, MXB, MXE, PKZ, and RSAD2, and in- lular localization of zebrafish NOD1 using an anti-NOD1 mAb flammatory cytokines, including IL-1b and IL-8, whereas no (26). Zebrafish NOD1 resided in the cytoplasm and membrane in significant effect was observed for SVCV-N, SVCV-G, and SVCV-P mock- and microbe-infected ZF4 cells by subcellular fractionation (Fig. 1F). In WT zebrafish larvae, the abundances of RIG-Ib and (Supplemental Fig. 1A, 1B). Interestingly, a fraction of endoge- IRF3 mRNA transcripts were lowered by SVCV infection at 2 dpi, nous NOD1 was also localized in the nucleus, which was asso- whereas most antiviral genes and inflammatory cytokine tran- ciated with chromatin (Supplemental Fig. 1C, 1D). SVCV scripts, including those of RIG-I, MDA5a, MAVS_tv1, IFN1, MXA, infection slightly increased the expression of NOD1 in the cyto- MXB, MXC, MXE, PKZ, RSAD2, IL-1b, and IL-8, were signifi- plasm and nucleus, whereas it decreased in chromatin at 6 hpi cantly elevated by 1 or 2 dpi SVCV infection (Supplemental Fig.

(Supplemental Fig. 1A, 1C, 1D). The localization of NOD1 in the 2C). All these results suggest that NOD1 knockout can impair the Downloaded from nuclei and chromatin is conserved both in piscine and mammalian SVCV-induced expression of IL-1b and IL-8 genes, but not for 2 2 cells (Supplemental Fig. 1). RLR pathway members. Notably, SVCV-infected NOD1-1IS / and NOD1-2IS2/2 zebraﬁsh exhibited reduced survival com- NOD1 regulates antiviral response and the expression of RLR pared with WT counterparts (Supplemental Fig. 2D, 2E). pathway members We also examined the effect of NOD1 knockdown on the ex-

To elucidate the importance of NOD1 in antiviral immunity, ZF4 pression of RLR pathway members and larvae survival. In the http://www.jimmunol.org/ cells transfected stably with NOD1 were infected separately with initial study, the expression of IRF3 was examined under mock or SVCV at various MOI. Cells transfected with NOD1 were much SVCV infection. As shown in Supplemental Fig. 2F, knock- more resistant to SVCV infection than the cells transfected with down of NOD1 significantly decreased the expression of IRF3. empty vector ptGFP1 (Supplemental Fig. 2A, 2B). Additionally, Similar to NOD1 knockout, NOD1 knockdown could not in- SVCV titers in cells transfected with NOD1 were significantly hibit SVCV-induced expression of RLR pathway members but lower than those in control cells (Fig. 1B). Therefore, NOD1 is exhibited reduced survival compared with control-MO counter- effective in restricting the replication of SVCV. parts (Supplemental Fig. 2G, 2H) and the decreased expression of To investigate the possible role of NOD1 in host defense, qRT- RLR pathway members in uninfected NOD1 knockdown larvae PCR were performed to quantify the expression of proin- (data not shown). by guest on September 28, 2021 flammatory cytokines and those genes involved in RLR-mediated antiviral signaling. NOD1 overexpression significantly elevated CARD is required by NOD1 to bind with SVCV transcript abundances of proinflammatory cytokines, including To determine whether piscine NOD1 acts as a PRR for viral RNA, IL-1b and IL-8, and those genes involved in RLR-mediated anti- we precipitated the full-length protein and those truncated proteins viral signaling, such as RIG-I, MDA5, MAVS, IRF3, TBK1, IFN1, with different domains of NOD1 from SVCV-infected EPC cells IFN3, MXA, MXB, MXC, MXE, PKZ, and RSAD2 (Fig. 1C). that were transfected with NOD1-FLAG, ΔCARD-NOD1-FLAG Previous studies have shown that ectopic overexpression of or LRRs-NOD1-FLAG (Fig. 2A), and amplified bound RNA with NOD1 led to the induction of NF-kB signaling and the production either SVCV specific primers or GAPDH primers (control). of proinflammatory cytokines (36, 37). To distinguish NOD1- Control with p3xFLAG was also added (Fig. 2B, 2C). Western dependent or cytokine-mediated upregulation of antiviral genes, blotting confirmed the expression of all transfected plasmids supernatant transfer experiments were performed in the naive ZF4 (Fig. 2B). The interaction of NOD1 with SVCV was observed. In cells. The results from qRT-PCR suggested that the upregulation contrast, GAPDH mRNA did not associate with NOD1 (Fig. 2C). of most antiviral genes involved in RLR-mediated signaling was The truncated NOD1 protein with both NACHT and LRR do- cytokine-mediated, whereas the upregulation of IFN3, IL-1b, and mains, or only LRR domains, failed to bind with SVCV (Fig. 2C). IL-8 was NOD1 dependent (Fig. 1D). To demonstrate the binding of CARD of NOD1 to SVCV, We further investigated the in vivo roles of NOD1 knockout and plasmids containing only CARD from NOD1 or MAVS were knockdown in the antiviral response and RLR-mediated signaling. constructed (Fig. 2D). The protein expression of CARD-NOD1- In uninfected zebrafish larvae, NOD1-1IS2/2 mutants exhibited FLAG was confirmed by Western blotting, together with the impaired expression of key genes involved in RLR-mediated expression of LRRs-NOD1-FLAG and CARD-MAVS-FLAG

