Foot-and-mouth disease virus (FMDV) negatively regulates ZFP36 protein expression to alleviate its antiviral activity.

Zinc finger protein 36 (ZFP36) is a key regulator of inflammatory and cytokine production. However, the interplay between swine zinc-finger protein 36 (sZFP36) and foot-and-mouth disease virus (FMDV) has not yet been reported. Here, we demonstrate that overexpression of sZFP36 restricted FMDV replication, while the knockdown of sZFP36 facilitated FMDV replication. To subvert the antagonism of sZFP36, FMDV decreased sZFP36 protein expression through its non-structural protein 3C protease (3Cpro). Our results also suggested that 3Cpro-mediated sZFP36 degradation was dependent on its protease activity. Further investigation revealed that both N-terminal and C-terminal-sZFP36 could be degraded by FMDV and FMDV 3Cpro. In addition, both N-terminal and C-terminal-sZFP36 decreased FMDV replication. Moreover, sZFP36 promotes the degradation of FMDV structural proteins VP3 and VP4 via the CCCH-type zinc finger and NES domains of sZFP36. Together, our results confirm that sZFP36 is a host restriction factor that negatively regulates FMDV replication.IMPORTANCEFoot-and-mouth disease (FMD) is an infectious disease of animals caused by the pathogen foot-and-mouth disease virus (FMDV). FMD is difficult to prevent and control because there is no cross-protection between its serotypes. Thus, we designed this study to investigate virus-host interactions. We first demonstrate that swine zinc-finger protein 36 (sZFP36) impaired FMDV structural proteins VP3 and VP4 to suppress viral replication. To subvert the antagonism of sZFP36, FMDV and FMDV 3Cpro downregulate sZFP36 expression to facilitate FMDV replication. Taken together, the present study reveals a previously unrecognized antiviral mechanism for ZFP36 and elucidates the role of FMDV in counteracting host antiviral activity.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

KIF5B-mediated internalization of FMDV promotes virus infection.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, which is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Antagonizes Type I Interferon Signaling via Degradation of Histone Deacetylase 5.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease of cloven-hoofed animals that hampers trade and production. To ensure effective infection, the foot-and-mouth disease virus (FMDV) evades host antiviral pathways in different ways. Although the effect of histone deacetylase 5 (HDAC5) on the innate immune response has previously been documented, the precise molecular mechanism underlying HDAC5-mediated FMDV infection is not yet clearly understood. In this study, we found that silencing or knockout of HDAC5 promoted FMDV replication, whereas HDAC5 overexpression significantly inhibited FMDV propagation. IFN-ß and IFN-stimulated response element (ISRE) activity was strongly activated through the overexpression of HDAC5. The silencing and knockout of HDAC5 led to an increase in viral replication, which was evident by decreased IFN-ß, ISG15, and ISG56 production, as well as a noticeable reduction in IRF3 phosphorylation. Moreover, the results showed that the FMDV capsid protein VP1 targets HDAC5 and facilitates its degradation via the proteasomal pathway. In conclusion, this study highlights that HDAC5 acts as a positive modulator of IFN-ß production during viral infection, while FMDV capsid protein VP1 antagonizes the HDAC5-mediated antiviral immune response by degrading HDAC5 to facilitate viral replication.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication.

Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host's antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-ß promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection.Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-ß:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2.

The role of Type I interferons in the pathogenesis of foot-and-mouth disease virus in cattle: A mathematical modelling analysis.

Type I interferons (IFN) are the first line of immune response against infection. In this study, we explore the interaction between Type I IFN and foot-and-mouth disease virus (FMDV), focusing on the effect of this interaction on epithelial cell death. While several mathematical models have explored the interaction between interferon and viruses at a systemic level, with most of the work undertaken on influenza and hepatitis C, these cannot investigate why a virus such as FMDV causes extensive cell death in some epithelial tissues leading to the development of lesions, while other infected epithelial tissues exhibit negligible cell death. Our study shows how a model that includes epithelial tissue structure can explain the development of lesions in some tissues and their absence in others. Furthermore, we show how the site of viral entry in an epithelial tissue, the viral replication rate, IFN production, suppression of viral replication by IFN and IFN release by live cells, all have a major impact on results.

