MSX1 Modulates RLR-Mediated Innate Antiviral Signaling by Facilitating Assembly of TBK1-Associated Complexes

This information is current as Liu-Ting Chen, Ming-Ming Hu, Zhi-Sheng Xu, Yu Liu and of October 1, 2021. Hong-Bing Shu J Immunol published online 18 May 2016 http://www.jimmunol.org/content/early/2016/05/17/jimmun ol.1600039 Downloaded from

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 18, 2016, doi:10.4049/jimmunol.1600039 The Journal of Immunology

MSX1 Modulates RLR-Mediated Innate Antiviral Signaling by Facilitating Assembly of TBK1-Associated Complexes

Liu-Ting Chen, Ming-Ming Hu, Zhi-Sheng Xu, Yu Liu, and Hong-Bing Shu

Recognition of viral dsRNA by the retinoic acid–inducible gene-1–like receptors (RLRs) triggers signaling cascades that lead to activation of the TBK1 kinase and transcription factor IFN regulatory factor 3, induction of downstream antiviral genes, and innate antiviral responses. In this study, we identiﬁed muscle segment homeobox1 (MSX1) as an important modulator of RLR- mediated signaling pathways. Knockdown or knockout of MSX1 signiﬁcantly impaired Sendai virus–triggered activation of TBK1 and IFN regulatory factor 3, induction of downstream antiviral genes, and cellular antiviral responses. Interestingly, MSX1 was translocated from the nucleus to cytoplasm, particularly mitochondria upon infection of Sendai virus. Biochemcially, MSX1 was important for assembly of TBK1/IKK-related kinase-associated protein 1/TNFR-associated factor-associated NF-kB activator complexes. Our results suggest that MSX1 is an important component of RLR-mediated signaling and reveal mechanisms on

innate immune responses against RNA viruses. The Journal of Immunology, 2016, 197: 000–000. Downloaded from

he innate immune response against virus begins with rec- TBK1 was first identified as an IkB kinase that mediates IKK and ognition of viral nucleic acids by specific pattern-recognition NF-kB activation in response to growth factors (10–12). It was later T receptors. Cytosolic dsRNA from viral genome or its rep- identified as a virus-activated kinase that coordinates with IKKε to lication intermediates are mainly recognized by retinoic acid– phosphorylate transcription factors IRF3 and IRF7, leading to in-