stably transfected with NOD1 or ptGFP1 were infected with SVCV with the MOI = 5, 0.5, and 0.05. Postinfection for 5 d at 20˚C, the supernatants from the cells with the MOI = 0.5 were collected for the determination of virus yield (output/input) by a standard plaque assay. (C) The expressions of antiviral genes and cytokines in ZF4 cells overexpressed with NOD1. The ZF4 cells overexpressed with ptGFP1 were used as a control group. (D) The expressions of antiviral genes and cytokines in the naive ZF4 cells incubated by the supernatant from NOD1-overexpressing cells. The naive ZF4 cells incubated by the supernatant from ptGFP1-overexpressing cells were used as a control group. (E) The expressions of antiviral genes and cytokines in the NOD1 knockout zebrafish without SVCV infection. The WT and NOD1-1IS2/2 zebrafish were collected at 5 and 10 dpf for RNA extraction and qRT-PCR. The WT zebrafish were used as a control group. (F) The expressions of antiviral genes and cytokines in the NOD1 knockout zebrafish with SVCV infection. The WT or NOD1-1IS2/2 zebrafish were collected at 1 and 2 dpi and used for qRT-PCR. The WT zebrafish infected with SVCV were used as a control group. For all qRT-PCR, data represent the means 6 the SEM (n = 3) and were tested for statistical significance using a two-tailed Student t test. *p , 0.05, **p , 0.01. ns, not significant. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. CARD is required by zebrafish NOD1 to bind with SVCV. (A) Schematics of WT and the truncated NOD1 proteins used in RNA binding studies. (B) The confirmation of protein expression of transfected plasmids. (C) The association of WT and the truncated NOD1 proteins with SVCV. (D) Schematics of the truncated CARD from NOD1 and MAVS. (E) The confirmation of protein expression of the truncated CARD and LRRs. (F) The association of the truncated NOD1 and MAVS proteins with SVCV. EPC cells were transfected with indicated plasmids and then infected with SVCV (MOI = 1) for 8 h. Lysates were analyzed by immunoblotting with the indicated Abs (B and E) or immunoprecipitated with Anti-FLAG M2-Agarose (C and F). Bound RNA was amplified with primers specific for SVCV-N, SVCV-G, actin, or GAPDH, followed by agarose gel electrophoresis.