Foot-and-Mouth Disease Virus VP3 Protein Acts as a Critical Proinflammatory Factor by Promoting Toll-Like Receptor 4-Mediated Signaling.

Foot-and-mouth disease virus (FMDV) infection in cloven-hoofed animals causes severe inflammatory symptoms, including blisters on the oral mucosa, hoof, and breast; however, the molecular mechanism underlying the inflammatory response is unclear. In this study, we provide the first evidence that the FMDV protein VP3 activates lipopolysaccharide-triggered Toll-like receptor 4 (TLR4) signaling. FMDV VP3 increased the expression of TLR4 by downregulating the expression of the lysozyme-related protein Rab7b. Additionally, Rab7b can interact with VP3 to promote the replication of FMDV. Our findings suggested that VP3 regulates the Rab7b-TLR4 axis to mediate the inflammatory response to FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) infection causes a severe inflammatory response in cloven-hoofed animals, such as pigs, cattle, and sheep, with typical clinical manifestations of high fever, numerous blisters on the oral mucosa, hoof, and breast, as well as myocarditis (tigroid heart). However, the mechanism underlying the inflammatory response caused by FMDV is enigmatic. In this study, we identified the VP3 protein of FMDV as an important proinflammatory factor. Mechanistically, VP3 interacted with TLR4 to promote TLR4 expression by inhibiting the expression of the lysozyme-related protein Rab7b. Our findings suggest that FMDV VP3 is a major proinflammatory factor in FMDV-infected hosts.

Ribosomal Protein L13 Participates in Innate Immune Response Induced by Foot-and-Mouth Disease Virus.

In addition to ribosomal protein synthesis and protein translation, ribosomal proteins also participate in tumorigenesis and tumor progression, immune responses, and viral replication. Here, we show that ribosomal protein L13 (RPL13) participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication. The overexpression of RPL13 promoted the induction and activation of the promoters of the nuclear factor-κB (NF-κB) and interferon-ß (IFN-ß) genes, and the expression and protein secretion of the antiviral factor IFN-ß and proinflammatory cytokine interleukin-6 (IL-6). The knockdown of RPL13 had the opposite effects. We also found that the FMDV 3Cpro protease interacts with RPL13, and that its activity reduces the expression of RPL13, thus antagonizing the RPL13-mediated antiviral activity. This study extends our knowledge of the extraribosomal functions of ribosomal proteins and provides new scientific information on cellular antiviral defenses and virus-antagonizing mechanisms.

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire.

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

The DDX23 Negatively Regulates Translation and Replication of Foot-and-Mouth Disease Virus and Is Degraded by 3C Proteinase.

DEAD-box helicase 23 (DDX23) is a host nuclear helicase, which is a part of the spliceosomal complex and involved in pre-mRNA splicing. To investigate whether DDX23, an internal ribosomal entry sites transacting factor (ITAF) affects foot-and-mouth disease virus (FMDV) replication and translation through internal ribosome entry site (IRES)-dependent manner. For this, we utilized a pull-down assay, Western blotting, quantitative real-time PCR, confocal microscopy, overexpression and small interfering RNA knockdown, as well as the median tissue culture infective dose. Our findings showed that FMDV infection inhibited DDX23 expression and the overexpression of DDX23 reduced viral replication, however, CRISPR Cas9 knockout/small interfering RNA knockdown increased FMDV replication. FMDV IRES domain III and IV interacted with DDX23, whereas DDX23 interacted with FMDV 3C proteinase and significantly degraded. The enzymatic activity of FMDV 3C proteinase degraded DDX23, whereas FMDV degraded DDX23 via the lysosomal pathway. Additionally, IRES-driven translation was suppressed in DDX23-overexpressing cells, and was enhanced in DDX23 knocked down. Collectively, our results demonstrated that DDX23 negatively affects FMDV IRES-dependent translation, which could be a useful target for the design of antiviral drugs.

