Foot-And-Mouth Disease Virus 3B Protein Interacts with Pattern

Foot-and-Mouth Disease Virus 3B Protein Interacts with Pattern Recognition Receptor RIG-I to Block RIG-I−Mediated Immune Signaling and Inhibit Host Antiviral This information is current as Response of September 25, 2021. Xiangle Zhang, Zixiang Zhu, Congcong Wang, Fan Yang, Weijun Cao, Pengfei Li, Xiaoli Du, Furong Zhao, Xiangtao Liu and Haixue Zheng J Immunol published online 11 September 2020 Downloaded from http://www.jimmunol.org/content/early/2020/09/10/jimmun ol.1901333 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2020/09/10/jimmunol.190133 Material 3.DCSupplemental

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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 September 11, 2020, doi:10.4049/jimmunol.1901333 The Journal of Immunology

Foot-and-Mouth Disease Virus 3B Protein Interacts with Pattern Recognition Receptor RIG-I to Block RIG-I–Mediated Immune Signaling and Inhibit Host Antiviral Response

Xiangle Zhang,*,1 Zixiang Zhu,*,1 Congcong Wang,* Fan Yang,* Weijun Cao,* Pengfei Li,* Xiaoli Du,* Furong Zhao,*,† Xiangtao Liu,*,‡ and Haixue Zheng*

Foot-and-mouth disease is a highly contagious disease of pigs, sheep, goats, bovine, and various wild cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) that has given rise to significant economic loss to global livestock industry. FMDV 3B protein is an important determinant of virulence of the virus. Modifications in 3B protein of FMDV considerably decrease virus yield. In the current study, we demonstrated the significant role of 3B protein in suppression of type I IFN production and host

antiviral response in both human embryonic kidney HEK293T cells and porcine kidney PK-15 cells. We found that 3B protein Downloaded from interacted with the viral RNA sensor RIG-I to block RIG-I–mediated immune signaling. 3B protein did not affect the expression of RIG-I but interacted with RIG-I to block the interaction between RIG-I and the E3 ubiquitin ligase TRIM25, which prevented the TRIM25-mediated, Lys63-linked ubiquitination and activation of RIG-I. This inhibition of RIG-I–mediated immune signaling by 3B protein decreased IFN-b, IFN-stimulated genes, and proinﬂammatory cytokines expression, which in turn promoted FMDV replication. All of the three nonidentical copies of 3B could inhibit type I IFN production, and the aa 17A in each copy of 3B was

involved in suppression of IFN-related antiviral response during FMDV infection in porcine cells. Together, our results indicate http://www.jimmunol.org/ the role of 3B in suppression of host innate immune response and reveal a novel antagonistic mechanism of FMDV that is mediated by 3B protein. The Journal of Immunology, 2020, 205: 000–000.

oot-and-mouth disease is a highly contagious disease of finally yields four mature structural proteins and 10 nonstructural pigs, sheep, goats, bovine, and various wild, cloven-hoofed proteins (2). Both the intermediate and mature viral proteins per- F animals that has caused significant economic loss to global form functions in the viral life cycle (3). A number of interactions livestock industry (1). The causative agent of the disease is foot- between the viral proteins and host proteins have been reported for and-mouth disease virus (FMDV), which belongs to the genus of understanding the functions of FMDV proteins and clarifying how Aphthovirus in family of Picornaviridae. FMDV includes a pos- FMDV manipulates the host machinery (4). by guest on September 25, 2021 itive single-strand RNA genome that encodes a large polyprotein. Innate immune system is the first line of defense against invading The polyprotein is processed into several intermediate proteins and pathogens, and the pathogen recognition receptors (PRRs) are responsible for detection of the pathogens and induction of host *State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Re- immune responses (5). PRRs mainly contain TLRs, RIG-I–like search Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China; receptors (RLRs), NOD-like receptors, and C-type lectin recep- † Institute of Oceanography, Minjiang University, Fuzhou, Fujian 350108, China; and tors. These PRRs sense different pathogen signatures and trigger ‡National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinary Re- search Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China the activation of different pathways (6, 7). RLRs, as one of the 1X.Z. and Z.Z. contributed equally to this work. major subsets of PRRs, play critical roles in the host defense against numerous viral infections (8). The activation of the Received for publication November 6, 2019. Accepted for publication August 10, 2020. downstream transcription factors of RLRs pathway drives type This work was supported by grants from the National Natural Sciences Foundation of China I IFN and proinflammatory cytokines production that induce (31802171), the Key Development and Research Foundation of Yunnan (2018BB004), the host antiviral signaling cascades (9). Chinese Academy of Agricultural Science and Technology Innovation Project (CAAS- Many picornaviruses suppress host innate immune system by XTCX2016011-01 and Y2017JC55), and the Central Public-Interest Scientific Institution Basal Research Fund (1610312016013 and 1610312016003). a variety of mechanisms to evade clearance by the host and Address correspondence and reprint requests to Prof. Haixue Zheng, State Key facilitate viral replication. For example, hepatitis A virus and Laboratory of Veterinary Etiological Biology and OIE/National Foot and Mouth FMDV proteolytically cleave host NF-kB essential modulator Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese (NEMO) by viral proteinase 3Cpro to counteract host innate Academy of Agricultural Sciences, Lanzhou 730046, Gansu, China. E-mail address: [email protected] immune response (10, 11). NEMO is a bridging adaptor that b k The online version of this article contains supplemental material. plays a critical role in activation of both IFN- and NF- B Abbreviations used in this article: CTD, C-terminal domain; FMDV, foot-and-mouth signaling pathways (12). The cleavage of NEMO significantly disease virus; hpi, hour postinfection; hpt, hour posttransfection; IFA, immunofluo- impairs IFN-b and proinflammatory cytokines production. The rescence microscopy; ISRE, IFN-stimulated response element; MAVS, mitochondrial L proteinase (Lpro) and 2A protein of most of picornaviruses antiviral signaling protein; NEMO, NF-kB essential modulator; PRR, pathogen recognition receptor; qPCR, quantitative real-time PCR; RIG-I KO, RIG-I knockout; cleave or decrease host translation initiation factor eIF4G to RLR, RIG-I–like receptor; SeV, Sendai virus; SVA, Senecavirus A; TCID50, 50% shut off host protein synthesis and promote viral replication tissue culture infective dose. (13–15). Moreover, the antagonistic role of other viral proteins This article is distributed under The American Association of Immunologists, Inc., in innate immune suppression is being recently unveiled, in- Reuse Terms and Conditions for Author Choice articles. dicating sophisticated immune suppression mechanisms and Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 strategies of picornaviruses (4, 16–19).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901333 2 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING

FMDV 3B (also known as VPg) is present in three similar but GST-O3B2, and GST-O3B3), the GST tag was inserted into pCDNA3.1 nonidentical copies (3B1, 3B2, and 3B3) (20), and there are no at the C terminus of the multiple cloning sites, and FMDV 3B sequence reports of naturally occurring FMDV strains with fewer than three was constructed into the pCDNA3.1-GST plasmid with the GST located at the C terminus. The deletion of the 3B1, 3B2, or 3B3 was introduced into copies of 3B (21). The three nonidentical copies are highly con- GST-3B through the PCR-based mutagenesis as described previously (30). served and all contain the position 3 Tyr (Y) residue (Fig. 2D) The GST-3B1–, GST-3B2–, and GST-3B3–expressing plasmids were also which is involved in the phosphodiester linkage to the viral ge- constructed through the PCR-based mutagenesis. A series of FLAG-tagged nome RNA. 3B is covalently attached to the 59 end of the viral 3B mutants constructs were generated by inserting the synthesized fragment into the FLAG–CMV-7.1 plasmid. IFN-stimulated response element genome RNA via the conserved Y residue by the viral RNA po- (ISRE), IFN-b, and NF-kB promoter luciferase reporter plasmids and pol lymerase (3D ), acting as a primer for the synthesis of the RNA pRL-TK internal control plasmids were described previously (16, 31). HA- during viral replication (22, 23). Although not all of the three tagged RIG-I, HA-tagged RIG-I (CARD) and various HA-tagged com- copies are essential for FMDV replication, the copy number of ponents including MAVS, TBK1, IRF3, and IRF7 expressing plasmids 3B is critically associated with the host range and the virulence used in this study were kind gifts from Professor H. Shu in Wuhan Uni- versity (16, 31). Myc-tagged RIG-I and HA-tagged K63 ubiquitin plasmids of FMDV. This may help to explain why all naturally isolated were described previously (32, 33). All the plasmids used in this study FMDVs have retained three copies of 3B (2). were veriﬁed by DNA sequencing analysis. The Lipofectamine 2000 re- FMDV has developed signiﬁcant ability to suppress host innate agent (Invitrogen) was used for plasmid transfection per the manufac- immune response by diverse strategies. The viral proteinases Lpro turer’s instructions. pro and 3C are the most well-known factors that present antago- RNA extraction and quantitative real-time PCR nistic role on host innate immune response (4, 24). Both Lpro and

