A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways.

The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.

Impaired interferon response in senecavirus A infection and identification of 3Cpro as an antagonist.

Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.

Picornavirus infection enhances aspartate by the SLC38A8 transporter to promote viral replication.

Foot-and-mouth disease, a class of animal diseases, is caused by foot-and-mouth disease virus (FMDV). The metabolic changes during FMDV infection remain unclear. Here, PK-15 cells, serum, and tonsils infected with FMDV were analyzed by metabolomics. A total of 284 metabolites in cells were significantly changed after FMDV infection, and most of them belong to amino acids and nucleotides. Further studies showed that FMDV infection significantly enhanced aspartate in vitro and in vivo. The amino acid transporter solute carrier family 38 member 8 (SLC38A8) was responsible for FMDV-upregulated aspartate. Enterovirus 71 (EV71) and Seneca Valley virus (SVV) infection also enhanced aspartate by SLC38A8. Aspartate aminotransferase activity was also elevated in FMDV-, EV71-, and SVV-infected cells, which may lead to reversible transition between the TCA cycle and amino acids synthesis. Aspartate and SLC38A8 were essential for FMDV, EV71, and SVV replication in cells. In addition, aspartate and SLC38A8 also promoted FMDV and EV71 replication in mice. Detailed analysis indicated that FMDV infection promoted the transfer of mTOR to lysosome to enhance interaction between mTOR and Rheb, and activated PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication. The mTORC1 signaling pathway was responsible for FMDV-induced SLC38A8 protein expression. For the first time, our data identified metabolic changes during FMDV infection. These data identified a novel mechanism used by FMDV to upregulate aspartate to promote viral replication and will provide new perspectives for developing new preventive strategies.

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-ß production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

The Foot-and-Mouth Disease Virus Lb Protease Cleaves Intracellular Transcription Factors STAT1 and STAT2 to Antagonize IFN-ß-Induced Signaling.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, one of the most highly infectious animal viruses throughout the world. The JAK-STAT signaling pathway is a highly conserved pathway for IFN-ß-induced antiviral gene expression. Previous studies have shown that FMDV can strongly suppress the innate immune response. Moreover, although STAT1 and STAT2 (STAT1/2) have been well established in JAK-STAT signaling-induced antiviral gene expression, whether FMDV proteins inhibit IFN-ß-induced JAK-STAT signaling remains poorly understood. In this study, we described the Lb leader protease (Lbpro) of FMDV as a candidate for inhibiting IFN-ß-induced signaling transduction via directly interacting with STAT1/2. We further showed that Lbpro colocalized with STAT1/2 to inhibit their nuclear translocation. Importantly, Lbpro cleaved STAT1/2 to inhibit IFN-ß-induced signal transduction, whereas the catalytically inactive mutant of LC51A (Lbpro with cysteine substituted with alanine at amino acid residue 51) had no effect on the stability of STAT1/2 proteins. The cleavage of the STAT1/2 proteins was also determined during FMDV infection in vitro. Lbpro could cleave the residues between 252 and 502 aa for STAT1 and the site spanning residues 140 - 150 aa (QQHEIESRIL) for STAT2. The in vivo results showed that Lbpro can cleave STAT1/2 in pigs. Overall, our findings suggest that FMDV Lbpro-mediated targeting of STAT1/2 may reveal a novel mechanism for viral immune evasion.

Knockout of the WD40 domain of ATG16L1 enhances foot and mouth disease virus replication.

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus-host interactions and potential antiviral strategies.

A ferritin-based nanoparticle displaying a neutralizing epitope for foot-and-mouth disease virus (FMDV) confers partial protection in guinea pigs.