MiR-4334-5p Facilitates Foot and Mouth Disease Virus Propagation by Suppressing Interferon Pathways via Direct Targeting ID1.

Emerging evidence indicates that the host microRNAs (miRNAs) are important intracellular regulators and play pivotal roles in intricate host-pathogen interaction networks. In our previous studies, ssc-microRNA-4334-5p (miR-4334-5p) was identified as a differentially expressed miRNA in microarray-based miRNAs profiling experiment, but whether miR-4334-5p regulates foot and mouth disease virus (FMDV) propagation is less understood. Here, we demonstrated that miR-4334-5p expression level was up-regulated shortly after FMDV infection, transfection of miR-4334-5p mimics promoted, while inhibitor transfection suppressed FMDV replication correspondingly. Further bioinformatic analysis and experimental study suggested ID1 was the direct target of miR-4334-5p, suppressing FMDV replication by regulating interferon (IFN) pathways. These findings shed light on microRNAs-ID1-interferon axis in regulating FMDV replication.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/ß) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/ß gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/ß gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692-694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVß6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity to determine their relative contributions to the reduction of IFN-α/ß gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-ß gene transcription.

NLRP3 inflammasome activation by Foot-and-mouth disease virus infection mainly induced by viral RNA and non-structural protein 2B.

Foot-and-mouth disease virus (FMDV) is a positive-strand RNA virus of the family Picornaviridae. Early studies show that some viruses of Picornaviridae, such as EMCV and EV71, induce NLRP3 inflammasome activation. Our current study demonstrates that FMDV induces the secretion of caspase-1 and interleukin 1 beta (IL-1ß), as well as activates the NLRP3 inflammasome in a dose- and time-dependent manner. Meanwhile, NLRP3 inflammasome can suppress FMDV replication during virus infection. Both FMDV RNA and viroporin 2B stimulate NLRP3 inflammasome activation. FMDV RNA triggers NLRP3 inflammasome through p-NF-κB/p65 pathway not dependent on RIG-I inflammasome. FMDV 2B activates NLRP3 inflammasome through elevation of intracellular ion, but not dependent on mitochondrial reactive oxygen species (ROS) and lysosomal cathepsin B. It further demonstrates that 2B viroporin activates NLRP3 inflammasome and induces IL-1ß in mice, which enhances the specific immune response against FMDV as an ideal self-adjuvant for FMD VLPs vaccine in guinea pigs. The results reveal a series of regulations between NLRP3 inflammasome complex and FMDV. Amino acids 140-145 of 2B is essential for forming an ion channel. By mutating the amino acid and changing the hydrophobic properties, the helical transmembrane region of the viroporin 2B is altered, so that the 2B is insufficient to trigger the activation of NLRP3 inflammasome. This study demonstrates the functions of FMDV RNA and 2B viroporin activate NLRP3 inflammasome and provides some useful information for the development of FMD vaccine self-adjuvant, which is also helpful for the establishment of effective prevention strategies by targeting NLRP3 inflammasome.

Mincle and STING-Stimulating Adjuvants Elicit Robust Cellular Immunity and Drive Long-Lasting Memory Responses in a Foot-and-Mouth Disease Vaccine.

Conventional foot-and-mouth disease (FMD) vaccines exhibit several limitations, such as the slow induction of antibodies, short-term persistence of antibody titers, as well as low vaccine efficacy and safety, in pigs. Despite the importance of cellular immune response in host defense at the early stages of foot-and-mouth disease virus (FMDV) infection, most FMD vaccines focus on humoral immune response. Antibody response alone is insufficient to provide full protection against FMDV infection; cellular immunity is also required. Therefore, it is necessary to design a strategy for developing a novel FMD vaccine that induces a more potent, cellular immune response and a long-lasting humoral immune response that is also safe. Previously, we demonstrated the potential of various pattern recognition receptor (PRR) ligands and cytokines as adjuvants for the FMD vaccine. Based on these results, we investigated PRR ligands and cytokines adjuvant-mediated memory response in mice. Additionally, we also investigated cellular immune response in peripheral blood mononuclear cells (PBMCs) isolated from cattle and pigs. We further evaluated target-specific adjuvants, including Mincle, STING, TLR-7/8, and Dectin-1/2 ligand, for their role in generating ligand-mediated and long-lasting memory responses in cattle and pigs. The combination of Mincle and STING-stimulating ligands, such as trehalose-6, 6'dibehenate (TDB), and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), induced high levels of antigen-specific and virus-neutralizing antibody titers at the early stages of vaccination and maintained a long-lasting immune memory response in pigs. These findings are expected to provide important clues for the development of a robust FMD vaccine that stimulates both cellular and humoral immune responses, which would elicit a long-lasting, effective immune response, and address the limitations seen in the current FMD vaccine.