(another FLAG-tagged CARD from MAVS) (Fig. 2E). The RSAD2, the inducible multiple of MDA5b–MAVS signaling is far truncated NOD1 protein with CARD was indeed associated below that achieved by MDA5a–MAVS signaling (Fig. 3A–D). with SVCV, but not for LRR domain from NOD1 and CARD of Furthermore, the overexpression of NOD1 markedly increased the MAVS (Fig. 2F). Furthermore, GAPDH and b-actin mRNAs activation of zebrafish IFN1 and IFN3 promoters induced by did not associate with the truncated NOD1 protein with CARD MDA5a–MAVS. However, the overexpression of NOD1 reduced (Fig. 2F). the activation of zebrafish IFN1 and IFN3 promoters induced by MDA5b–MAVS (Fig. 3E, 3F). Furthermore, comicroinjection NOD1 and MDA5a cooperate to induce the transcription of of NOD1 and MDA5a in zebrafish cooperatively increased the downstream antiviral genes abundance of all tested antiviral genes including IRF3, IFN1, NOD1 deficiency impaired the expression of MDA5 and MAVS MXE, and RSAD2, whereas comicroinjection of NOD1 and (Supplemental Fig. 3A–C), and vice versa (Fig. 1C). This MDA5b in zebrafish failed to cooperatively increase the expres- prompted us to determine whether fish NOD1 and MDA5/MAVS sion of most downstream antiviral genes, except for a weak effect signaling have a cooperative effect in antiviral defense. Because on IFN1 at 24 h (Fig. 3G–J). piscine MDA5a and MDA5b have been shown to mediate the MDA5a and MDA5b contain the common two CARDs and a antiviral immune response against SVCV infection (24), we next DExDc in the N terminus, whereas the HELICc and the RD in the investigated the roles of NOD1 and MDA5 isoforms in MAVS- C terminus are absent for MDA5b. We next determined whether mediated signaling. Although both MDA5a–MAVS and MDA5b– the HELICc and RD in the C terminus of MDA5a were pivotal MAVS induced significant expression of IRF3, IFN1, MXE, and for the coordinate induction of antiviral genes mediated by NOD1 8 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. The effect of MDA5 isoforms and MAVS or NOD1 on the expression of antiviral genes. (A) The effect of MDA5a–MAVS and MDA5b– MAVS on the expression of IRF3. (B) The effect of MDA5a–MAVS and MDA5b–MAVS on the expression of IFN1. (C) The effect of MDA5a–MAVS and MDA5b–MAVS on the expression of MXE.(D) The effect of MDA5a–MAVS and MDA5b–MAVS on the expression of RSAD2. (E) Overexpression of NOD1 increased zebraﬁsh IFN1 and IFN3 promoter activities induced by MDA5a–MAVS. (F) Overexpression of NOD1 (Figure legend continues) The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 4. The effect of NOD1 on the poly(I:C) binding and SVCV replication mediated by MDA5 isoforms. (A) NOD1 enhanced the binding of MDA5a to poly(I:C). (B) NOD1 had no significant effect on the binding of MDA5b to poly(I:C). For (A) and (B), EPC cells were transfected with the indicated plasmids. Cell lysates were incubated with poly(I:C) beads. Bound proteins were analyzed by immunoblotting with anti-MDA5 Ab. The cell by guest on September 28, 2021 lysates were analyzed by immunoblotting with the indicated Abs. The expression ratio was quantified by Quantity One. Each Western blot is representative of at least four independent experiments. (C) NOD1 enhanced the inhibition of SVCV-N expression mediated by MDA5a. (D) NOD1 enhanced the inhibition of SVCV-G expression mediated by MDA5a. ZF4 cells stably transfected with NOD1 or ptGFP1 were transfected with empty plasmid, MDA5a, or MDA5b. At 36-h transfection, these cells were infected with SVCV and collected at 24 hpi. Data represent the mean 6 SEM (n = 3), and statistical significance was assessed using a one-way ANOVA, followed by a Tukey test. The asterisk above the error bars indicates statistical significance using the group microinjected with empty plasmid as the control group. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket. **p , 0.01. ns, not significant. and MDA5a. The overexpression of HELICc or RD alone only MDA5a or MDA5b may be due to the influence of the HELICc increased the expression of IRF3 and/or IFN1. However, with the and RD in the C terminus of MDA5a. addition of NOD1, the RD cooperatively increased the expression of IRF3, IFN1, MXE, and RSAD2, especially the expression of NOD1 and MDA5a cooperate to bind dsRNA and inhibit IFN1 with a 324-fold increase against the MDA5-RD alone and a viral replication 2959-fold increase against the MDA5-RD in combination with To directly test the hypothesis that NOD1 may modulate the NOD1 (Fig. 3K–N). Comicroinjection of NOD1 and the HELICc binding of the MDA5 isoforms to their ligand poly(I:C), pulldown of MDA5a also cooperatively increased the expression of IFN1, assays were performed in EPC cells with poly(I:C) beads and MXE, and RSAD2 (Fig. 3L–N). Altogether, all these data sug- blotted with anti-GFP Ab. NOD1 was found to bind to poly(I:C) gest that the inverse relationships observed between NOD1 and in the absence of MDA5 isoforms (Supplemental Fig. 3D).

suppressed zebrafish IFN1 and IFN3 promoter activities induced by MDA5b–MAVS. (G) The effect of NOD1 and MDA5 isoforms on the expression of IRF3. (H) The effect of NOD1 and MDA5 isoforms on the expression of IFN1. (I) The effect of NOD1 and MDA5 isoforms on the expression of MXE. (J) The effect of NOD1 and MDA5 isoforms on the expression of RSAD2. (K) The RD or HELICc of MDA5 on the expression of IRF3 with or without NOD1 expression. (L) The RD or HELICc of MDA5 on the expression of IFN1 with or without NOD1 expression. (M) The RD or HELICc of MDA5 on the expression of MXE with or without NOD1 expression. (N) The RD or HELICc of MDA5 on the expression of RSAD2 with or without NOD1 expression. For (A)–(D) and (G)–(N), every plasmid was diluted to 100 ng/ml and microinjected into zebrafish embryos with the indicated combinations (1:1). Fifty to sixty embryos were collected at 24 and/or 48 h postmicroinjection and used for qRT-PCR. For (E) and (F), EPC cells seeded overnight in 24-well plates were transiently transfected with various indicated plasmids with indicated DNA concentration, together with 25 ng Renilla and 250 ng IFN1 or IFN3 reporter plasmids. Data represent the mean 6 SEM (n = 3), and statistical significance was assessed using a one-way ANOVA, followed by a Tukey test. The asterisk above the error bars indicates statistical significance using the group microinjected with empty plasmid as the control group. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket. **p , 0.01, *p , 0.05. ns, not significant. 10 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. The effect of NOD1 on the formation of MDA5 isoform(s)–MAVS and MAVS–TRAF3 complexes. (A) The protein sizes of MDA5a and MDA5b were determined by Western blotting using MDA5 Ab. The protein samples were from EPC cells transfected with the expression plasmids of pcDNA3.1-MDA5a and ptGFP1-MDA5b. (B) Endogenous association of NOD1 and MDA5. The ZF4 cells were either left uninfected or infected with SVCV for 24 h. Cell lysates were immunoprecipitated with anti-NOD1–conjugated agarose beads. The immunoprecipitate (Figure legend continues) The Journal of Immunology 11