3Cpro cleave eIF4G to shut off host protein synthesis (13, 25). In Total RNA was extracted from the cell culture using TRIzol Reagent Downloaded from (Invitrogen) according to the manufacturer’s instructions. The first-strand addition, they suppress IFN production by impairing the activation cDNA was synthesized using the M-MLV reverse transcriptase (Promega) of RLRs signaling pathway (26). In the current study, we identi- and random hexamer primers (TaKaRa Bio). The synthesized cDNAs were fied FMDV nonstructural protein 3B as a new antagonistic factor subjected to quantitative real-time PCR (qPCR) analysis. The relative of host innate immune response. 3B suppressed the expression of amounts of cDNAs were determined using the SYBR Premix Ex Taq reagents IFN-b and antiviral genes during FMDV infection and efficiently (TaKaRa Bio) and a QuantStudio 5 Real-Time PCR instrument (Applied Biosystems) following the manufacturer’s protocol. Relative mRNA levels 2DD promoted viral replication. We found that 3B interacted with RIG- were calculated using 2 CT method (CT indicates threshold cycle) as http://www.jimmunol.org/ I and impaired the interaction between RIG-I and the adaptor described previously (32). The house keeping gene GAPDH was used as protein mitochondrial antiviral signaling protein (MAVS), which an internal control in qPCR analysis. The qPCR primers used in this study is critical for IFN-b production and antiviral genes expression. were shown in Table I. The lysine 63 (K63)-linked ubiquitination of RIG-I is crucial for Reporter gene assays the interaction between RIG-I and MAVS. Our results showed that HEK293T cells were cultured on 24-well plates, and the monolayer cells the K63-linked ubiquitination of RIG-I was significantly inhibited by were transfected with the indicated host protein expressing plasmids, empty 3B. The interaction of 3B with RIG-I blocked the RIG-I–TRIM25 vector, or viral protein expressing plasmids, in the presence of 100 ng per interaction, preventing the TRIM25-mediated K63-linked ubiq- well reporter plasmid and 10 ng per well internal control Renilla luciferase uitination and activation of RIG-I. Therefore, we determined that 3B reporter plasmid pRL-TK (normalization of the transfection efficiency). by guest on September 25, 2021 targeted the viral sensor RIG-I to disrupt its activation, leading to a The Lipofectamine 2000 reagent was used for plasmid transfection. The empty vector plasmids were used in the transfection experiments to ensure blockade of RIG-I–mediated immune signaling and host antiviral the cells receive the same amounts of total plasmids. Where indicated, the response. transfected cells were mock-infected or infected with SeV (100 hemaggluti- nating activity units per milliliter) for 16 h at 24 h posttransfection (hpt). The Materials and Methods dual-luciferase activities were measured using the Promega Dual-Luciferase Viruses and cells Reporter Assay System (Promega) according to the manufacturer’s protocol. As for the critical components of RIG-I pathway induced FMDV strain O/BY/CHA/2010 (GenBank number: JN998085), which was luciferase reporter assay, HEK293T cells were cotransfected with Flag isolated from a pig in China in 2010 (27), was used in this study. Sendai vector or Flag-3B–encoding plasmids and the plasmids expressing a set virus (SeV), a model RNA virus routinely used to activate type I IFN of components of RIG-I pathway, together with the ISRE-driven lu- pathway in cell culture, was obtained from H. Shu’s Laboratory in Wuhan ciferase reporter plasmid and the internal control pRL-TK reporter University (16, 28). Human embryonic kidney HEK293T cells and porcine plasmid. Luciferase activity was detected and analyzed at 24 hpt. The kidney PK-15 cells were cultured in in DMEM (Invitrogen), supple- dual-luciferase activities were then measured using the Promega Dual- mented with 10% heated-inactivated FBS at 37˚C in a humidified 5% Luciferase Reporter Assay System. CO2 incubator. Western blotting and coimmunoprecipitation assays Abs Briefly, the cells were harvested at the indicated time and lysed in the lysis The commercial Abs used in this study include anti-FLAG mouse Ab buffer supplemented with various proteinase inhibitors. The cell lysates (catalog no. F1804; Sigma-Aldrich), anti-FLAG rabbit Ab (catalog no. were collected in the precold tubes. Equal amounts of samples were 701629; Invitrogen), anti-HA mouse Ab (catalog no. 26183; Invitrogen), subjected to 10% SDS-PAGE and analyzed for the expression of different anti-HA rabbit Ab (catalog no. 3724; Cell Signaling Technology), anti-Myc proteins. The nitrocellulose membranes (Pall) were used to retain proteins, mouse Ab (catalog no. sc-40; Santa Cruz Biotechnology), anti–RIG-I rabbit and the Ab–Ag complexes were generated by incubation of various Abs. Ab (catalog no. ab180675; Abcam), anti–RIG-I mouse Ab (catalog no. sc- The generated Ab–Ag complexes were detected by ECL detection reagents 376845; Santa Cruz Biotechnology), anti-MAVS rabbit Ab (catalog no. (Thermo Fisher Scientific). b-actin was used as a loading control to 24930; Cell Signaling Technology), anti-ubiquitin (linkage-specific K63) demonstrate equal protein sample loading. For the coimmunoprecipitation rabbit Ab (catalog no. ab179434; Abcam), anti-GST rabbit Ab (catalog no. assays, the cell lysates were immunoprecipitated with indicated Abs, and 2625; Cell Signaling Technology), and anti–b-actin mouse Ab (catalog no. the immunoprecipitated samples were subjected to immunoblotting anal- sc-8432; Santa Cruz Biotechnology). Anti-3B rabbit polyclonal Ab was ysis as described previously (32). prepared by our laboratory. Immunofluorescence microscopy and confocal imaging Plasmids HEK293T cells were cultured into Nunc glass-bottom dishes, and trans- The FLAG-tagged 3B plasmid was constructed by our laboratory previ- fected with 2 mg of FLAG-3B and 2 mg of HA–RIG-I plasmids for 24 h. ously, and the cDNA sequence of FMDV 3B was inserted into pCAGGS The cells were collected, washed with PBS, and then fixed with 4% vector with a C-terminal FLAG tag (29). For GST-3B and GST-3B paraformaldehyde in PBS at room temperature for 0.5 h. Then the fixed single-copy–deletion mutants expressing plasmids (including GST-O3B1, cells were washed with ice-cold PBS three times. The 0.2% Triton X-100 The Journal of Immunology 3 was used to permeabilize the cells for 10 min. Five