inducible gene-1 (RIG-I) and melanoma differentiation-associated duction of type I IFNs (13–17). With the exception of plasmacytoid http://www.jimmunol.org/ gene 5 (MDA5). Both RIG-I and MDA5 contain two N-terminal dendritic cells, most cells deficient for both TBK1 and IKKε fail to tandem caspase activation and recruitment domains and a produce type I IFNs in response to viral infection (14, 17). C-terminal DexD/H box RNA helicase domain, and thus they are Due to the vital role of TBK1 in virus-induced signaling, the collectively called RIG-I–like receptors (RLRs). The binding of molecular events underlying TBK1 activation have been heavily RLR with viral dsRNA induces conformational changes and olig- investigated. Multiple molecules have been reported to regulate omerization of RLR that activate virus-triggered signaling adaptor activation of TBK1 to ensure suitable immune responses against (VISA, also known as MAVS, IPS-1, and Cardif) on the mito- invaded viruses. Suppressor of IKKε functions as a physiological chondrial and peroxisomal membranes. VISA forms a signalsome suppressor of TBK1, as well as IKKε, by sequestering IKKε/TBK1 by recruiting and activating multiple signaling components, in- in inactive complexes (18). Nod-like receptor family pyrin domain- by guest on October 1, 2021 cluding WDR5, MITA (also known as STING), TAK1, TBK1, containing 4 promotes degradation of TBK1 by recruiting the E3 and/or IKKε kinases, leading to activation of transcription factors ligase d 4 (DTX4) to TBK1 and promoting K48-linked poly- IFN regulatory factor (IRF)3 and NF-kB and induction of type I ubiquitination of TBK1 (19). Dual-specificity tyrosine phosphory- IFNs (IFN-a and IFN-b) and inflammatory cytokines (1–9). Type lation–regulated kinase 2 phosphorylates TBK1 at Ser527,leadingto I IFNs further activate the JAK–STAT signal transduction path- recruitment of Nod-like receptor family pyrin domain-containing 4 ways, leading to transcriptional induction of a wide range of down- and DTX4 to degrade TBK1 (20). Glycogen synthase kinase 3b stream antiviral genes. potentiates activation of TBK1 by promoting TBK1 self-association and self-phosphorylation at S172 (21). Several adaptor molecules are also reported to modulate activation of TBK1, including TNFR- College of Life Sciences, Medical Research Institute, Collaborative Innovation Cen- ter for Viral Immunology, Wuhan University, Wuhan 430072, China associated factor-associated NF-kB activator (TANK) and IKK- related kinase-associated protein 1 (NAP1). Deficiency of TANK ORCIDs: 0000-0001-9962-5286 (L.-T.C.); 0000-0001-9667-7662 (M.-M.H.); 0000- 0001-7034-0666 (Z.-S.X.). or NAP1 inhibits Sendai virus (SeV)–induced activation of IRF3, Received for publication January 7, 2016. Accepted for publication April 22, 2016. suggesting that these adaptor proteins are required for RLR- This work was supported by Ministry of Science and Technology of China Grants mediated signaling. NAP1 connects RLRs with TBK1 to activate 2014CB910103 and 2012CB910201 and National Natural Science Foundation of IRF3 (22). Consistently, TANK forms complexes with VISA, China Grants 31521091, 91429304, and 31370867. TRAF3, and TBK1 (23). Therefore, NAP1 and TANK are adaptors Address correspondence and reprint requests to Dr. Hong-Bing Shu, College of Life linking TBK1 with the upstream signaling complexes, leading to Sciences, Medical Research Institute, Collaborative Innovation Center for Viral Immu- nology, Wuhan University, Wuhan 430072, China. E-mail address: [email protected] phosphorylation and activation of TBK1 (22, 23). The online version of this article contains supplemental material. Muscle segment homeobox1 (MSX1) is a member of the MSX transcription factors that play key roles in development by re- Abbreviations used in this article: HA, hemagglutinin; IRF, IFN regulatory factor; ISRE, IFN-stimulated regulatory element; MDA5, melanoma differentiation- pressing gene expression through interacting with components of associated gene 5; MSX1, muscle segment homeobox1; NAP1, IKK-related the core transcription complexes as well as other homeoproteins kinase-associated protein 1; NDV, Newcastle disease virus; poly (I:C), polyinosinic- polycytidylic acid; qPCR, quantitative PCR; RIG-I, retinoic acid–inducible gene-1; (24, 25). MSX1 plays important roles in multiple signaling path- RLR, RIG-I–like receptor; SeV, Sendai virus; TANK, TNFR-associated factor- ways, including Wnt/b-catenin and D-Notch pathways (26–28). associated NF-kB activator; TRAF, TNFR-associated factor; VISA, virus-triggered Mutations of MSX1 in both humans and mice have been associ- signaling adaptor; VSV, vesicular stomatitis virus. ated with impaired development of cranial neural crest-derived Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 structures, oral clefts, and nonsyndromic oligodontia (24, 29–31).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600039 2 MSX1 REGULATES RLR SIGNALING

However, the roles of MSX1 in RLR-mediated signaling path- HCT116 were transfected by lipofectamine 2000 (Invitrogen). Empty ways have not been previously studied. control plasmid was added to ensure that each transfection received the In this study, we identified MSX1 as an important regulator of same amount of total DNA. To normalize for transfection efficiency, pRL- TK (Renilla luciferase) reporter plasmid (0.02 mg) was added to each RLR-mediated signaling pathways. Knockdown or knockout of transfection. Luciferase assays were performed using a dual-specific lu- MSX1 significantly inhibited RLR-mediated activation of IRF3, ciferase assay kit (Promega, Madison, WI). Firefly luciferase activities induction of downstream antiviral genes, and cellular antiviral were normalized on the basis of Renilla luciferase activities. responses. MSX1 was translocated from the nucleus to the cyto- Quantitative real-time PCR plasm upon viral infection and important for assembly of TBK1- associated complexes. Our findings reveal a previously unidentified Total RNA was isolated from cells using TRIzol reagent (Invitrogen). After reverse transcription with an oligo deoxy-thymine primer using a RevertAid function of MSX1 and provide insight into the mechanisms of First Strand cDNA Synthesis Kit (Fermentas), aliquots of products were innate antiviral responses. subjected to quantitative PCR (qPCR) analysis to measure mRNA expression levels of tested genes. GAPDH was used as a reference gene. Human gene- specific primer sequences were as follows: GAPDH,59-GACAAGCTTCC- Materials and Methods CGTTCTCAG-39 (forward) and 59-GAGTCAACGGATTTGGTCGT-39 (re- Reagents and Abs verse); IFNB1,59-TTGTTGAGAACCTCCTGGCT-39 (forward) and 59- 9 ISG56 9 Mouse mAbs against Flag and b-actin (Sigma-Aldrich), hemagglutinin TGACTATGGTCCAGGCACAG-3 (reverse); ,5-TCATCAGGTCA- 9 9 9 (HA) (Convance), and b-tubulin (Invitrogen); rabbit mAbs against LMNB1 AGGATAGTC-3 (forward) and 5 -CCACACTGTATTTGGTGTCTAGG-3 ISG15 9 9 9 (Protein Tech), TBK1, and p-TBK1(S172) (Abcam); AIF, IRF3, MSX1, (reverse); ,5-AGGACAGGGTCCCCCTTGCC-3 (forward) and 5 - 9 RANTES 9 STAT1, and TANK (Santa Cruz Biotechnology); p-IRF3(S396) and p-STAT1 CCTCCAGCCCGCTCACTTGC-3 (reverse); ,5-GGCAGCCC- 9 9 9 (Y701) (CST) and VISA (Bethyl); human rIFN-b and rIFN-g (R&D Sys- TCGCTGTCATCC-3 (forward) and 5 -GCAGCAGGGTGTGGTGTCCG-3