Basal Level p53 Suppresses Antiviral Immunity against Foot-and-Mouth Disease Virus.

Tumor suppressor protein p53 (p53) is a master transcription factor that plays key roles in cell cycle arrest, apoptosis, senescence, and metabolism, as well as regulation of innate immunity during virus infection. In order to facilitate their replication and spreading, viruses have evolved to manipulate p53 function through different strategies, with some requiring active p53 while others demand reduction/inhibition of p53 activity. However, there are no clear-cut reports about the roles of p53 during the infection of foot-and-mouth disease virus (FMDV), the causative agent of a highly contagious foot-and-mouth disease (FMD) of cloven-hoofed animals. Here we showed that p53 level was dynamically regulated during FMDV infection, being degraded at the early infection stage but recovered to the basal level at the late stage. Cells depleted of p53 showed inhibited FMDV replication and enhanced expression of the immune-related genes, whereas overexpression of p53 didn't affect the viral replication. Viral challenge assay with p53 knockout mice obtained similar results, with viral load decreased, histopathological changes alleviated, and lifespan extended in the p53 knockout mice. Together, these data demonstrate that basal level p53 is required for efficient FMDV replication by suppressing the innate immunity.

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication.

Foot-and-mouth disease virus (FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1 (DDX1), which interacted with FMDV 3D. The DDX1-3D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay (IFA) in porcine kidney 15 (PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-ß activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.

Foot-and-Mouth Disease Virus Antagonizes NOD2-Mediated Antiviral Effects by Inhibiting NOD2 Protein Expression.

The role of nucleotide-binding oligomerization domain 2 (NOD2) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that FMDV infection activated NOD2-mediated beta interferon (IFN-ß) and nuclear factor-κB (NF-ĸB) signaling pathways. NOD2 inhibited FMDV replication in the infected cells. FMDV infection triggered NOD2 transcription, while it reduced the abundance of NOD2 protein. Our results revealed that FMDV 2B, 2C, and 3C proteinase (3Cpro) were responsible for the decrease in NOD2 protein levels. 3Cpro is a viral proteinase that can cleave multiple host proteins and limit protein synthesis. Our previous studies determined that FMDV 2B suppressed protein expression of RIG-I and LGP2. Here, we found that 3Cpro and 2B also decreased NOD2 expression. However, this is the first report that 2C induced the reduction of NOD2 protein levels. We determined that both 2B- and 2C-induced decreases in NOD2 were independent of the cleavage of host eukaryotic translation initiation factor 4 gamma (eIF4G), induction of cellular apoptosis, or proteasome, lysosome, and caspase pathways. The interactions between NOD2 and 2B or 2C were observed in the context of viral infection. The carboxyl-terminal amino acids 105 to 114 and 135 to 144 of 2B were essential for the reduction of NOD2, while the residues 105 to 114 were required for the interaction. Amino acids 116 to 260 of the carboxyl terminus of 2C were essential for the interaction, while truncated 2C mutants did not reduce NOD2. These data suggested novel antagonistic mechanisms of FMDV that were mediated by 2B, 2C, and 3Cpro proteins.IMPORTANCE NOD2 was identified as a cytoplasmic viral pattern recognition receptor in 2009. Subsequently, many viruses were reported to activate NOD2-mediated signaling pathways. This study demonstrated that FMDV infection activated NOD2-mediated IFN-ß and NF-ĸB signaling pathways. Host cells have developed multiple strategies against viral infection; however, viruses have evolved many strategies to escape host defenses. FMDV has evolved multiple mechanisms to inhibit host type I IFN production. Here, we showed that NOD2 suppressed FMDV replication during viral infection. FMDV 2B, 2C, and 3Cpro decreased NOD2 protein expression by different mechanisms to promote viral replication. This study provided new insight into the immune evasion mechanisms mediated by FMDV and identified 2B, 2C, and 3Cpro as antagonistic factors for FMDV to evade host antiviral responses.