percent BSA in PBS genome of O/BY/CHA/2010 (mutation of the 691–696 region in 2C from was used to block the cells for 1 h at 37˚C. The cells were then incubated 59-ATTGAC-39→59-ATCGAT-39) to generate a unique ClaI restriction with proper Abs as described previously (32). The stained cells were an- enzyme site. A unique SbfI restriction enzyme site was present in the C alyzed and imagined with a Nikon eclipse 80i fluorescence microscope and terminus after the viral poly (A) in our reverse-genetics system. A ∼3120 NIS Elements F 2.30 software. bp of fragment was obtained by digestion of the r3B-FMDV infectious clone using the ClaI and SbfI restriction enzymes. The obtained frag- 50% tissue culture infective dose assay ment was then inserted into the POK12 plasmid, and the aa A17E mutation in each copy of 3B was then introduced into the fragment at The samples were collected at the indicated hours postinfection (hpi). The the indicated position by the PCR-based mutagenesis as described 50%tissuecultureinfectivedose(TCID50) assay was carried out as described previously (30). After confirmation of the A→E mutation in all of the previously (34). Briefly, the monolayer cells were grown in the 96-well plates. 21 29 three nonidentical copies of 3B, the ClaI and SbfI restriction enzymes The virus suspension was diluted with 10 –10 serial dilutions and were used to generate the fragment bearing the 3B-A/E mutation. The m 50- l portion of the diluent was added to the cell cultures. Each dilution collected fragment was then insertedintother3B-FMDVinfectious was repeated in eight wells. The cells were maintained until cytopathic clone instead of the original region. All of the constructed plasmids effects were clearly observed. The TCID50 values were then calculated were sequenced. The viral rescue and identification was then performed by the Reed-Muench method. as described previously (35). Construction of the r3B-FMDV and r3B-A/E-FMDV Statistical analysis infectious clones All the data are represented as the mean with SE from three in- The construction strategy for the r3B-FMDV and r3B-A/E-FMDV independent experiments. A Student t test was used for a comparison fectious clones was based on the reverse-genetics system that has been of three independent experiments. All tests were two-sided. A p value developed by our laboratory previously (35). A synonymous substitution , 0.05 was considered to be statistically significant (designated by a ledtonochangesinaminoacidsinthe2C was introduced into the viral single asterisk). A p value , 0.01 was considered to be statistically Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. FMDV 3B protein suppressed the activation of type I IFN and NF-kB pathways. HEK293T cells were transfected with 0, 75, 150, or 300 ng of FLAG-3B–expressing plasmids together with 100 ng of ISRE- (A), IFN-b promoter– (B) or NF-kB promoter–driven (C) luciferase reporter plasmids and 10 ng of the internal control plasmid pRL-TK for 24 h, and the cells were mock-infected or infected with SeV for another 16 h. The luciferase activity was determined by the dual-luciferase assay. (D–F) HEK293T cells were transfected with 0, 100, 200, or 400 ng of FLAG-VP2–expressing plasmids together with 100 ng of ISRE- (D), IFN-b promoter– (E) or NF-kB promoter–driven (F) luciferase reporter plasmids and 10 ng of the internal control plasmid for 24 h, and the cells were mock-infected or infected with SeV for another 16 h. The luciferase activity was determined by the dual-luciferase assay. All the experiments were repeated three times with similar results. The data represent results from one of the triplicate experiments. *p , 0.05 considered significant, **p , 0.01 considered highly significant. n.s., not significant. 4 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING highly significant (designated by a double asterisk). n.s. indicated not undermine SeV-induced IFN-b promoter (Fig. 1E) and NF-kB significant. promoter activation (Fig. 1F). This indicated that FMDV 3B Results significantly suppressed RLR pathway signaling. FMDV 3B blocked SeV-induced IFN-b and NF-kB FMDV 3B considerably impaired the expression of pathways signaling IFN-stimulated genes and proinflammatory cytokines FMDV 3B protein plays important roles in viral replication (21). To examine whether FMDV 3B protein antagonizes IFN-b and Our previous study found that FMDV 3A suppressed SeV- IFN-stimulated genes (ISGs) expression (Table I), HEK293T cells triggered IFN-b promoter activation (29). To identify the sup- were transfected with empty vector or 3B-expressing plasmids and pressive role of 3B on host innate immune pathways signaling, then infected with SeV. The cell supernatants and cells were the SeV that is routinely used to induce type I IFNs was used as collected separately for the ELISA and qPCR detection of an agonist of innate immune pathway, and SeV-induced activation IFN-b expression as previously described (36). The expression of IFN-b promoter, ISRE, and NF-kB promoter was evaluated in levels three ISGs (ISG56, ISG54, and MX1) were also mea- 3B-overexpressing cells. HEK293T cells were transfected with sured. Overexpression of 3B protein considerably inhibited vector plasmids or increasing amounts of FLAG-3B–expressing IFN-b and ISGs expression (Fig. 2A, 2B). The activation of plasmids together with ISRE-, IFN-b promoter–, or NF-kB NF-kB pathway induces the expression of a set of proin- promoter–driven reporter plasmids and the internal control flammatory cytokines that are involved in innate immunity plasmid pRL-TK. The transfected cells were infected with SeV (37). 3B had been shown to inhibit SeV-induced NF-kBpro- to activate type I IFN pathway or NF-kB pathway, and the lu- moter activation (Fig. 1C). To confirm 3B-mediated suppres- Downloaded from ciferase activity was measured. Overexpression of 3B protein sive role on NF-kB pathway, the expression levels of several significantly suppressed SeV-induced ISRE (Fig. 1A), IFN-b cytokines, including TNF-a, IL-6, and IL-1b were measured in promoter (Fig. 1B), and NF-kB promoter (Fig. 1C) activation, the cells transfected with vector or 3B-expressing plasmids and showing a dose-dependent manner. The expression of FMDV infected by SeV. SeV infection induced weakly but statistically 3B protein was detected by immunoblotting using an anti-FLAG significantly TNF-a,IL-6,andIL-1b expression in the vector- a b