CXCL10 9 9 Downloaded from tems); and polyinosinic-polycytidylic acid [poly(I:C)] (InvivoGen) were (reverse); ,5-GGTGAGAAGAGATGTCTGAATCC-3 (forward) 9 9 MSX1 9 purchased from the indicated companies. Mouse anti-MSX1, rabbit anti- and 5 -GTCCATCCTTGGAAGCACTGCA-3 (reverse); and ,5-AG- 9 9 MSX1, and mouse anti-NAP1 antisera were raised against the indicated ACTAGCCAGCCAGGAAGATGAA-3 (forward) and 5 -CCAGAGCAAAT- 9 recombinant proteins. HSV-1 (KOS), SeV, vesicular stomatitis virus (VSV), GTTTTGTTGTTGAA-3 (reverse). and Newcastle disease virus (NDV) were previously described (9). Generation and transfection of human primary macrophages Constructs Generation and transfection of human primary macrophages were performed, as previously described (36). The human CD14+ monocytes were IFN-stimulated regulatory element (ISRE), the IFN-b and IRF1 promoter + http://www.jimmunol.org/ purchased from ALLCELLS. For generation of macrophages, the CD14 luciferase reporter plasmids, mammalian expression plasmids for HA- or ε monocytes were treated with recombinant M-CSF (50 ng/ml; PeproTech) Flag-tagged RIG-I-N, MDA5, IRF3, VISA, TRAF3, IKK , TBK1, and its for 6 d. Differentiated macrophages were transfected with small interfering truncation mutants were previously described (21, 32, 33). HA-tagged RNA by lipofectamine 2000. Thirty-six hours after transfection, cells were pRK-NAP1 and TANK, Flag- or HA-tagged pRK-MSX1, and its trunca- infected with SeV or HSV-1 for the indicated times before qPCR or im- tion mutants were constructed by standard molecular biology techniques. munoblot assays were performed. MSX1 and its truncation mutants were also constructed in the retroviral vector pMSCV that contains puromycin marker. Subcellular fractionation DNA oligonucleotides Isolation of cytoplasmic and nuclear fraction. HCT116 cells infected with SeV or left uninfected were washed with PBS and lysed by douncing for 40 The following oligonucleotides were used to stimulate cells: HSV120: 59- times in 1 ml cytosol extraction reagent (ApplyGen). The homogenate was by guest on October 1, 2021 AGACGGTATATTTTTGCGTTATCACTGTCCCGGATTGGACACGGT- centrifuged at 500 3 g for 5 min. The supernatant was saved as cytoplasm. CTTGTGGGATAGGCATGCCCAGAAGGCATATTGGGTTAACCCC- The pellet was washed twice with 500 ml nuclear extraction reagent TTTTTATTTGTGGCGGGTTTTTTGGAGGACTT-39. (ApplyGen) and centrifuged twice at 4000 3 g for 5 min to precipitate the nuclei. The precipitates were analyzed by standard immunoblot procedures. RNAi experiments Isolation of mitochondrial and cytosolic fractions. HCT116 cells infected Double-strand oligonucleotides corresponding to the target sequences were with SeV or left uninfected were washed with PBS and lysed by douncing cloned into the pSuper-Retro RNAi plasmid (Oligoengine). Small inter- for 40 times in a homogenization buffer (10 mM Tris-HCl [pH 7.4], 10 mM fering RNA corresponding to the same target sequences were purchased potassium chloride, 2 mM magnesium chloride, and 250 mM saccharose). from GenePharma. The following sequences were targeted for human The homogenate was centrifuged at 500 3 g for 10 min. The supernatant MSX1 cDNA: 1) 59-AGAAGATGCGCTCGTCAAA-39;2)59-TACAA- was centrifuged at 5000 3 g for 10 min to precipitate mitochondria. The GAGGCAGAGCTGGA-39; and 3) 59-GAGACGCAGGTGAAGATAT-39. supernatant from this step was cytosol. The precipitates were analyzed by standard immunoblot procedures. CRISPR-Cas9 VSV plaque assays The protocols for genome engineering using the CRISPR-Cas9 system were described by Ran et al. (34). pGL-U6-gRNA and pST1374-Cas9-D10A plas- The cells were grown in 24-well plates and infected with VSV (multiplicity of infection = 0.1) for 1 h. Then cells were washed with PBS, and full medium was mids were provided by X.-D. Zhang (Wuhan University). The MSX1 gRNA 26 target sequence was 59-AGGCGCTCATGGCCGACCAC-39. The identifica- added for another 24 h. The supernatants were diluted to 10 for infection of tion primer sequences for MSX1 were 59-TGGAGGCGCTCATGGCCGACC- Vero cells seeded in 24-well plates. After 1-h infection, 3% methylcellulose 39 (forward) and 59-TTGACGAGCGCATCTTCTGG-39 (reverse). was overlaid, and the plates were incubated for 2 d. The overlay was then removed, and cells were fixed with 4% paraformaldehyde for 20 min and Fluorescent confocal microscopy stained with 1% crystal violet for 30 min. The plaques were counted, averaged, and multiplied by the dilution factor to determine the viral titer as PFU/ml. HEK293 cells were transfected with Flag-MSX1 by standard calcium phosphate precipitation. Twenty-four hours after transfection, the cells Statistical analysis were left uninfected or infected with SeV for 4 h. Cells were then stained with t p , the MitoTrack Red (Invitrogen) following protocols recommended by the All experiments were analyzed by the Student test. A value 0.05 was manufacturer. After fixation by 4% paraformaldehyde, cells were per- considered significant. meabilized by 0.1% (v/v) Triton X-100 and incubated with primary Abs, followed by AlexaFluor 488 secondary Abs (Molecular Probes). The cells Results were observed by confocal microscopy. Knockdown of MSX1 inhibits RLR-mediated activation of ISRE b Transfection and reporter assays and the IFN- promoter Transfection and reporter assays were performed, as previously described In our preliminary studies for identification of proteins that activate (35). HEK293 cells were seeded on 24-well plates and transfected on the STAT3 by expression screens, we identified MSX1 as a candidate following day by standard calcium phosphate precipitation. HeLa and protein. However, further analysis failed to validate endogenous The Journal of Immunology 3