The ability of NOD1 to enhance the MDA5a–dsRNA interaction confirmed to interact with the CARDs of MDA5, but not with the was also observed (Fig. 4A). Conversely, NOD1 did not signifi- CARD of MAVS (Supplemental Fig. 3H). cantly enhance the MDA5b–dsRNA interaction (Fig. 4B). Finally, whether NOD1 interfered with the formation of the Based on the stable cell lines transfected with ptGFP1-NOD1 MDA5 isoform(s)–MAVS complex was investigated through or empty vector ptGFP1, we ectopically expressed MDA5a, competition binding experiments. The results from co-IP suggest MDA5b, or ptGFP1 in NOD1- overexpressing or ptGFP1- that NOD1 inhibits the assembly of the MDA5b–MAVS_tv1 overexpressing ZF4 cells and determined the influence of complex (Fig. 5C). In contrast, the addition of NOD1 significantly these proteins (and their interactions) on SVCV replication. strengthened the association between MDA5a and MAVS_tv1 Compared with the corresponding control group transfected (Fig. 5D). The decreased association of MDA5b–MAVS_tv1 with empty vector, NOD1 and MDA5a, but not MDA5b, sig- complex and increased association of MDA5a–MAVS_tv1 com- nificantly decreased the expression of SVCV-N and SVCV-G at plex were further confirmed by the coexpression of NOD1, 24 hpi. Moreover, the cooperative repression of SVCV genes by MDA5a, MDA5b, and MAVS_tv1 (Fig. 5E). NOD1 and MDA5a was observed in ZF4 cells (Fig. 4C, 4D). In mammals, the association of TRAF3 with MAVS is essential Compared with the corresponding control group transfected for IFN induction. We further determined the effect of NOD1 on with empty vector, the decreased 4.17-fold, 18.00-fold, and the MAVS–TRAF3 signalosome. Co-IP experiments showed 51.90-fold for SVCV-N were observed in the group transfected thatTRAF3interactedwithMAVS_tv1(Fig.5F)andNOD1 with MDA5a alone, NOD1 alone, and the combination of (Supplemental Fig. 3I) and that the interaction between TRAF3 NOD1 and MDA5a (Fig. 4C). The decreased 3.93-fold, 23.29- and MAVS was markedly increased upon addition of NOD1 fold, and 64.15-fold for SVCV-G were observed in the group (Fig. 5F). In the case of coexpression of NOD1, MDA5b, MAVS_tv1, Downloaded from transfected with MDA5a alone, NOD1 alone, and the combi- and TRAF3, the decreased association of MDA5b–MAVS_tv1 nation of NOD1 and MDA5a (Fig. 4D). complex and increased association of MAVS_tv1–TRAF3 complex were further confirmed (Fig. 5G). NOD1 differentially regulates the formation of MDA5 isoform(s)–MAVS complex and targets TRAF3 to promote the MDA5 isoforms differentially regulate NOD1 expression formation of MAVS–TRAF3 complex

During the pulldown assays for input proteins, we repeatedly http://www.jimmunol.org/ We next investigated whether NOD1 targeted the MDA5–MAVS observed that NOD1 expression was much lower after cotrans- complex to regulate immune responses. Because of the fact that fection with MDA5b and higher after cotransfection with MDA5a. the polyclonal Ab of MAVS failed to recognize the endogenous That ﬁnding prompted us to investigate the effect of MDA5 iso- expression of MAVS, we ﬁrst determined whether NOD1 inter- forms on NOD1 expression. We found that the overexpression of acted with MAVS-FLAG using an anti-FLAG Ab. The interaction MDA5a at low dosage increased the expression of NOD1 but was between MDA5a and MAVS_tv1 was examined as a positive unchanged at high dosage (Fig. 6A). The overexpression of control. The association of MDA5a with MAVS_tv1 was readily MDA5b led to considerable loss of overexpressed NOD1 in a detected by IP analysis, but NOD1 did not interact with dosage-dependent manner (Fig. 6B). The NOD1 loss induced by