Ab. We also evaluated the effect of FMDV VP2 on SeV-induced transfected cells, and the expression of TNF- ,IL-6,andIL-1 http://www.jimmunol.org/ ISRE, IFN-b promoter, and NF-kB promoter activation, which was considerably decreased in the 3B-overexpressing cells showed that a high amount of VP2 weakly but statistically sig- (Fig. 2C). FMDV 3B includes three nonidentical copies (3B1, niﬁcantly inhibited ISRE activation (Fig. 1D), although it did not 3B2, and 3B3) (Fig. 2D). The effect of 3B1, 3B2, and 3B3 on

Table I. The qPCR primers used in this study

Gene Primers (59 → 39)

Human IFN-b Forward: 59-GACATCCCTGAGGAGATTAAG-39 by guest on September 25, 2021 Reverse: 59-ATGTTCTGGAGCATCTCATAG-39 Human ISG56 Forward: 59-CCACAAAAAATCACAAGCCATTT-39 Reverse: 59-CAGGGCAAGGAGAACCTTAATATATC-39 Human ISG54 Forward: 59-ACGGTATGCTTGGAACGATTG-39 Reverse: 59-AACCCAGAGTGTGGCTGATG-39 Human MX1 Forward: 59-TCTTCATGCTCCAGACGTAC-39 Reverse: 59-CCAGCTGTAGGTGTCCTTG-39 Human TNF-a Forward: 59-AGAGGGAGAGAAGCAACTACA-39 Reverse: 59-GGGTCAGTATGTGAGAGGAAGA-39 Human IL-6 Forward: 59-TGACCCAACCACAAATGC-39 Reverse: 59-AGGAACTCCTTAAAGCTGCG-39 Human IL-1b Forward: 59-CAAAGGCGGCCAGGATATAA-39 Reverse: 59-CTAGGGATTGAGTCCACATTCAG-39 Human MAVS Forward: 59-ATGGTGCTCACCAAGGTGTCTG-39 Reverse: 59-TCTCAGAGCTGCTGTCTAGCCA-39 Human RIG-I Forward: 59-AGTGAGCATGCACGAATGAA-39 Reverse: 59-GGGATCCCTGGAAACACTTT-39 Human GAPDH Forward: 59-CGGGAAGCTTGTGATCAATGG-39 Reverse: 59-GGCAGTGATGGCATGGACTG-39 Porcine IFN-b Forward: 59-GCTAACAAGTGCATCCTCCAAA-39 Reverse: 59-AGCACATCATAGCTCATGGAAAGA-39 Porcine ISG56 Forward: 59-AAATGAATGAAGCCCTGGAGTATT-39 Reverse: 59-AGGGATCAAGTCCCACAGATTTT-39 Porcine ISG15 Forward: 59-GATCGGTGTGCCTGCCTTC-39 Reverse: 59-CGTTGCTGCGACCCTTGT-39 Porcine MX1 Forward: 59-CATCTGTAAAACTCTGCCCCTGT-39 Reverse: 59-CATCTTCCCGCTTTCATCCT-39 Porcine MAVS Forward: 59-AAAGTGCCTACTGGCTTGCT-39 Reverse: 59-TGCTGGAGTCTCCTTTTCAGG-39 Porcine RIG-I Forward: 59-TTCAACTCCCAGTGTATGAGCAGC-39 Reverse: 59-TGATGGAATTGTCCCATTGGTAAG-39 Porcine GAPDH Forward: 59-ACATGGCCTCCAAGGAGTAAGA-39 Reverse: 59-GATCGAGTTGGGGCTGTGACT-39 FMDV Forward: 59-CACTGGTGACAGGCTAAGG-39 Reverse: 59-CCCTTCTCAGATTCCGAGT-39 SVA Forward: 59-AGAATTTGGAAGCCATGCTCT-39 Reverse: 59-GAGCCAACATAGARACAGATTGC-39 The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. FMDV 3B inhibited the expression of IFN-b, ISGs, and proinflammatory cytokines. (A) HEK293T cells were transfected with vector or 3B- expressing plasmids for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. IFN-b in the harvested supernatants was detect with an ELISA. (B) The expression of IFN-b, ISG56, ISG54, and MX1 mRNA in the collected cells was detected by qPCR. (C) The expression of proinflammatory cytokines TNF-a, IL-6, and IL-1b mRNA was detected by qPCR. (D) Schematic representation of 3B protein with three nonidentical copies. (E) HEK293T cells were transfected with GST-3B, GST-3B single-copy–deletion mutants expressing plasmids or GST vector for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. The expression of IFN-b was detected by qPCR. The expression of GST-3B, GST-3B deletion mutants, and GST was detected by Western blotting. (F) PK-15 cells were transfected with GST-3B, GST-3B single-copy–deletion mutants expressing plasmids or GST vector for 24 h, and the cells were then mock-infected or infected with SeV for another 24 h. The expression of IFN-b was detected by qPCR. (G) HEK293T cells were transfected with GST vector, GST-3B single-copy mutants or GST-3B–expressing plasmids for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. The expression of IFN-b was detected by qPCR. The expression of GST, GST-3B1, GST-3B2, GST-3B3, and GST-3B was detected by Western blotting. All the experiments were repeated three times with similar results. The data represent results from one of the triplicate experiments. *p , 0.05 considered significant, **p , 0.01 considered highly significant.

IFN-b production was also investigated in both HEK293T and expressing GST-tagged 3B1, 3B2, or 3B3 and evaluated whether PK-15 cells. The results showed that deletion of a single copy of a single copy of 3B could suppress IFN-b production. The re- 3B did not subvert 3B-mediated suppressive effect on IFN-b sults showed that the expression of any copy of 3B inhibited production (Fig. 2E, 2F). It showed a similar outcome by using SeV-induced IFN-b production (Fig. 2G). These results further three deletion mutants. We further generated the constructs confirmed that FMDV 3B protein antagonized host innate 6 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING immune signaling and inhibit the expression of downstream 3B protein, HEK293T cells were cotransfected with empty vector antiviral genes. or FLAG-3B–expressing plasmids and a series of plasmids expressing the components of type I IFN pathway, including RIG- b FMDV 3B suppressed IFN- and ISGs expression and I (CARD) (the CARD domain of RIG-I), MAVS, RIG-I/MDA5 promoted viral replication (full-length RIG-I together with MDA5), TBK1, IRF3, or IRF7, 3B suppressed SeV-induced innate immune signal transduction. To together with ISRE luciferase reporter plasmid and the internal investigate the viral replication status in 3B-overexpressing cells, control plasmid pRL-TK. The component protein-induced activation PK-15 cells were transfected with empty vector or FLAG-3B– of ISRE luciferase activity was evaluated at 24 h after transfection. expressing plasmids and then mock-infected or infected with Overexpression of these components significantly activated ISRE FMDV for 12 h. The viral RNA levels and virus yields were de- reporter system (Fig. 4). However, 3B inhibited the activation of tected by qPCR and TCID50 assay respectively as described pre- ISRE reporter system driven by MAVS, RIG-I(CARD), and RIG-I/ viously (17, 29). The results showed that overexpression of 3B MDA5 (Fig. 4A). In contrast, TBK1, IRF3, or IRF7-induced acti- significantly promoted FMDV replication (Fig. 3A). The ex- vation of ISRE reporter system was not affected by 3B (Fig. 4B). pression levels of IFN-b and ISGs (ISG56, ISG15, and MX1) Consequently, this confirmed that FMDV 3B targeted MAVS or its were also determined by qPCR analysis, which showed that 3B upstream molecule RIG-I. decreased the expression of IFN-b and ISGs (Fig. 3B). FMDV FMDV 3B did not affect the mRNA and protein expression of infection in PK-15 cells led to very low expression of ISGs. This MAVS and RIG-I implied that FMDV infection blocked the production of IFNs and subsequent IFN signaling in the PK-15 cells, therefore To investigate whether FMDV 3B suppressed the expression of Downloaded from resulted in low expression of ISGs. An ∼50-fold increase in MAVS or RIG-I, HEK293T cells were transfected with empty viral titers was observed on the basis of an 8-fold increase in vector or increasing amounts of FLAG-3B–expressing plasmids. intracellular FMDV RNA in Fig. 3A. FMDV 3B inhibited ex- The expression levels of MAVS or RIG-I mRNA and endogenous pression of ISGs. These ISGs performed antiviral functions MAVS or RIG-I proteins were evaluated at 36 hpt. Both the through multiple mechanisms. Some of the ISGs possibly have MAVS mRNA (Fig. 5A) and MAVS protein levels (Fig. 5B) were

affected the assembly/release of the virus. We also investigated not changed in the 3B-overexpressing cells compared with the http://www.jimmunol.org/ the effect of 3B on the replication level of another picornavirus vector-transfected cells. Similarly, we did not ﬁnd the decrease of Senecavirus A (SVA), which showed that overexpression of 3B RIG-I mRNA (Fig. 5C) or RIG-I protein levels (Fig. 5D) in 3B- also promoted SVA replication (Fig. 3C). These results indi- overexpressing cells. The effect of 3B on endogenous expression cated that 3B suppressed IFN-b andISGsexpressionwhich of MAVS or RIG-I in PK-15 cells was also examined. PK-15 cells promoted viral replication. were transfected with increasing amounts of FLAG-3B–expressing plasmids. The expression levels of MAVS or RIG-I mRNA FMDV 3B protein targeted at MAVS or upstream of MAVS and endogenous MAVS or RIG-I proteins were examined at 36 FMDV 3B suppressed both SeV-induced IFN-b and NF-kB pro- hpt. The results were consistent with those noted above. There- moter activation (Fig. 1). This suggested that 3B targeted the RIG- fore, 3B did not affect the expression of MAVS (Fig. 5E) and RIG- by guest on September 25, 2021 I signaling pathway at or upstream of MAVS. To conﬁrm this I mRNA (Fig. 5F) and endogenous MAVS (Fig. 5G) and RIG-I inhibitory effect and the potential components targeted by FMDV proteins (Fig. 5H). These results suggested that 3B might affect