MSX1 as a regulator of STAT3 activation (data not shown). In the MSX1 signiﬁcantly inhibited SeV-triggered activation of the IFN-b course of those studies, we examined whether MSX1 is involved in promoter and ISRE (Fig. 1B), but had little effect on IFN-g– other signaling pathways and identiﬁed a role of MSX1 in virus- triggered activation of the IRF1 promoter in HEK293 cells triggered signaling. As shown in Fig. 1A, we constructed three (Fig. 1C). Consistently, knockdown of MSX1 markedly inhibited MSX1-RNAi plasmids, which could knockdown the expression of SeV-induced transcription of IFNB1, ISG56, ISG15, and RANTES MSX1 to various degrees. In reporter assays, knockdown of genes (Fig. 1D), but not IFN-g–induced transcription of IRF1 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Knockdown of MSX1 inhibits RLR-mediated activation of ISRE and the IFN-b promoter. (A) In the upper panels, HEK293 cells (4 3 105) were cotransfected with expression plasmids for Flag-MSX1 and HA-b-actin (0.1 mg each) as well as the indicated RNAi plasmids (2 mg each). Twenty- four hours after transfection, cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, HEK293 cells (1 3 107) were transfected with control or the indicated MSX1-RNAi plasmids (10 mg each) for 48 h. Cell lysates were then analyzed by immunoblots with anti-MSX1 or anti–b-actin. (B and C) HEK293 cells (1 3 105) were transfected with the indicated MSX1-RNAi (1 mg each) and reporter (0.1 mg) plasmids. Forty-eight hours after transfection, cells were left uninfected or infected with SeV (B) or treated with IFN-g (100 ng/ml) (C) for 12 h before luciferase assays were performed. (D and E) HEK293 cells (4 3 105) were transfected with control or the indicated MSX1-RNAi plasmids (2 mg). Forty-eight hours after transfection, cells were left uninfected or infected with SeVor treated with IFN-g (100 ng/ml) for 10 h, and then total RNA was extracted for qPCR analysis. (F) Cells were transfected with the IFN-b promoter reporter (0.1 mg) plasmids. Forty-eight hours later, cells were retransfected with poly(I:C) (1 mg) or control buffer with Lipofectamine 2000 for 12 h before luciferase assays were performed. (G) The experiments were performed in HCT116 and HeLa cells similarly as in (B). (H) HeLa cells (1 3 105) were transfected with the indicated MSX1-RNAi (1 mg each) and the IFN-b promoter (0.1 mg). Forty-eight hours after transfection, cells were left uninfected or infected with HSV-1 for 12 h before luciferase assays were performed. (I) THP-1 cells were transduced with control or the indicated MSX1-RNAi by retroviral-mediated gene transfer. Cells were left uninfected or infected with HSV-1 for 8 h, and then analyzed by qPCR. (J) Human monocyte-derived macrophages (2 3 105) were transfected with the indicated small interfering RNA by Lipofectamine 2000. Thirty- six hours after transfection, cells were left uninfected or infected with SeV or HSV-1 for the indicated times, and then analyzed by qPCR. Graphs show mean 6 SD, n =3.*p , 0.05, **p , 0.01. 4 MSX1 REGULATES RLR SIGNALING gene (Fig. 1E). Furthermore, knockdown of MSX1 inhibited ac- inhibit HSV-1–triggered activation of the IFN-b promoter in HeLa tivation of the IFN-b promoter induced by transfection of the cells (Fig. 1H). Furthermore, qPCR experiments confirmed that RNA analog poly(I:C)-high m.w. and poly(I:C)-low m.w. transcription of downstream IFNB1, ISG56, ISG15, and RANTES (Fig. 1F). In these experiments, the decreases of transcription or genes induced by HSV-1 was not inhibited in MSX1 RNAi- promoter activation induced by SeV or tranfected poly(I:C) were transduced THP-1 cells (Fig. 1I). Previously, it has been demon- correlated with the knockdown efficiencies of the respective strated that transfected dsDNA (120 mers) representing the ge- MSX1-RNAi plasmids. Similar results were obtained in HCT116 nomes of HSV-1 (HSV120) was efficient at inducing IFN-b and HeLa cells, respectively (Fig. 1G, Supplemental Fig. 1A), response (37). As shown in Supplemental Fig. 1B, knockdown of suggesting that MSX1 plays an important role in virus-triggered MSX1 did not have marked effects on HSV120-induced tran- induction of downstream antiviral genes in different cell types. We scription of downstream genes, including IFNB1, ISG56, ISG15, then determined whether MSX1 is involved in DNA virus- and CXCL10 genes. More importantly, knockdown of MSX1 triggered signaling. Unexpectedly, knockdown of MSX1 did not inhibited SeV- but not HSV-1–induced transcription of IFNB1, Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Knockout of MSX1 inhibits RLR-mediated induction of downstream antiviral genes. (A) Genotyping of MSX1-deficient and control HEK293 cells. (B) Expression of MSX1 protein in MSX1-deficient and control HEK293 cells was analyzed by immunoblots with the indicated Abs. (C–E) MSX1-deficient and control HEK293 cells (4 3 105) were left uninfected or infected with SeV (C), VSV (D), or NDV (E) for the indicated times, and then total RNA was extracted for qPCR analysis. (F) Cells were transfected with poly(I:C) (2 mg) or control buffer for the indicated times, and then total RNA was extracted for qPCR analysis. (G) The indicated cells were treated with IFN-g (100 ng/ml) for 10 h before qPCR analysis. (H) MSX1-deficient HEK293 cells were reconstituted with MSX1 by retroviral-mediated gene transfer. Cells were left uninfected or infected with SeV for 12 h, and then analyzed by qPCR. Expression of the transduced proteins was detected by immunoblot with the indicated Abs. Graphs show mean 6 SD, n =3.*p , 0.05, **p , 0.01. The Journal of Immunology 5