MAVS_tv1 (Supplemental Fig. 3E). Next, we determined whether MDA5b overexpression was blocked by the proteasome inhibitor by guest on September 28, 2021 NOD1 interacted with MDA5 isoforms using an NOD1 mAb and MG132 (Fig. 6C); thus, we concluded that MDA5b might trigger an MDA5 polyclonal Ab. The sizes of MDA5a and MDA5b were proteasomal degradation of NOD1. confirmed by Western blotting (Fig. 5A). Co-IP experiments MDA5a and MDA5b contain the common two CARDs and a demonstrated that endogenous NOD1 weakly interacted with DExDc in the N terminus, whereas the HELICc and RD in MDA5 isoforms in mock-infected ZF4 cells, but the interaction the C terminus are absent for MDA5b. Therefore, we wanted was increased following SVCV infection (Fig. 5B). to determine if the N-terminal domains were important for Domain mapping analysis was further performed to determine MDA5b-induced degradation of NOD1 but the opposite for the which domains of MDA5 are responsible for its interaction with C-terminal domains of MDA5a. We found that the DExDc in NOD1. We generated a series of MDA5 truncations tagged with the N terminus triggered NOD1 degradation, which equated GFP, including CARD1 (MDA5 truncation containing the first to the degradation of MDA5b by NOD1. Different from DExDc, CARD domain), CARD2 (MDA5 truncation containing the second the HELICc and RD in the C terminus of MDA5a increased the CARD domain), DExDc (MDA5 truncation containing DExDc), expression of NOD1, especially RD (Fig. 6D). Besides, the and RD (MDA5 truncation containing RD) (Supplemental Fig. 3F). HELICc or/and RD can inhibit protein degradation of NOD1 These truncations were expressed in EPC cells along with NOD1- mediated by MDA5b (Fig. 6E). FLAG. As shown in Supplemental Fig. 3G, NOD1 failed to interact with the middle DExDc and the C-terminal RD. In The roles of NOD1 in enhancing the complex formation of the contrast, an obvious association of NOD1 with the N-terminal normal form of MDA5–MAVS and the binding of MDA5 to CARDs of MDA5 was observed, especially the first CARD poly(I:C) are evolutionarily conserved across species (Supplemental Fig. 3G). To eliminate the possibility of forming To analyze and test the functional complementarity between human hetero-oligomers by CARDs, we added the CARD from MAVS as NOD1 and piscine NOD1, several key molecules including NOD1, a negative control in the immunoprecipitations. Again, NOD1 was MDA5, MAVS, and TRAF3 were cloned from human cell lines.

and lysates were analyzed by immunoblotting with anti-NOD1 and anti-MDA5 Abs. (C) NOD1 inhibited the interaction between MDA5b and MAVS. (D) NOD1 enhanced the interaction between MDA5a and MAVS. (E) The decreased formation of the MDA5b–MAVS complex and the increased formation of the MDA5a–MAVS complex were further confirmed in the case of coexpression of NOD1, MDA5a, MDA5b, and MAVS_tv1. (F) NOD1 enhanced the interaction between MAVS and TRAF3. (G) The decreased formation of the MDA5b–MAVS complex and increased formation of the MAVS–TRAF3 complex were further confirmed in the case of coexpression of NOD1, MDA5b, MAVS_tv1, and TRAF3. For (C)–(G), co-IP was performed with anti- FLAG–conjugated agarose beads in EPC cells. The cell lysates and bound proteins were analyzed by immunoblotting with the indicated Abs. The expression ratio was quantified by Quantity One. Each Western blot is representative of approximately two to four independent experiments. 12 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. The effect of MDA5 isoforms and domains on the protein expression of NOD1. (A) The effect of MDA5a on the protein expression of NOD1. (B) The effect of MDA5b on the protein expression of NOD1. For (A) and (B), HEK293T cells seeded in six-well plates were transfected with various indicated plasmids. After 24 h posttransfection, cell lysates were analyzed by immunoblotting using the indicated Abs. +, 250 ng; ++, 500 ng; ++++, 1000 ng. (C) The degradation of MDA5b on the NOD1 by a ubiquitination pathway. HEK293T cells seeded in six-well (Figure legend continues) The Journal of Immunology 13

At least seven splicing isoforms of human NOD1 were obtained zebrafish already have an impaired immune system. Therefore, and confirmed by sequencing (Supplemental Fig. 4A). However, NOD1-deficient larvae were susceptible to various pathogens only one form of MDA5 was found to exist in human HEK293T and environmental changes, which led to abnormal mass mor- and Hela cell lines (Supplemental Fig. 4B). tality in the breeding process. In mammals, it has been well established that MDA5/RIG-I RIG-I and MDA5 are key PRRs for initiating intracellular an- interacts with MAVS to activate the recruitment of downstream tiviral immunity and are controlled by many positive and negative TRAF3. In addition, a previous study has showed that, like their regulators such as RNF125, RNF135, TRIM25, and CYLD through mammalian orthologs, zebrafish NOD1 and NOD2 proteins have differential ubiquitination and phosphorylation (23, 43–45). Al- conserved antibacterial roles (38). Similar to zebrafish NOD1, though MDA5 encodes an RD-like motif analogous to the RIG-I human NOD1 did enhance the complex formation of MDA5 RD, this C-terminal region, also referred as the CTD, CTR, or and MAVS with or without poly(I:C) stimulation (Fig. 7A, MDA5_C, does not impose autoregulation of MDA5 signaling as Supplemental Fig. 4C). However, in contrast to zebrafish NOD1, it does for RIG-I (1). In fact, unlike RIG-I, when ectopically human NOD1 did not affect the formation of the MAVS–TRAF3 expressed, WT MDA5 induces constitutive signaling without the complex with or without poly(I:C) stimulation (Supplemental need for an RNA ligand to stimulate its activation (46). Further- Fig. 4D, 4E). We next performed poly(I:C) pulldown assays. more, several studies have shown that the MDA5 C-terminal re- Similar to zebrafish NOD1, human NOD1 itself can bind to poly(I: gion does not function as an RD (46, 47). In teleost fish, the RD of C) and also markedly enhance the binding of MDA5 to poly(I:C) grass carp MDA5 played a positive role in MDA5-mediating (Fig. 7B). immune response to both grass carp reovirus and bacterial