BACKGROUND: Foot-and-mouth disease (FMD) is a devastating disease affecting cloven-hoofed animals, that leads to significant economic losses in affected countries and regions. Currently, there is an evident inclination towards the utilization of nanoparticles as powerful platforms for innovative vaccine development. Therefore, this study developed a ferritin-based nanoparticle (FNP) vaccine that displays a neutralizing epitope of foot-and-mouth disease virus (FMDV) VP1 (aa 140-158) on the surface of FNP, and evaluated the immunogenicity and protective efficacy of these FNPs in mouse and guinea pig models to provide a strategy for developing potential FMD vaccines. RESULTS: This study expressed the recombinant proteins Hpf, HPF-NE and HPF-T34E via an E. coli expression system. The results showed that the recombinant proteins Hpf, Hpf-NE and Hpf-T34E could be effectively assembled into nanoparticles. Subsequently, we evaluated the immunogenicity of the Hpf, Hpf-NE and Hpf-T34E proteins in mice, as well as the immunogenicity and protectiveness of the Hpf-T34E protein in guinea pigs. The results of the mouse experiment showed that the immune efficacy in the Hpf-T34E group was greater than the Hpf-NE group. The results from guinea pigs immunized with Hpf-T34E showed that the immune efficacy was largely consistent with the immunogenicity of the FMD inactivated vaccine (IV) and could confer partial protection against FMDV challenge in guinea pigs. CONCLUSIONS: The Hpf-T34E nanoparticles stand out as a superior choice for a subunit vaccine candidate against FMD, offering effective protection in FMDV-infected model animals. FNP-based vaccines exhibit excellent safety and immunogenicity, thus representing a promising strategy for the continued development of highly efficient and safe FMD vaccines.

FMDV 3A Antagonizes the Effect of ANXA1 to Positively Modulate Viral Replication.

The RIG-I-like receptor signaling pathway is crucial for producing type I interferon (IFN-I) against RNA viruses. The present study observed that viral infection increased annexin-A1 (ANXA1) expression, and ANXA1 then promoted RNA virus-induced IFN-I production. Compared to ANXA1 wild-type cells, ANXA1-/- knockout cells showed IFN-ß production decreasing after viral stimulation. RNA virus stimulation induced ANXA1 to regulate IFN-ß production through the TBK1-IRF3 axis but not through the NF-κB axis. ANXA1 also interacted with JAK1 and STAT1 to increase signal transduction induced by IFN-ß or IFN-γ. We assessed the effect of ANXA1 on the replication of foot-and-mouth disease virus (FMDV) and found that ANXA1 inhibits FMDV replication dependent on IFN-I production. FMDV 3A plays critical roles in viral replication and host range. The results showed that FMDV 3A interacts with ANXA1 to inhibit its ability to promote IFN-ß production. We also demonstrated that FMDV 3A inhibits the formation of ANXA1-TBK1 complex. These results indicate that ANXA1 positively regulates RNA virus-stimulated IFN-ß production and FMDV 3A antagonizes ANXA1-promoted IFN-ß production to modulate viral replication. IMPORTANCE FMDV is a pathogen that causes one of the world's most destructive and highly contagious animal diseases. The FMDV 3A protein plays a critical role in viral replication and host range. Although 3A is one of the viral proteins that influences FMDV virulence, its underlying mechanisms remain unclear. ANXA1 is involved in immune activation against pathogens. The present study demonstrated that FMDV increases ANXA1 expression, while ANXA1 inhibits FMDV replication. The results also showed that ANXA1 promotes RNA virus-induced IFN-I production through the IRF3 axis at VISA and TBK1 levels. ANXA1 was also found to interact with JAK1 and STAT1 to strengthen signal transduction induced by IFN-ß and IFN-γ. 3A interacted with ANXA1 to inhibit ANXA1-TBK1 complex formation, thereby antagonizing the inhibitory effect of ANXA1 on FMDV replication. This study helps to elucidate the mechanism underlying the effect of the 3A protein on FMDV replication.

The Insufficient Activation of RIG-I-Like Signaling Pathway Contributes to Highly Efficient Replication of Porcine Picornaviruses in IBRS-2 Cells.