ISG56, ISG15,andCXCL10 genes in human primary macrophages (Fig. lJ). These results suggest that MSX1 is specifically involved in RLR- but not DNA sensor-mediated antiviral signaling pathways. Knockout of MSX1 inhibits RLR-mediated induction of type I IFNs To confirm the role of MSX1 in RLR-mediated signaling, we generated MSX1-deficient HEK293 cells using CRISPR-Cas9 technology (34). Three independent MSX1-deficient clones were obtained and confirmed at both DNA and protein levels (Fig. 2A, 2B). The cells were infected with SeV for the indicated times, and transcription of downstream IFNB1, ISG56, ISG15, and RANTES genes was significantly decreased in all three MSX1-deficient clones compared with their control cells (Fig. 2C). Similarly, the transcription of downstream genes induced by VSV or NDV infection, or transfected poly(I:C) was markedly inhibited in all three MSX1-deficient clones (Fig. 2D–F). In contrast, IFN-g–induced transcription of IRF1 gene was comparable in MSX1- deficient and control cells (Fig. 2G). Reconstitution of MSX1 but Downloaded from not its truncation mutants into the three MSX1-deficient clones restored SeV-induced transcription of downstream genes (Fig. 2H, Supplemental Fig. 2). Collectively, these results confirm that MSX1 is indispensable for RLR-mediated induction of downstream genes. Because results from all three different MSX1- deficient clones of HEK293 cells were similar in experiments http://www.jimmunol.org/ described above, we used the #1 knockout clone and its control cells in the following experiments. MSX1 is required for cellular antiviral responses Because MSX1 is important for RLR-mediated induction of downstream antiviral genes, we examined whether MSX1 plays a FIGURE 3. MSX1 is required for cellular antiviral responses. (A) functional role in innate antiviral responses. MSX1-deficient and MSX1-deficient and control HEK293 cells were infected with NDV control cells were left untransfected or transfected with poly(I:C), (multiplicity of infection [MOI] of 0.1) for 1 h. The cells were washed by guest on October 1, 2021 followed by infection with GFP-tagged NDV. The cells were then twice by PBS and cultured with DMEM containing 2% FBS for 48 h harvested for analyzing by flow cytometry. As monitored by the before flow cytometry. (B and C) The MSX1 knockout cells without re- expression levels of GFP protein, the replication of NDV was constitution or reconstituted with wild-type or mutant MSX1 were infected significantly enhanced in MSX1-deficient cells compared with the with VSV (MOI = 0.1) for 1 h. The cells were washed twice by PBS and control cells (Fig. 3A). Furthermore, in plaque assays, higher level cultured with DMEM containing 2% FBS for 18 h. The culture medium of VSV titer was detected in MSX1-deficient than in the control was harvested for measurement of VSV production by plaque assays. The cells (Fig. 3B). In addition, reconstitution with MSX1 but not graph shows mean 6 SD, n =3.*p , 0.05. its truncation mutants restored the antiviral activity of MSX1- deficient cells (Fig. 3C). These data demonstrate that MSX1 is DNA virus-triggered signaling. Consistently, knockdown of MSX1 vital for cellular antiviral responses. inhibited SeV- but not HSV-1–induced phosphorylation of TBK1 and IRF3 in human primary macrophages (Fig. 4C, 4D). Deficiency of MSX1 inhibits SeV-induced phosphorylation of TBK1 and IRF3 MSX1 translocates from the nucleus to the cytosol and Activation of RLRs leads to phosphorylation of TBK1 and IRF3. mitochondria following viral infection Knockdown of MSX1 markedly decreased SeV-triggered phos- It has been demonstrated that MSX1 mainly locates in the nucleus phorylation of TBK1 and IRF3 (Fig. 4A), but had little effects and functions as a transcription factor (38). MSX1 deficiency af- on IFN-b–orIFN-g–triggered phosphorylation of STAT1 fects SeV-triggered phosphorylation of TBK1 and IRF3, sug- (Supplemental Fig. 3A, 3B). Consistently, phosphorylation of gesting that MSX1 acts in the cytoplasm in innate antiviral TBK1 and IRF3 induced by SeV in MSX1-deficient cells was responses. We therefore investigated the cellular localization of decreased in comparison with control cells (Fig. 4B), whereas MSX1 before and after viral infection. Cell fractionation experi- IFN-g–induced phosphorylation of STAT1 was comparable be- ments indicated that a major fraction of MSX1 was located in the tween MSX1-deficient and control cells (Supplemental Fig. 3C). nucleus and only trace amount of MSX1 was detected in the cy- Reconstitution of MSX1 in MSX1-deficient cells restored phos- toplasm in uninfected cells (Fig. 4E). Interestingly, upon viral phorylation of TBK1 and IRF3 induced by SeV (Fig. 4B).These infection, the cytoplasmic level of MSX1 was increased, where- results not only suggest that MSX1 participates in RLR- but not as nuclear MSX1 was decreased, with total level of MSX1 IFN-induced signaling, but also indicate that MSX1 functions as unchanged (Fig. 4E). Because mitochondrion is a critical plat- an upstream protein of TBK1 or targets TBK1 directly. Addi- form for RLR-mediated signaling, we further examined whether tionally, knockdown of MSX1 had no marked effects on HSV-1– MSX1 is translocated to the mitochondria following viral infection. induced phosphorylation of TBK1 and IRF3 in THP-1 cells The results showed that MSX1, as well as TBK1, accumulated in (Supplemental Fig. 3D), suggesting that MSX1 is not involved in the mitochondrial fraction following viral infection (Fig. 4F). 6 MSX1 REGULATES RLR SIGNALING