Among the human NOD1 splicing isoforms we cloned, one pathogen-associated molecular pattern molecules. The deletion of Downloaded from sequence is representative of the human NOD1 isoform 35 helicase or the RD reduced the inductive effect of MDA5 on the (GenBank accession no XP_011513387; https://www.ncbi.nlm. expression of IFN1, IL-1b, and Mx1 genes (47). In accordance nih.gov/protein/XP_011513387). Compared with human NOD1 with the RD of grass carp MDA5, the RD of zebraﬁsh MDA5a normal form, NOD1 isoform 35 lacks 84 aa at the C terminus also increased the expression of IFN1. This lack of RD function (Supplemental Fig. 4F). Similar to human NOD1 normal form, was suggested to reﬂect an important role for MDA5 constitu-

NOD1 isoform 35 enhanced the binding of MDA5 to poly(I:C) tive signaling in amplifying the production of ISGs and in the host http://www.jimmunol.org/ (Supplemental Fig. 4F) and the complex formation of MDA5 response (46). and MAVS without or with poly(I:C) stimulation (Supplemental In contrast to RSV infection, murine norovirus, poly(I:C), and Fig. 4G, 4H). type I IFN signaling induce the expressions of both NOD1 and NOD2 in bone marrow–derived macrophages, indicating that Discussion NODs are IFN-stimulated genes (48). It was also shown that ec- Although two studies have reported that NOD1 participates in topic overexpression of NOD1 led to the induction of NF-kB the innate antiviral immune response (17, 20), the mechanisms signaling (49). These are likely reasons why NOD1 over- behind this are still largely unknown. In the current study, we expression increases the upregulation of many antimicrobial fac- identified NOD1 as a host PRR that can sense SVCV and tors and RLR pathway members. In this study, to our knowledge, by guest on September 28, 2021 dsRNA. The in vitro importance of NOD1 in antiviral responses our results first showed that the upregulation of most antiviral was demonstrated by its ability to lower viral titers and to genes involved in RLR-mediated signaling, which included protect transfected cells against SVCV infection. The in vivo MDA5 isoforms, may be cytokine mediated. Previous studies have role of NOD1 in antiviral responses was evident from the in- demonstrated the mutual regulation between NOD2 and RIG-I creased viral loads and enhanced susceptibility to SVCV in- both in teleost fish and mammals (50, 51). However, little is fection in the NOD1 knockout zebrafish for two different known regarding the functional correlation between NOD1 and mutants, NOD1-1IS2/2 and NOD1-12S2/2 (26). MDA5, especially the function of their isoforms. The key finding In plants, it has been reported that nuclear shuttling of NLRs is in this study is that several lines of evidence confirm the mutual involved in innate immune recognition by NLR-related proteins, regulation existing between NOD1 and MDA5 isoforms. First, suggesting that these proteins may act in the nucleus in a currently co-IP experiments indicated that NOD1 was associated with unknown manner (39). In mammals, transcriptional functions of MDA5 isoforms at both exogenous and endogenous levels. NLRs were well established for CIITA and NLRC5, and their Second, NOD1 significantly strengthened the association be- nuclear locations were even suggested to be pivotal for tran- tween MDA5a and MAVS, but inhibited the assembly of the scriptional regulation of the inflammasome or MHC genes (40– MDA5b–MAVS complex. Third, NOD1 and MDA5a cooper- 42). In this study, NOD1 was detected in the nuclei and chromatin ated to induce the transcription of all tested antiviral genes, of piscine and mammalian cells. However, the mechanism by bind dsRNA, and inhibit viral replication, whereas NOD1 and which NOD1 comes to occur in the nuclei and chromatin of MDA5bfailedtodothese.Fourth,MDA5aincreasedthepro- these cells is unknown at present. The reduced expression of tein expression of NOD1; however, MDA5b mediated the these genes encoding proinflammatory cytokines and essential proteasomal degradation of NOD1. All these findings together antiviral components in uninfected NOD1-deficient larvae might demonstrate that cumulative effects and antagonism exist be- imply that NOD1 has a regulatory function and that these tween NOD1 and MDA5 isoforms.