FIGURE 3. Overexpression of 3B promoted FMDV replication by suppression of IFN-b and ISGs expression. PK-15 cells were transfected with empty vector or FLAG-3B–expressing plasmids for 24 h, and the cells were then infected with 0.5 multiplicity of infection (MOI) of FMDV for another 12 h. (A)

The viral RNA levels were measured by qPCR and virus yields were determined by TCID50 assay. (B) The expression of IFN-b, ISG56, ISG15, and MX1 was determined by qPCR. (C) PK-15 cells were transfected with empty vector or FLAG-3B–expressing plasmids for 24 h, and the cells were then infected with 0.5 MOI of SVA for another 12 h. The SVA RNA levels were measured by qPCR. All the experiments were repeated three times with similar results. The data represent results from one of the triplicate experiments. *p , 0.05 considered signiﬁcant, **p , 0.01 considered highly signiﬁcant. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 4. Effects of FMDV 3B protein on type I IFN pathway signaling induced by various components of RIG-I pathway. HEK293T cells were cotransfected with empty vector or FLAG-3B–expressing plasmids and RIG-I (CARD)–, MAVS-, RIG-I/MDA5– (full-length RIG-I together with MDA5), TBK1-, IRF3-, or IRF7-expressing plasmids with ISRE luciferase reporter plasmid and the internal control plasmid pRL-TK. The luciferase activity was determined at 24 hpt. (A) The effect of 3B on RIG-I (CARD), MAVS or RIG-I/MDA5–mediated signaling. (B) The effect of 3B on TBK1, IRF3, or IRF7 by guest on September 25, 2021 mediated signaling. All the experiments were repeated three times with similar results. The data represent results from one of the triplicate experiments. **p , 0.01 considered highly significant. the function of MAVS or RIG-I by other manner rather than de- 3B protein interacted with RIG-I protein (Fig. 6C, 6D). The creasing their expression. interaction between 3B and endogenous RIG-I in the context of viral infection was subsequently examined. PK-15 cells were FMDV 3B interacted with RIG-I infected by FMDV for 12 h, and the coimmunoprecipitation To investigate the possible interaction between 3B protein and assay was performed. The results showed that FMDV 3B also the components of RIG-I–mediated immune signal pathway, efficiently interacted with endogenous RIG-I in FMDV- HEK293T cells were cotransfected with FLAG-3B and the infected cells (Fig. 6E). This confirmed that FMDV 3B pro- empty vector or the plasmids expressing the HA-tagged com- tein interacted with host RIG-I protein during viral infection. The ponents of RIG-I–mediated immune signal pathway. The results PK-15 cells were also cotransfected with vector or FLAG-3B– showed that RIG-I but not MAVS was coprecipitated with expressing plasmids and HA-tagged porcine RIG-I (HA-pRIG-I)– or FLAG-3B (Supplemental Fig. 1), suggesting that there was an HA-tagged porcine MDA5 (HA-pMDA5)–expressing plasmids interaction between FMDV 3B and RIG-I protein. To evaluate for 36 h, and the coimmunoprecipitation assay was performed. whether 3B protein and RIG-I share similar subcellular locations, The results showed that FMDV 3B protein interacted with the indirect immunofluorescence microscopy (IFA) assay was car- porcine RIG-I protein but not MDA5 (Fig. 6F, 6G). The ex- ried out. PK-15 cells were transfected with Myc–RIG-I–expressing pression level of IFN-b and viral replication status in FMDV- plasmids for 24 h, and then followed by mock-infection or infected RIG-I knockout (RIG-I KO) PK-15 cells and wild-type FMDV infection for 12 h. The subcellular localization of 3B PK-15 cells were determined and compared with reveal the and Myc–RIG-I was investigated. FMDV 3B and Myc–RIG-I essential antiviral role of RIG-I against FMDV. Knockout of showed remarkable colocalization in the cytoplasm (Fig. 6A). RIG-I considerably decreased IFN-b expression and promoted The colocalization of FMDV 3B and endogenous RIG-I in PK- FMDV replication as well as viral yields (Fig. 6H). Further- 15 cells was further investigated, which also showed that more, RIG-I KO PK-15 cells were transfected with vector FMDV 3B colocalized with RIG-I (Fig. 6B). The interaction of plasmids or increasing amounts of FLAG-3B–expressing 3B protein with endogenous RIG-I was further evaluated. PK- plasmids for 24 h, and followed FMDV infection for 12 h. The 15 cells were transfected with vector or FLAG-3B–expressing replication of FMDV was then evaluated. Overexpression of 3B plasmids for 36 h, and the coimmunoprecipitation assay was did not enhance FMDV replication in the RIG-I KO PK-15 cells performed. Both the immunoprecipitation experiment and the (Fig. 6I). As expected, we also found that overexpression of 3B reverse immunoprecipitation experiment showed that FMDV also did not promote FMDV replication in BHK-21 cells (Fig. 6J), 8 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING Downloaded from

FIGURE 5. FMDV 3B did not affect the mRNA and protein expression levels of RIG-I and MAVS. (A–D) HEK293T cells were transfected with 0, 0.5, 1, http://www.jimmunol.org/ or 2 mg of FLAG-3B–expressing plasmids for 36 h, the expression levels of endogenous MAVS mRNA (A), MAVS protein (B), RIG-I mRNA (C), and RIG- I protein (D) were determined. (E–H) PK-15 cells were transfected with 0, 0.5, 1, or 2 mg of FLAG-3B–expressing plasmids for 36 h, the expression levels of endogenous MAVS mRNA (E), MAVS protein (F), RIG-I mRNA (G), and RIG-I protein (H) were determined. The empty vector was used in the whole transfection process to ensure that the cells received the same amount of total plasmids. All the experiments were repeated three times with similar results. *p , 0.05 considered significant. which have a deficient IFN signal transduction system (38, 39). 3B protein significantly suppressed the K63-linked ubiquitination These results indicated that RIG-I was critical for suppressing of RIG-I in a concentration-dependent manner (Fig. 7B). The FMDV replication during FMDV infection and 3B-mediated effect of FMDV infection on poly(I:C)–induced K63-linked by guest on September 25, 2021 antagonistic effect against RIG-I was favorable for FMDV ubiquitination of RIG-I was examined as well. PK-15 cells were replication. transfected with poly(I:C) for 12 h and then infected by FMDV for 12 h. The K63-linked ubiquitination of RIG-I was then FMDV 3B inhibited the RIG-I/MAVS complex formation and detected. FMDV infection also inhibited poly(I:C)–induced K63- the ubiquitination of RIG-I linked ubiquitination of RIG-I (Fig. 7C). These results indi- The interaction between RIG-I and MAVS is essential for signal cated that FMDV 3B prevented the K63-linked ubiquitination transduction of RIG-I–mediated immune signal pathway (40). 3B of RIG-I, resulting in the suppression of RIG-I–MAVS com- was determined to interact with RIG-I. Therefore, to investigate plex formation. whether 3B protein impaired the interaction between RIG-I and MAVS, HEK293T cells were transfected with increasing amounts FMDV 3B inhibited the interaction between TRIM25 of FLAG-3B expressing plasmids together with Myc–RIG-I– and and RIG-I HA-MAVS–expressing plasmids and then infected with SeV. The The interaction of TRIM25 with RIG-IisessentialforTRIM25- transfectants were immunoprecipitated with anti-Myc Ab and mediated K63-linked ubiquitination of RIG-I. 3B inhibited the subjected to Western blotting analysis. RIG-I efficiently pulled K63-linked ubiquitination of RIG-I. To decipher the mecha- down MAVS. However, the amount of MAVS that bound to nism by which 3B interaction with RIG-I leads to the sup- RIG-I gradually decreased as the increasing expression of 3B pression of K63-linked ubiquitination of RIG-I, we evaluated protein (Fig. 7A). These results suggested that FMDV 3B protein whether 3B interfered with the interaction between RIG-I and sequestered the RIG-I–MAVS interaction in a dose-dependent TRIM25. HEK293T cells were cotransfected with Myc–RIG-I, manner. HA-TRIM25, and vector or FLAG-3B–expressing plasmids. The interaction of RIG-I with MAVS is dependent upon K63- The transfectants were immunoprecipitated with anti-Myc Ab linked ubiquitination of RIG-I, which is essential for RIG-I– and subjected to Western blotting analysis. Coimmunopreci- mediated immune signaling (41). The ubiquitin ligase TRIM25 pitation revealed that RIG-I interacted with TRIM25. How- positively mediates K63-linked ubiquitination of RIG-I (42). To ever, the amount of TRIM25 that bound to RIG-I clearly examine whether 3B protein blocked the K63-linked ubiq- decreased in the presence of 3B protein (Fig. 8A). Consistent uitination of RIG-I, HEK293T cells were transfected with with its ability to suppress the K63-linked ubiquitination of Myc–RIG-I–, FLAG-TRIM25–, HA–Ub-K63 (HA-tagged K63 RIG-I, 3B inhibited the RIG-I–TRIM25 interaction in a dose- ubiquitin)–expressing plasmids together with different amounts dependent manner (Fig. 8B). We also investigated whether 3B of FLAG-3B–expressing plasmids and then infected with SeV. interacted with TRIM25, and no interaction was observed The transfectants were immunoprecipitated with anti-Myc Ab between 3B and TRIM25 (Fig. 8C). These results collectively and subjected to Western blotting analysis. Overexpression of indicated that the interaction of 3B with RIG-I blocked the The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/