Seneca Valley virus (SVV) or commonly known as senecavirus A, is one of the picornavirus that is associated with vesicular disease and neonatal mortality in swine herds. Our previous study found that SVV replicates extremely faster in porcine Instituto Biologico-Rim Suino-2 (IBRS-2) cells than that in porcine kidney-15 (PK-15) cells. However, the underlying mechanism remains unknown. In this study, we comprehensively compared the expression features between IBRS-2 cells and PK-15 cells in response to SVV infection by an unbiased high-throughput quantitative proteomic analysis. We found that the innate immune response-related pathways were efficiently activated in PK-15 cells but not in IBRS-2 cells during SVV infection. A large amount of interferon (IFN)-stimulated genes were induced in PK-15 cells. In contrast, no IFN-stimulated genes were induced in IBRS-2 cells. Besides, we determined similar results in the two cell lines infected by another porcine picornavirus foot-and-mouth disease virus. Further study demonstrated that the Janus kinase signal transducer and activator of transcription signaling pathway was functioning properly in both IBRS-2 and PK-15 cells. A systematic screening study revealed that the aberrant signal transduction from TANK-binding kinase 1 to IFN regulatory factor 3 in the retinoic acid-inducible gene I-like receptor signaling pathway in IBRS-2 cells was the fundamental cause of the different innate immune response manifestation and different viral replication rate in the two cell lines. Together, our findings determined the different features of IBRS-2 and PK-15 cell lines, which will help for clarification of the pathogenesis of SVV. Besides, identification of the underlying mechanisms will provide new targets and an insight for decreasing the viral clearance rate and probably improve the oncolytic effect by SVV in cancer cells.

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.

Foot-and-Mouth Disease Virus Structural Protein VP1 Destroys the Stability of TPL2 Trimer by Degradation TPL2 to Evade Host Antiviral Immunity.

Tumor progression locus 2 (TPL2) is a serine/threonine kinase that belongs to the mitogen-activated protein 3 kinase (MAP3K) family, and it plays an important role in pathogen infection. The trimer complex of TPL2, p105, and ABIN2 is essential for maintenance of TPL2 steady-state levels and host cell response to pathogens. Foot-and-mouth disease virus (FMDV) is a positive-strand RNA virus of the family Picornaviridae that encodes proteins capable of antagonizing host immune responses to achieve infection. The VP1 protein of FMDV is a multifunctional protein that can bind host cells and induce an immune response as well as cell apoptosis. However, the role and mechanisms of TPL2 in FMDV infection remain unknown. Here, we determined that FMDV infection could inhibit TPL2, p105, and ABIN2 at the transcription and protein levels, while VP1 could only inhibit TPL2, p105 and ABIN2 at protein level. TPL2 inhibited the replication of FMDV in vivo and in vitro, the 268 to 283 amino-acid region in the TPL2 kinase domain was essential for interaction with VP1. Moreover, VP1 promoted K48-linked polyubiquitination of TPL2 and degraded TPL2 by the proteasome pathway. However, VP1-induced degradation of p105 and ABIN2 was independent of proteasome, autophagy, lysosome, and caspase-dependent pathways. Further studies showed that VP1 destroyed the stability of the TPL2-p105-ABIN2 complex. Taken together, these results revealed that VP1 antagonized TPL2-meditated antivirus activity by degrading TPL2 and destroying its complex. These findings may contribute to understand FMDV-host interactions and improve development of a novel vaccine to prevent FMDV infection.Importance Virus-host interactions are critical for virus infection. This study was the first to demonstrate the antiviral effect of host TPL2 during FMDV replication by increasing production of interferons and antiviral cytokines. Both FMDV and VP1 protein can reduce host TPL2, ABIN2 and p105 to destroy TPL2-p105-ABIN2 trimer complex. VP1 interacted with TPL2 and degrade TPL2 via proteasome pathway to repress TPL2-mediated antivirus activity. This study provided new insights into FMDV immune evasion mechanisms, elucidating new informations regarding FMDV counteraction of host antivirus activity.

Profiling and functional analysis of differentially expressed circular RNAs identified in foot-and-mouth disease virus infected PK-15 cells.

Circular RNAs (circRNAs) are a new type of endogenous noncoding RNA that exhibit a variety of biological functions. However, it is not clear whether they are involved in foot-and-mouth disease virus (FMDV) infection and host response. In this study, we established circRNA expression profiles in FMDV-infected PK-15 cells using RNA-seq (RNA-sequencing) technology analysis. The biological function of the differentially expressed circRNAs was determined by protein interaction network, Gene Ontology (GO), and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment. We found 1100 differentially expressed circRNAs (675 downregulated and 425 upregulated) which were involved in various biological processes such as protein ubiquitination modification, cell cycle regulation, RNA transport, and autophagy. We also found that circRNAs identified after FMDV infection may be involved in the host cell immune response. RNA-Seq results were validated by circRNAs qRT-PCR. In this study, we analyzed for the first time circRNAs expression profile and the biological function of these genes after FMDV infection of host cells. The results provide new insights into the interactions between FMDV and host cells.