plates were transfected with 200 ng NOD1-FLAG and 500 ng ptGFP1-MDA5b plasmids. After 24 h posttransfection, cells were treated with DMSO or MG132 (+, 10 mM; ++, 20 mM; +++, 40 mM) for 6 h or left untreated. Following this, cell lysates were analyzed by immunoblotting using the indicated Abs. (D) The DExDc of MDA5 was essential for degradation NOD1. (E) The RD and/or HELICc of MDA5 inhibited the protein degradation of NOD1 mediated by MDA5b. For (D) and (E), HEK293T cells seeded in six-well plates were transfected with various indicated plasmids. After 24 h posttransfection, cell lysates were analyzed by immunoblotting using the indicated Abs. Western blotting was representative of approximately two to three independent experiments. 14 NOD1 AND MDA5 NORMAL FORM INDUCE ANTIVIRAL RESPONSE Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. The effect of human NOD1 on the formation of hMDA5–hMAVS and the binding of hMDA5 and poly(I:C). (A) Human NOD1 enhanced the interaction between hMDA5 and hMAVS with poly(I:C) stimulation. HEK293T cells were cotransfected with the indicated plasmids and then stimulated by poly(I:C) for 6 h. Co-IP was performed with anti-FLAG–conjugated agarose beads. Bound proteins were analyzed by immunoblotting with anti-HA and anti-FLAG Abs. The cell lysates were analyzed by immunoblotting with the indicated Abs. (B) Human NOD1 enhanced the binding of hMDA5 to poly(I:C). HEK293T cells were transfected with the indicated plasmids. Cell lysates were incubated with poly(I:C) beads. Bound proteins were analyzed by immunoblotting with anti-hNOD1 and anti-FLAG Abs. The cell lysates were analyzed by immunoblotting with the indicated Abs. For (A) and (B), the expression ratio was quantiﬁed by Quantity One. Each Western blot is representative of at least three independent experiments. (C) Proposed model il- lustrating the intracellular innate immune pathways modulated by NOD1 and MDA5 isoforms.

A previous study showed that the recognition of dsRNA by the possibility that NOD1 binds SVCV RNA through an inter- human NOD1 was independent of the LRR domain, because a mediate binder is not ruled out. mutant lacking the entire LRR domain was as efﬁcient in dsRNA Based on the experimental data derived from this study and binding as the WT protein (20). In this study, we show that LRR previous reports, a working model to explain how NOD1 positively domains have no effect on SVCV binding, whereas the CARD regulates the production of ISGs and innate immune responses is domain is required by NOD1 to bind with SVCV. Because iso- presented. MAVS is the pivotal adaptor protein of the RLR- lating NOD1-FLAG or CARD-NOD1-FLAG from cells will also mediated antiviral signaling pathway. Under normal physiologi- pull down possible intermediate binding proteins such as MDA5, cal conditions, piscine MDA5a and MDA5b form a complex with The Journal of Immunology 15

MAVS. Although MDA5b also enhances the protection of trans- 7. Kufer, T. A., J. H. Fritz, and D. J. Philpott. 2005. NACHT-LRR proteins (NLRs) in bacterial infection and immunity. Trends Microbiol. 13: 381–388. fected cells against SVCV infection and induces a significantly 8. Kumar, S., H. Ingle, D. V. Prasad, and H. Kumar. 2013. Recognition of bacterial higher level of IFN activation by adding MAVS (24), the accu- infection by innate immune sensors. Crit. Rev. Microbiol. 39: 229–246. mulative effect of MDA5b–MAVS signaling in inducing the 9. Van Gorp, H., A. Kuchmiy, F. Van Hauwermeiren, and M. Lamkanfi. 2014. NOD-like receptors interfacing the immune and reproductive systems. FEBS J. transcription of IRF3 and ISGs is far below that achieved by 281: 4568–4582. MDA5a–MAVS signaling. SVCV infection induces the expression 10. Coutermarsh-Ott, S., K. Eden, and I. C. Allen. 2016. 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Supplemental Fig. 1. Subcellular localization of zebrafish and human NOD1. (A-D) Western blot analysis after subcellular fractionation using anti-NOD1 antibody (zebrafish NOD1) on cytoplasm (A), membrane (B), nucleus (C) and chromatin (D) extracts from unstimulated ZF4 cells and ZF4 cells infected with E. piscicida and SVCV. For A-D, zebrafish ZF4 cells seeded overnight were infected with E. piscicida and SVCV at the MOI=1 or left untreated. (E-H) Western blot analysis after subcellular fractionation using anti-NOD1 antibody (human NOD1) on cytoplasm (E), membrane (F), nucleus (G) and chromatin (H) extracts from unstimulated Hela cells and Hela cells stimulated with LPS or poly I:C. For E-H, Hela cells seeded overnight were stimulated with LPS or poly I:C or left untreated. For A-H, cells were collected at 6 h after treatment, and used for subcellular fractionation. Tubulin served as a cytoplasmic marker, histone H3 and HDAC1 used as a nuclear marker. Each Western blot is representative of at least 3 independent experiments. The expression ratio of NOD1 relative to unstimulated control was quantified by Quantity One.