FIGURE 4. Deficiency of MSX1 inhibits RLR-mediated phosphor- ylationofTBK1andIRF3.(A) HEK293 cells (4 3 105) were transfected with control or the indicated MSX1-RNAi plasmids (2 mg). Forty-eight hours after transfection, cells were left uninfected or infected with SeV for the indicated times before immunoblot analysis was performed with the indicated Abs. (B) MSX1-deficient or reconstituted HEK293 cells were left uninfected or infected with SeV for the indicated times before immunoblot analysis was performed with the indicated Abs. (C and D) Human monocyte-derived macrophages (1 3 106) were transfected with the indicated small interfering RNA by Downloaded from Lipofectamine 2000. Thirty-six hours after transfection, cells were left uninfected or infected with SeV or HSV-1 for the indicated times before immunoblot analysis was performed with the indicated Abs. (E and F) HCT116 cells (5 3 107) were http://www.jimmunol.org/ left uninfected or infected with SeV for the indicated times. The cells were fractionated, and the subcellular fractions, including cytoplasm and nucleus (E) and mitochondria (F), were equilibrated to equal vol- umes and analyzed by immunoblots with the indicated Abs. (G) HEK293 3 5 cells (1 10 ) were transfected with by guest on October 1, 2021 Flag-tagged MSX1. Twenty hours after transfection, cells were infected with SeV for 4 h or left uninfected and then incubated with MitoTrack Red for 1 h. Cells were then fixed with 4% paraformaldehyde, and immunostaining was performed before confocal microscopy. The line graphs show the relative intensities of the bands, which were quantitated by densitometry using ImageJ and normalized to b-actin levels.