FIGURE 6. FMDV 3B targeted RIG-I to suppress RIG-I–mediated antiviral effect. (A) PK-15 cells were transfected with 2 mg of Myc–RIG-I–expressing plasmids for 24 h. The cells were then mock-infected or infected by FMDV for 12 h and subjected to IFA analysis using the anti-Myc and anti-3B Abs. (B) PK-15 cells were mock-infected or infected by FMDV for 12 h and subjected to IFA analysis using the anti–RIG-I and anti-3B Abs. The Pearson correlation coefﬁcient was analyzed using the Image-Pro Plus 6.0 software. (C and D) PK-15 cells were transfected with 8 mg of vector or FLAG-3B–expressing by guest on September 25, 2021 plasmids for 36 h. The cells were collected and subjected to immunoprecipitation experiments and immunoblotting analysis. The lysates were immunoprecipitated using anti–RIG-I Ab (C) or anti-FLAG Ab (D) and subjected to Western blotting analysis using the indicated Abs. (E) PK-15 cells were mock-infected or infected by FMDV for 12 h and collected. The lysates were immunoprecipitated using anti–RIG-I Ab and subjected to Western blotting analysis using the indicated Abs. (F) HEK293T cells were cotransfected with 5 mg of FLAG-3B and 5 mg of HA vector, HA–pRIG-I–expressing plasmids (expressing porcine RIG-I) for 36 h. The cells were then lysed and subjected to immunoprecipitation experiments and immunoblotting analysis. (G) HEK293T cells were cotransfected with 5 mg of FLAG-3B and 5 mg of HA vector, HA-pMDA5–expressing plasmids (expressing porcine MDA5) for 36 h. The cells were then lysed and subjected to immunoprecipitation experiments and immunoblotting analysis. (H) RIG-I KO PK-15 cells and wild-type PK-15 cells were infected by FMDV for 0, 6, or 12 h respectively. The expression of IFN-b was detected by qPCR (left panel). The expression of RIG-I and viral protein VP1 was detected by Western blotting (middle panel). The viral yields in the supernatants at 12 hpi were determined by TCID50 assay (right panel). (I) PK-15 cells were transfected with 0, 1, or 2 mg of FLAG-3B–expressing plasmids for 24 h. The cells were then infected by FMDV for 12 h, and the viral RNA levels were measured by qPCR. (J) BHK21 cells were transfected with 0, 1, or 2 mg of FLAG-3B–expressing plasmids for 24 h. The cells were then infected by FMDV for 10 h, and the viral RNA levels were measured by qPCR. All the experiments were repeated three times with similar results. n.s., not signiﬁcant.

RIG-I–TRIM25 interaction, inhibiting K63-linked ubiquitination The previous data showed 3B inhibited the RIG-I (CARD)– of RIG-I. triggered IFN-b promoter activation (Fig. 4A). These results suggested that the interaction of 3B with RIG-I–CARD domain FMDV 3B interacted with the CARD and DEAD helicase blocked the K63-linked ubiquitination of RIG-I and suppressed domains of RIG-I RIG-I–mediated immune signaling. CARD domain is involved in RIG-I–MAVS complex formation and the K63-linked ubiquitination of RIG-I. To investigate the The 17A in each copy of FMDV 3B was involved in suppression b binding domain of RIG-I that is responsible for its interaction with of IFN- production 3B, a series of constructs expressing the CARD, DEAD Helicase, FMDV 3B binds to the 59 end of viral genomic RNA via the or C-terminal domain (CTD) of RIG-I were generated (Fig. 9A). conserved Y residue, acting as a primer for the synthesis of the The interaction between these mutants and 3B was evaluated. RNA during viral replication (22, 23). To investigate whether HEK293T cells were cotransfected with HA vector, HA–RIG-I, the binding activity of 3B prevented viral RNA recognition by HA–RIG-I–CARD, HA–RIG-I–DEAD Helicase, or HA–RIG-I– RIG-I, we generated a construct expressing mutated 3B (3B-Y3A) CTD– and FLAG-3B–expressing plasmids. The transfectants were bearing the Y to A mutations at the conserved sites (Fig. 10A). To immunoprecipitated with anti-FLAG Ab and subjected to Western evaluate the suppressive role of 3B-Y3A on host innate immune blotting analysis. Coimmunoprecipitation revealed that both the pathway signaling, SeV-induced activation of IFN-b promoter and CARD and DEAD Helicase of RIG-I interacted with 3B (Fig. 9B). IFN-b mRNA expression were analyzed in the presence or 10 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING

FIGURE 7. FMDV 3B suppressed the interaction between RIG-I and MAVS, and the K63-linked ubiquitination of RIG-I. (A) HEK293T cells were cotransfected with 4 mg of Myc–RIG-I and 4 mg of HA vector or HA-MAVS–expressing plasmids with 0, 3, or 5 mg of FLAG-3B–expressing plasmids for 24 h. The transfected cells were then infected with SeV for another 16 h and subjected to coimmunoprecipitation (Co-IP) assay. The lysates were immunoprecipitated with anti-Myc Ab and detected using the indicated Abs. (B) HEK293T cells were cotransfected with Myc–RIG-I, FLAG vector or FLAG-TRIM25, and HA vector or HA–Ub-K63–expressing plasmids with increasing amount of FLAG-3B–expressing plasmids for 36 h, following SeV infection for another 12 h. The cells were lysed and immunoprecipitated with anti-Myc Ab and Downloaded from detected using the indicated Abs. WCLs were detected using the indicated Abs. (C)PK-15 cells were mock-transfected or transfected with poly poly(I:C) for 12 h and followed by FMDV infection for another 12 h. The cells were lysed and immunoprecipitated