Supplemental Fig. 2. The effect of piscine NOD1 in the antiviral immune responses. (A) In vitro antiviral role of zebrafish NOD1 determined by crystal violet staining assay. (B) In vitro antiviral role of zebrafish NOD1 determined by optical density at 562 nm (OD562). ZF4 cells stably transfected with NOD1 or ptGFP1 were infected with SVCV with the MOI=5, 0.5 and 0.05. After infection for 5 d at 20 °C, the plates were fixed and then stained with crystal violet and photographed. The absorbance was read at a 562-nm wavelength. (C) The expression of antiviral genes in the WT zebrafish detected by qRT-PCR at 1 and 2 dpi. (D) NOD1-1IS−/− zebrafish were more sensitive to SVCV infection compared with the WT based on the survival rate. (E) NOD1-2IS−/− zebrafish were more sensitive to SVCV infection compared with the WT based on the survival rate. For C-E, the hatched larvae (4 dpf) from WT, NOD1-1IS−/− and NOD1-2IS−/− zebrafish were exposed to 2 × 106 PFU/ml SVCV. (F) Effect of NOD1 knockdown on the expression of IRF3. (G) Effect of NOD1 knockdown on gene expression during SVCV infection of zebrafish embryos. For F and G, embryos were injected with zfNOD1-MO and control-MO at the one-cell or two-cell stage, and then injected with SVCV or medium at 24 hpf. For each condition, 15~20 embryos were lysed in Trizol at 6 hpi and 24 hpi to prepare total RNAs. (H) Knockdown of NOD1 increased the mortality of zebrafish embryos infected with SVCV. Embryos were injected with zfNOD1-MO and control-MO at the one-cell or two-cell stage, and then infected with SVCV at 24 hpf. N = 36-45 embryos per group.

Supplemental Fig. 3. The effect of piscine NOD1 in the MDA5-MAVS-TRAF3 signaling. (A) NOD1 deficiency decreased the transcription of MDA5a. (B) NOD1 deficiency decreased the transcription of MDA5b. (C) NOD1 deficiency decreased the transcription of MAVS_tv1. For A-C, 30~50 larvae from WT and NOD1-1IS−/− zebrafish were collected at 5 and 7 dpf, and used for qRT-PCR. (D) NOD1 binded to poly(I:C). Cell lysates from EPC cells transfected with NOD1-FLAG or empty vector were incubated with poly(I:C) beads. The immunoprecipitate and lysates were analyzed by immunoblotting with the indicated antibodies. Each Western blot was representative of at least 3 independent experiments. (E) NOD1 failed to interact with MAVS. (F) Schematics of wild type and truncated MDA5 used in co-immunoprecipitation studies. (G) NOD1 and MDA5 domain interacted in vitro. (H) NOD1 interacted with the CARDs of MDA5, but not with the CARD of MAVS. (I) NOD1 and TRAF3 interacted. For E, G-I, EPC cells were co-transfected with the indicated plasmids, and co-immunoprecipitation was performed with anti-FLAG conjugated agarose beads. The immunoprecipitate and lysates were analyzed by immunoblotting with the indicated antibodies. Western Blotting was representative of 2~4 independent experiments.

Supplemental Fig. 4. The effect of human NOD1 in the MDA5-MAVS-TRAF3 signaling. (A) The agarose gel electrophoresis of NOD1 isoforms. (B) The agarose gel electrophoresis of MDA5. For A and B, HEK293T and Hela cells were used for RNA extraction and RT-PCR. (C) Human NOD1 enhanced the interaction between hMDA5 and hMAVS without poly(I:C) stimulation. (D) Human NOD1 had no effect on the interaction between hMAVS and hTRAF3 without poly(I:C) stimulation. (E) Human NOD1 had no effect on the interaction between hMAVS and hTRAF3 with poly(I:C) stimulation. (F) Human NOD1 isoform X5 enhanced the binding of hMDA5 to poly(I:C). HEK293T cells were transfected with the indicated plasmids. Cell lysates were incubated with poly(I:C) beads. Bound proteins were analyzed by immunoblotting with anti-hNOD1 and anti-FLAG antibodies. The cell lysates were analyzed by immunoblotting with the indicated antibodies. Each Western blot was representative of at least 3 independent experiments. (G) The effect of human NOD1 isoform X5 on the formation of MDA5-MAVS complex without poly(I:C) stimulation. (H) The effect of human NOD1 isoform X5 on the formation of MDA5-MAVS complex with poly(I:C) stimulation. For C-E, G and H, HEK293T cells were co-transfected with the indicated plasmids, and then stimulated by poly(I:C) for 6 h or left untreated. Co-immunoprecipitation was performed with anti-FLAG conjugated agarose beads. Bound proteins were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies. The cell lysates were analyzed by immunoblotting with the indicated antibodies. The expression ratio was quantified by Quantity One. Each Western blot was representative of at least 3 independent experiments.