Confocal microscopy further confirmed that MSX1 was translocated the TBK1 level. Further results from coimmunoprecipitation assays from the nucleus to a fraction of mitochondria upon SeV infection showed that overexpressed MSX1 strongly interacted with TBK1, (Fig. 4G). These results suggest that MSX1 is translocated from the but not TRAF3, IKKε, NAP1, or TANK (Fig. 5B). Endogenous nucleus to the cytosol and mitochondria upon viral infection. MSX1 was constitutively associated with TBK1, and this interaction was increased following SeV infection (Fig. 5C). Domain MSX1 regulates RLR-mediated IRF3 activation at the TBK1 mapping analysis indicated that the N-terminal domain of MSX1 level (1–172) and the C-terminal coiled-coil containing domain of TBK1 To investigate the molecular mechanisms on the role of MSX1 in (451–729) are required for their association (Fig. 5D, 5E). RLR-mediated signaling, we examined the effects of MSX1 deficiency on activation of ISRE mediated by overexpression of RIG-I, MSX1 regulates assembly of TBK1-associated complexes MDA5, VISA, TBK1, IKKε, and IRF3. The results showed that Because activation of TBK1 is related to several adaptor proteins, MSX1 deficiency significantly inhibited RIG-I–, MDA5-, VISA-, such as NAP1 and TANK, we wondered whether MSX1 is involved and TBK1-, but not IKKε- or IRF3-mediated activation of ISRE in assembly of TBK1-associated complexes. The results showed (Fig. 5A). Together with the results that MSX1 deficiency in- that the interaction between TBK1 and NAP1, TANK, or IRF3 was hibited SeV-induced phosphorylation of TBK1 (Fig. 4A–C), we markedly impaired in MSX1-knockdown cells, but TBK1 oligo- reasoned that MSX1 might regulate RLR-mediated signaling at merization and VISA–TBK1 association were not markedly af- The Journal of Immunology 7

FIGURE 5. MSX1 regulates RLR-mediated signaling at the TBK1 level. (A) MSX1-deﬁcient and control cells (1 3 105) were transfected with the indicated expression plasmids (0.1 mg each) for 24 h before luciferase assays. (B) HEK293 cells (1 3 107) were transfected with the indicated plasmids (5 mg each) for 20 h, and then coimmunoprecipitation and immunoblot analysis were performed with the indicated Abs. (C) HEK293 cells (4 3 107) were left uninfected or infected with SeV for the indicated times. Endogenous coimmunoprecipitation and immunoblotting analysis were performed with the indicated Downloaded from Abs. (D and E) Domain mapping of MSX1-TBK1 association. HEK293 cells (1 3 107) were transfected with Flag-MSX1 (1 mg) and HA-TBK1 or its truncation mutants (5 mg) (D), or HA-TBK1 (4 mg) and Flag-MSX1 E or its truncation mutants (5 mg) ( ). http://www.jimmunol.org/ Coimmunoprecipitation and immunoblot analysis were performed with the indicated Abs after transfection for 20 h. The graph shows mean 6 SD, n =3.**p , 0.01.

fected (Fig. 6A). Furthermore, MSX1 deﬁciency impaired SeV- These data suggest that MSX1 plays an important role in RLR- by guest on October 1, 2021 induced association between endogenous TBK1 and NAP1, mediated signaling through promoting assembly of TBK1- TANK, or IRF3, but not VISA, in untransfected cells (Fig. 6B). associated complexes.