with anti–RIG-I Ab and detected using the http://www.jimmunol.org/ indicated Abs. WCLs were detected using the indicated Abs. All the experiments were repeated three times with similar results. WCL, whole-cell lysate. absence 3B-Y3A. The results showed that 3B-Y3A retained the to RIG-I. It suggested that the A–E mutation in 3B possibly just activity to inhibit SeV-induced RLR pathway signaling and IFN-b disrupted the activity of 3B to inhibit RIG-I–mediated signal expression (Fig. 10B). We also found that FMDV VP2 did not transduction. RIG-I, as an ISG, also has a direct antiviral ac- affect SeV-induced IFN-b expression (Fig. 10C). To confirm the tivity, other mechanisms might also be involved in 3B–RIG-I by guest on September 25, 2021 suppressive role of 3B-Y3A on IFN-b production during FMDV interaction. infection, PK-15 cells were transfected with empty vector or To confirm the function of the residues of 17A in 3B, a FLAG–3B-Y3A–expressing plasmids and then mock-infected or recombinant virus bearing the 3B-A/E instead of 3B was gen- infected with FMDV for 12 h. The mRNA expression levels of erated, and the parental wild-type virus was named as r3B- IFN-b and ISGs (ISG56 and MX1) were then measured. Over- FMDV and the recombinant virus was named r3B-A/E-FMDV expression of 3B-Y3A inhibited the expression of IFN-b and ISGs (Fig. 11A). The two viruses showed similar viral titers in the in FMDV-infected cells as well (Fig. 10D). These data suggested BHK-21 cells (Fig. 11B). We further evaluated the replication that the RNA-binding activity of 3B was independent for 3B to status of the two viruses in PK-15 cells. PK-15 cells were in- suppress RIG-I–mediated signal transduction. fected with equal amount of r3B-FMDV and r3B-A/E-FMDV, The expression of any of the single copy of 3B inhibited SeV- respectively, the expression of viral RNA, IFN-b, and ISGs were induced IFN-b production (Fig. 2G). This indicated that all of measured at 12 hpi. r3B-A/E-FMDV showed a decreased rep- the three copies of 3B possessed the activity to inhibit IFN-b lication ability compared with r3B-FMDV. However, it induced production. Therefore, we aligned the amino acid sequences of increased IFN-b and ISGs expression (Fig. 11C). These results 3B1, 3B2, as well as 3B3 and identified five conserved regions/ confirmed that the 17A in each copy of FMDV 3B was involved sites among the three copies (Fig. 10E). These regions or sites in suppression of IFN-b production. were then mutated respectively, and their activity to suppress SeV-induced IFN-b production was subsequently evaluated. Discussion Mutation of the GP-1 (the first two residues), GP-2 (the fifth to It is well known that type I IFNs initiate a series of signaling sixth residues), LKV (the 13–15th residues), or E (the last one cascades and then induce the expression of hundreds of ISGs, residue) of the three copies did not abolish the activity of 3B to which play important roles not only in host innate immune re- inhibit IFN-b production. However, mutation of the 17th ala- sponses but also in adaptive immune responses (43, 44). Various nine to glutamicacid (A/E) completely abolished the activity of viruses have been found to subvert the host innate immune re- 3B to inhibit IFN-b production (Fig. 10F). The interaction be- sponse by disruption of the function of the PRRs, adaptor factors, tween single 3B and RIG-I was investigated as well, all of the and critical components of the antiviral pathway for IFN-b sig- three copies of 3B interacted with RIG-I (Fig. 10G). The in- naling (11, 19, 24, 43). Modifications in viral 3B protein of FMDV teraction between the other 3B mutants and RIG-I was further decrease virus yield in different cell lines, suggesting the impor- investigated, which showed that all of the mutants interacted tant role of 3B in FMDV replication (45). Our previous study with RIG-I (Fig. 10H). These results indicated multiple sites in found that FMDV 3B inhibited SeV-triggered IFN-b promoter 3B were involved in 3B–RIG-I interaction. The 3B-A/E mutant activation (29). In this study, we extensively probed the link be- that lost the potential to inhibit IFN-b transcription also bound tween the capacity of FMDV 3B protein to inhibit the function of The Journal of Immunology 11

FIGURE 8. FMDV 3B blocked the interaction of RIG-I with TRIM25. (A) HEK293T cells were cotransfected with 4 mgofMyc– RIG-I and 4 mgofHAvectororHA-TRIM25– expressing plasmids with 0 or 4 mgof FLAG-3B–expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-Myc Ab and detected using anti- Myc, anti-HA, and anti-FLAG Abs, respectively. WCLs were detected using the indicated Abs. (B) The dose-dependent assay of (A) by transfection of 0, 1, 3, or 5 mg of FLAG-3B–expressing plasmids. (C) HEK293T cells were cotransfected with 5 mgofMyc-TRIM25orMyc–RIG-I, and 5 mgofFLAGvectororFLAG-3B– expressing plasmids for 36 h. The cell ly- Downloaded from sates were immunoprecipitated with anti- Myc Ab and detected using anti-Myc and anti-FLAG Abs, respectively. WCLs were detected using the indicated Abs. All the experiments were repeated three times with similar results. WCL, whole-cell lysate. http://www.jimmunol.org/

targets in the host innate immune signaling pathway and its ca- Therefore, RIG-I is the target of FMDV 3B to attenuate host an- pacity to suppress antiviral response. We found that FMDV 3B tiviral response. significantly suppressed SeV-induced IFN-b and NF-kB pathways RIG-I, as a critical cytosolic sensor for IFN-b pathway, plays a signaling, thus, considerably decreasing the expression of IFN-b, critical role in host antiviral response. Upon viral infection, RIG-I ISGs, and proinflammatory cytokines. Further investigation iden- is redistributed and polyubiquitinated to bind the adaptor protein by guest on September 25, 2021 tified that 3B blocked the K63-linked ubiquitination of RIG-I, in MAVS, which initiates the host antiviral immune signaling (41). turn, leading to the decreased interaction of RIG-I and MAVS. Given the importance of RIG-I in antiviral immune signaling, it is