The Pseudoknot Region of the 5' Untranslated Region Is a Determinant of Viral Tropism and Virulence of Foot-and-Mouth Disease Virus.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is characterized by genetic instability and different antigenic properties. The nonstructural protein 3A is a primary determinant of the tropism and virulence of Cathay topotype FMDVs. However, several other determinants are also speculated to be involved in viral tropism and virulence. Deletion of 43 nucleotides (nt) in the pseudoknot (PK) region of the 5' untranslated region (UTR) has been found to coexist with the identified 3A deletion in Cathay topotype FMDV genomes. In this study, we isolated an O/ME-SA/PanAsia lineage FMDV strain, O/GD/CHA/2015, that includes an 86-nt deletion in the PK region and shows a porcinophilic phenotype. To investigate the potential role of the PK region in viral pathogenicity, we generated a recombinant FMDV strain with an incomplete PK region and compared its virulence and pathogenesis to the intact FMDV strain in swine and bovines. Deletion of the 86 nt in the PKs had no major effects on the pathogenicity of the virus in swine but significantly attenuated its ability to infect bovine cells and cattle, indicating that the PK region is a newly discovered determinant of viral tropism and virulence. The role of the 43-nt deletion existing in the Cathay topotype FMDV was also investigated by evaluating the infection properties of genetically engineered viruses. Consistently, the 43-nt deletion in the PK region significantly decreased the pathogenicity of the virus in bovines. Overall, our findings suggest that the PK region deletion occurred naturally in the FMDV genome and that the PK region is highly associated with viral host range and functions as a novel determinant for FMDV pathogenesis.IMPORTANCE This study demonstrates that the deletion in the PK region occurred naturally in the FMDV genome. The isolated O/ME-SA/PanAsia lineage FMDV with an 86-nt deletion in the PK region showed a pig-adapted characteristic that could cause clinical signs in swine but not bovines. Compared to the wild-type FMDV strain, which possesses full infection capacity in both swine and bovines, the recombinant virus with the 86-nt deletion in the PK region is deficient in causing disease in bovines. Deletion of the previously reported 43 nt in the PK region also led to significantly decreased pathogenicity of FMDV in bovines. This study indicates that the PK region is a novel determinant of the tropism and virulence of FMDV.

Deciphering foot-and-mouth disease (FMD) virus-host tropism.

The pattern of interactions between foot and mouth disease (FMD) viral protein 1 (VP1) with susceptible and resistant host integrins were deciphered. The putative effect of site-directed mutation on alteration of interaction is illustrated using predicted and validated 3D structures of VP1, mutated VP1 and integrins of Bos taurus, Gallus and Canis. Strong interactions were observed between FMDV-VP1 protein motifs at conserved tripeptide, Arg-Gly-Asp 143RGD145 and at domain 676SIPLQ680 in alpha-integrin of B. taurus. Notably, in-silico site-directed mutation in FMDV-VP1 protein led to complete loss of interaction between FMD-VP1 protein and B. taurus integrin, which confirmed the active role of arginine-glycine-aspartic acid (RGD) domain. Interestingly, in-vitro analysis demonstrates the persistence of the putative tropism site 'SIPLQ' in different cattle breeds undertaken. Thus, the attempt to decipher the tropism of FMDV at host receptor level interaction might be useful for future FMD control strategies through development of mimetic marker vaccines and/or host receptor manipulations. Communicated by Ramaswamy H. Sarma.

Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation.

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (Lpro) or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV Lpro interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2α, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously, we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.