FIGURE 6. MSX1 is required for assembly of TBK1-associated complexes. (A) HEK293 cells were transduced with control or the indicated MSX1-RNAi by retroviral- mediated gene transfer. Cells were transfected with the indicated expression plasmids (2 mg each) for another 20 h, and then coimmunoprecipitation and immunoblot analysis were performed with the indicated Abs. The bar graphs show the relative intensities of the bands, which were quantitated by densitometry using ImageJ and normalized to the indicated protein enrich- ment levels. (B) MSX1-deﬁcient and control cells (4 3 105) were left uninfected or infected with SeV for the indicated times. The cell lysates were subjected to immunopre- cipitation and immunoblot analysis with the indicated Abs. The line graphs show the relative intensities of the bands, which were quantitated by densitometry using ImageJ and normalized to the indicated protein en- richment levels. 8 MSX1 REGULATES RLR SIGNALING

Discussion In conclusion, we identified MSX1 as an important component of In this study, we identified MSX1 as a modulator of RLR-mediated RLR-mediated antiviral signaling. Upon viral stimulation, MSX1 is innate antiviral signaling pathways. MSX1 deficiency significantly associated with TBK1 and promotes assembly of TBK1-associated impaired dsRNA, but not DNA virus-triggered activation of IRF3, complexes, leading to IRF3 activation and induction of downstream induction of downstream antiviral genes, and cellular antiviral re- antiviral genes. Our studies provide insights into the regulatory sponses. Mechanistically, MSX1 interacted with TBK1 and pro- mechanism of RLR-mediated innate antiviral responses. moted assembly of TBK1-associated complexes upon viral infection. NAP1 and TANK are adaptor proteins that specifically associate Acknowledgments with TBK1 and participate in RLR-induced signaling. They contain a We thank Dr. Xiao-Dong Zhang (Wuhan University) for technical help. conserved TBK1-binding domain, which mediates the constitutive association with TBK1 (22, 23, 39, 40). It has been reported that Disclosures NAP1 and TANK bind to the C-terminal coiled-coil domain in The authors have no financial conflicts of interest. TBK1 and compete for binding with TBK1 (41). Interestingly, we identified that MSX1 also interacted with the coiled-coil domain of TBK1 (Fig. 5D) and facilitated the association between TBK1 and References 1. Kawai, T., K. Takahashi, S. Sato, C. Coban, H. Kumar, H. Kato, K. J. Ishii, NAP1 or TANK (Fig. 6A). In addition, MSX1 deficiency impaired O. Takeuchi, and S. Akira. 2005. IPS-1, an adaptor triggering RIG-I- and Mda5- the association of TBK1 with NAP1 or TANK (Fig. 6B). The mediated type I interferon induction. Nat. Immunol. 6: 981–988. simplest explanation for our results is that MSX1 mediates the as- 2. Meylan, E., J. Curran, K. Hofmann, D. Moradpour, M. Binder, R. Bartenschlager, and J. Tschopp. 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway sociation of TBK1 with NAP1 or TANK. Domain mapping analysis and is targeted by hepatitis C virus. Nature 437: 1167–1172. Downloaded from identified that the N-terminal domain of MSX1 interacted with 3. Seth, R. B., L. Sun, C. K. Ea, and Z. J. Chen. 2005. Identification and charac- terization of MAVS, a mitochondrial antiviral signaling protein that activates TBK1, whereas none of MSX1 truncation mutants restored its NF-kappaB and IRF 3. Cell 122: 669–682. function in RLR-mediated antiviral signaling. It is possible that the 4. Xu, L. G., Y. Y. Wang, K. J. Han, L. Y. Li, Z. Zhai, and H. B. Shu. 2005. VISA is N-terminal domain of MSX1 is only required for its association an adapter protein required for virus-triggered IFN-beta signaling. Mol. Cell 19: 727–740. with TBK1, but not sufficient for activation of the signaling. 5. Lei, C. Q., Y. Zhang, M. Li, L. Q. Jiang, B. Zhong, Y. H. Kim, and H. B. Shu. 2015. ECSIT bridges RIG-I-like receptors to VISA in signaling events of innate

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