FIGURE 9. Identiﬁcation of the binding regions of RIG-I with FMDV 3B. (A)Sche- matic representation of HA-tagged full-length RIG-I and a series of truncated RIG-I constructs. (B) HEK293T cells were cotransfected with 5 mgofFLAG-3Band5mgofHA vector, HA–RIG-I, HA–RIG-I–CARD, HA–RIG-I–DEAD Helicase, or HA–RIG- I–CTD for 36 h. The cells were lysed and immunoprecipitated with anti-FLAG Ab and detected using anti-FLAG and anti- HA Abs. WCLs were detected using the indicated Abs. All the experiments were repeated three times with similar results. WCL, whole-cell lysate. 12 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 10. The RNA-binding activity of 3B was independent for 3B to suppress RIG-I–mediated immune signaling. (A) Schematic representation of FLAG-tagged wild-type 3B and 3B-Y3A mutant constructs. (B) HEK293T cells were transfected with 0, 75, 150, or 300 ng of FLAG-3B-Y3A–expressing plasmids together with 100 ng of IFN-b promoter–driven luciferase reporter plasmids and 10 ng of the internal control plasmid pRL-TK for 24 h, and the cells were mock-infected or infected with SeV for another 16 h. The luciferase activity was determined by the dual-luciferase assay (left panel). HEK293T cells were transfected with vector or FLAG–3B-Y3A expressing plasmids for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. IFN-b mRNA levels in the collected cells was detected by qPCR (middle panel). The right panel is the dose-dependent assay of the middle panel. (C) HEK293T cells were transfected with vector plasmids or increasing amounts of FLAG-VP2–expressing plasmids for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. IFN-b mRNA levels in the collected cells was detected by qPCR. (D) PK-15 cells were transfected by guest on September 25, 2021 with vector or FLAG-3B-Y3A–expressing plasmids for 24 h, and the cells were then mock-infected or infected with FMDV for another 12 h. The mRNA levels of IFN-b, ISG56, and MX1 in the collected cells was detected by qPCR. (E) The alignment of FMDV 3B1, 3B2, and 3B3 aa sequences. The red box represented the conserved regions or sites among the three nonidentical copies. (F) HEK293T cells were transfected with vector, FLAG-3B, or the indicated 3B mutants expressing plasmids for 24 h, and the cells were then mock-infected or infected with SeV for another 16 h. IFN-b mRNA levels in the collected cells was detected by qPCR. The expression of the 3B protein and 3B mutants were detected by Western blotting. (G) HEK293T cells were cotransfected with 5 mg of Myc–RIG-I and 5 mg of GST vector, GST-3B1, GST-3B2, or GST-3B3 for 36 h. The cells were then lysed and subjected to immunoprecipitation experiments and immunoblotting analysis. (H) HEK293T cells were cotransfected with 5 mg of Myc–RIG-I and 5 mg of FLAG vector, FLAG-3B or the indicated 3B mutants expressing plasmids for 36 h. The cells were then lysed and subjected to immunoprecipitation experiments and immunoblotting analysis. All the experiments were repeated three times with similar results. *p , 0.05 considered significant, **p , 0.01 considered highly significant. n.s., not significant. not surprising that viruses have evolved a variety of strategies that TRIM25 with RIG-I, then resulting in the decreased K63-linked target RIG-I to antagonize IFN response. Many studies have ubiquitination of RIG-I and RIG-I–MAVS complex formation to reported that viral proteins target RIG-I by different mechanisms suppress IFN production and prevent an efficient host immune. to block IFN pathway activation. For example, poliovirus 3C Influenza virus NS1 suppresses the K63-linked ubiquitination of proteinase cleaves RIG-I during viral replication to attenuate host RIG-I by interacting with the E3 ubiquitin ligase TRIM25 and antiviral response (46). Ebola virus VP35 protein binds dsRNA to blocking TRIM25 multimerization (49). This suggests that the prevent the sensing of viral RNA by RIG-I (47). Human respiratory disruption of RIG-I–TRIM25 interaction is a target for different syncytial virus NS2 protein binds to RIG-I to inhibit the RIG-I–MAVS viruses to impair host antiviral response. The DEAD helicase interaction (48). This suggests that RIG-I–mediated activation process domain is involved in RIG-I filament formation and pathway ac- can be disrupted by viral proteins in various ways. tivation. The interaction of 3B with DEAD helicase domain might The N-terminal CARD domain of RIG-I is critical for MAVS also interfere with the function of RIG-I by other unknown binding and downstream signaling (9). In this study, we identified mechanisms. The very recently prominent papers have proposed that FMDV 3B protein impaired the K63-linked ubiquitination of that the E3 ligase RIPLET promotes RIG-I–mediated immune RIG-I to interfere with RIG-I–MAVS complex formation and signaling through both Ub-dependent and -independent manners, antagonize downstream signaling. TRIM25 interacts with the which may be more important than TRIM25 (50, 51). In this CARD domain of RIG-I, and this interaction effectively induces study, we confirmed that 3B inhibited the K63-linked ubiq- the K63-linked ubiquitination of RIG-I, leading to its interaction uitination of RIG-I. Overexpression of TRIM25 promoted the with MAVS (42). FMDV 3B interacted with the CARD domain of K63-linked ubiquitination of RIG-I, and 3B blocked TRIM25- RIG-I and blocked the interaction of the E3 ubiquitin ligase induced K63-linked ubiquitination of RIG-I. In addition, 3B The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 11. The 17A in each copy of FMDV 3B was involved in suppression of IFN-b production. (A) Schematic representation of the recombinant viruses r3B-FMDV (parental virus) and r3B-A/E-FMDV (containing the 3B-A/E instead of 3B). (B) The viral titers of r3B-FMDV and r3B-A/E-FMDV in

BHK-21 cells were determined by the TCID50 assay. (C) PK-15 cells were mock-infected or infected with r3B-FMDVor r3B-A/E-FMDV for 12 h. The viral RNA, IFN-b, ISG56, MX1, and ISG15 mRNA expression levels were measured by qPCR. interacted with both the CARD and DEAD helicase domains. transcripts and protein levels of MAVS, and we did not observe There might be an alternative model that 3B binds RIG-I and the interaction between 3B and MAVS. The involved mecha- keeps it in a “locked” state, which will make RIG-I inaccessible nism will be further investigated. Besides, our data showed for any of E3 ligases including the RIPLET. that FMDV 3B could not completely abolish the SeV-induced A recent study reported that the host zinc-finger protein RLRspathwayactivation(Figs.1,2).However,3B,indeed, ZCCHC3 promotes the K63-linked ubiquitination of RIG-I (52). efficiently inhibited RIG-I function and promoted FMDV Although it remains unknown whether FMDV 3B protein affects replication (Figs. 3, 4). The IFN-b expression could also be the ZCCHC3-mediated K63-linked ubiquitination of RIG-I, the detected in the FMDV-infected RIG-I KO cells. Therefore, we current study identified a new strategy for FMDV to suppress RIG- speculated that other PRRs and pathways might have also been I–mediated immune signaling by inhibiting the activation of RIG- involved in the initiation of host innate immune response during I. Apart from sensing of viral RNA, RIG-I also serves as an ISG FMDV infection. RIG-I KO decreased IFN-b production almost and functions as a direct antiviral effector (53, 54). For example, by 50%, and this still left many of IFNs in the system, which RIG-I promotes the disassembly of the viral polymerase complex may induce substantial expression of ISGs. Multiple antagonistic to suppress influenza virus replication (55). RIG-I counteracts mechanisms are used by different viral proteins during FMDV the interaction between hepatitis B virus polymerase and pre- infection. Some of the viral proteins target the components of genomic RNA to suppress viral replication (56). The direct RLRs pathway, and some of the viral proteins target the JAK- antiviral function of RIG-I in FMDV-infected cells should be STAT pathway or even directly interfere with the functions of further investigated. Whether 3B protein is also involved in at- several ISGs. There might be a lot of synergy among different tenuation of the antiviral function of RIG-I should be exploited. In viral proteins and a viral replication cascade effect. Therefore, this study, we also found that 3B suppressed the adaptor protein the effect of the RIG-I KO on FMDV replication is very signifi- MAVS-mediated ISRE activation. However, 3B did not affect the cant. 3B effectively blocked RIG-I–mediated immune signaling. 14 FMDV 3B PROTEIN BLOCKS RIG-I–MEDIATED IMMUNE SIGNALING

Whether 3B could also target other PRRs or other antiviral pro- 10. Wang, D., L. Fang, K. Li, H. Zhong, J. Fan, C. Ouyang, H. Zhang, E. Duan, R. Luo, Z. Zhang, et al. 2012. Foot-and-mouth disease virus 3C protease cleaves teins remains unknown. NEMO to impair innate immune signaling. J. Virol. 86: 9311–9322. FMDV is distinguished from other members of the Picorna- 11. Wang, D., L. Fang, D. Wei, H. Zhang, R. Luo, H. Chen, K. Li, and S. Xiao. 2014. viridae by the presence of three copies of 3B (2). Therefore, the Hepatitis A virus 3C protease cleaves NEMO to impair induction of beta interferon. J. Virol. 88: 10252–10258. role of 3B in different picornaviruses might not be completely 12. Kato, H., K. Takahasi, and T. Fujita. 2011. RIG-I-like receptors: cytoplasmic same. FMDV 3B protein plays important roles in viral replication. sensors for non-self RNA. Immunol. Rev. 243: 91–98. Deletion of the 3B3 sequence within the full-length infectious 13. Glaser, W., and T. Skern. 2000. 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Supplemental Figure 1. The interaction of FMDV 3B with the components of RIG-I mediated signal pathway. (A-B) HEK293T cells were co-transfected with 5 μg of FLAG-3B and 5 μg of HA vector, HA-RIG-I, HA-MAVS, HA-TBK1, HA-IRF3 or HA-IRF7 expressing plasmids for 36 h. The cells were then lysed and subjected to immunoprecipitation experiments and immunoblotting analysis. (A) The lysates were immunoprecipitated using anti-FLAG antibody and detected using the anti-HA and anti-FLAG antibodies, and the whole cell lysates (WCLs) were detected by the indicated antibodies. (B) The lysates were also immunoprecipitated using anti-HA antibody and detected by the anti-HA and anti-FLAG antibodies, and the WCLs were detected by the indicated antibodies as input.