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.

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 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-ß, 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 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.

Development and application of a colloidal-gold dual immunochromatography strip for detecting African swine fever virus antibodies.

African swine fever (ASF) is an acute and highly contagious infectious disease caused by the African swine fever virus (ASFV). Currently, there is no vaccine against ASF worldwide, and no effective treatment measures are available. For this reason, developing a simple, rapid, specific, and sensitive serological detection method for ASFV antibodies is crucial for the prevention and control of ASF. In this study, a 1:1 mixture of gold-labeled p30 and p72 probes was used as the gold-labeled antigen. The p30 and p72 proteins and their monoclonal antibodies were coated on a nitrocellulose membrane (NC) as a test (T) line and control (C) line, respectively. A colloidal-gold dual immunochromatography strip (ICS) for ASFV p30 and p72 protein antibodies was established. The results showed that the colloidal-gold dual ICS could specifically detect ASFV antibodies within 5-10 min. There was no cross-reaction after testing healthy pig serum; porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease type A virus (FMDV-A), foot-and-mouth disease type O virus (FMDV-O), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) positive sera. A positive result was obtained only for the positive control P1. The sensitivity of the test strips was 1:256, which was equivalent to that of commercially ELISA kits. Their coincidence rate with the two commercial ASFV ELISA antibodies detection kits was higher than 98%. The test strips were stably stored at 18-25 °C and 4 °C for 4 and 6 months, respectively. The colloidal-gold dual ICS prepared in this study had high sensitivity and specificity and were characterized by rapid detection, simple operation, and easy interpretation of results. Therefore, they are of great significance to diagnose, prevent, and control African swine fever. KEY POINTS: • We establish an antibody detection that is quick and can monitor an ASF infection. • We observe changes in two protein antibodies to dynamically monitor ASF infection. • We use diversified detection on a single test strip to detect both antibodies.

Foot-and-Mouth Disease Virus 3A Protein Causes Upregulation of Autophagy-Related Protein LRRC25 To Inhibit the G3BP1-Mediated RIG-Like Helicase-Signaling Pathway.

Foot-and-mouth disease virus (FMDV) is one of the most notorious pathogens in the global livestock industry. To establish an infection, FMDV needs to counteract host antiviral responses. Several studies have shown how FMDV suppresses the type I interferon (IFN) response; however, whether FMDV modulates the integrated autophagy and innate immunity remains largely unknown. Here, the porcine Ras-GAP SH3-binding protein 1 (G3BP1) was shown to promote the retinoic acid-inducible gene I (RIG-I)-like helicase (RLH) signaling by upregulating the expression of RIG-I and melanoma differentiation-associated gene 5 (MDA5). FMDV nonstructural protein 3A interacted with G3BP1 to inhibit G3BP1 expression and G3BP1-mediated RLH signaling by upregulating the expression of autophagy-related protein LRRC25. In addition, 3A proteins of other picornaviruses, including Seneca Valley virus (SVV) 3A, enterovirus 71 (EV71) 3A, and encephalomyocarditis virus (EMCV) 3A, also showed similar actions. Taking the data together, we elucidated, for the first time, a novel mechanism by which FMDV has evolved to inhibit IFN signaling and counteract host innate antiviral responses by autophagy.IMPORTANCE We show that foot-and-mouth disease virus (FMDV) 3A inhibits retinoic acid-inducible gene I (RIG-I)-like helicase signaling by degrading G3BP1 protein. Furthermore, FMDV 3A reduces G3BP1 by upregulating the expression of autophagy-related protein LRRC25. Additionally, other picornavirus 3A proteins, such as Seneca Valley virus (SVV) 3A, enterovirus 71 (EV71) 3A, and encephalomyocarditis virus (EMCV) 3A, also degrade G3BP1 by upregulating LRRC25 expression. This study will help us improve the design of current vaccines and aid the development of novel control strategies to combat FMD.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Interacts with Host Ribosomal Protein SA To Maintain Activation of the MAPK Signal Pathway and Promote Virus Replication.

Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting that RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated the mitogen-activated protein kinase (MAPK) pathway and demonstrated that MAPK pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPK pathway activation during FMDV infection and displayed an antiviral function. FMDV VP1 interacted with RPSA to abrogate the RPSA-mediated suppressive role in MAPK pathway activation. Together, our study indicated that MAPK pathway activation was required for FMDV replication and that host RPSA played a negatively regulatory role on MAPK pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPK signal pathway.IMPORTANCE Identification of virus-cell interactions is essential for making strategies to limit virus replication and refine the models of virus replication. This study demonstrated that FMDV utilized the MAPK pathway for viral replication. The host RPSA protein inhibited FMDV replication by suppressing the activation of the MAPK pathway during FMDV infection. FMDV VP1 bound to RPSA to repress the RPSA-mediated regulatory effect on MAPK pathway activation. This study revealed an important implication of the MAPK pathway for FMDV infection and identified a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and maintain the activation of the MAPK pathway, elucidating new information regarding the signal reprogramming of host cells by FMDV.

Genetic Determinants of Altered Virulence of Type O Foot-and-Mouth Disease Virus.

Under different circumstances, the alteration of several viral genes may give an evolutionary advantage to the virus to maintain its prevalence in nature. In this study, a 70-nucleotide deletion in the small fragment (S fragment) of the viral 5'-untranslated region (5'-UTR) together with one amino acid insertion in the leader protein (Lpro) that naturally occurred in several serotype O foot-and-mouth disease virus (FMDV) strains in China was identified. The properties of two field serotype O FMDV strains, with or without the 70-nucleotide deletion in the S fragment and the amino acid insertion in Lpro, were compared in vitro and in vivo Clinical manifestations of FMD were clearly observed in cattle and pigs infected by the virus without the mutations. However, the virus with the mentioned mutations caused FMD outcomes only in pigs, not in cattle. To determine the role of the 70-nucleotide deletion in the S fragment and the single amino acid insertion in Lpro in the pathogenicity and host range of FMDV, four recombinant viruses, with complete genomes and a 70-nucleotide deletion in the S fragment, a single amino acid insertion in Lpro, or both mutations, were constructed and rescued. It showed that deletion of 70 nucleotides in the S fragment or insertion of one amino acid (leucine) at position 10 of Lpro partly decreased the viral pathogenicity of Mya-98 lineage virus in cattle and pigs. However, the virus with dual mutations caused clinical disease only in pigs, not in cattle. This suggested that the S fragment and Lpro are significantly associated with the virulence and host specificity of FMDV. The naturally occurring dual mutation in the S fragment and Lpro is a novel determinant of viral pathogenicity and host range for serotype O FMDV.IMPORTANCE FMD is probably the most important livestock disease in the world due to the severe economic consequences caused. The alteration of several viral genes may give the virus selective advantage to maintain its prevalence in nature. Here, we identified that a 70-nucleotide deletion in the S fragment combined with a single leucine insertion in the leader protein (Lpro) is a novel determinant of restricted growth on bovine cells, which significantly contributes to the altered virulence of serotype O FMDV in cattle. A synergistic and additive effect of the 70-nucleotide deletion in the S fragment and the single leucine insertion in Lpro on the virulence and host specificity of the virus was determined. These results will benefit efforts to understand the vial pathogenicity mechanism and molecular characteristics of FMDV.

Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion.

The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious and devastating viral disease of domestic and wild cloven-hoofed animals. FMD affects livestock and animal products' national and international trade, causing severe economic losses and social consequences. Currently, inactivated vaccines play a vital role in FMD control, but they have several limitations. The genetic code expansion technology provides powerful strategies for generating premature termination codon (PTC)-harbouring virus as a live but replication-incompetent viral vaccine. However, this technology has not been explored for the design and development of new FMD vaccines. In this study, we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery. We demonstrated that the transgenic cells stably integrated the orthogonal pyltRNA/pylRS pair into the genome and enabled efficient, homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells. Next, we constructed 129 single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance, location, and potential functions of those mutated sites. Amber stop codons individually substituted the selected amino acid codons in four viral proteins (3D, L, VP1, and VP4) of FMDV. We successfully rescued PTC-FMDV mutants, but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication. Our findings highlight that the genetic code expansion technology for the generation of PTC-FMD vaccines needs to be further improved and that the genetic stability of amber codons during the packaging and replication of FMDV is a concern.

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.