Foot-and-mouth Disease Virus 2B Research Articles

Foot-and-mouth disease virus non-structural protein 2B downregulates the RLR signaling pathway via degradation of RIG-I and MDA5.

Foot-and-mouth disease virus (FMDV) is a single-stranded, positive-sense RNA virus containing at least 13 proteins. Many of these proteins show immune modulation capabilities. As a non-structural protein of the FMDV, 2B is involved in the rearrangement of the host cell membranes and the disruption of the host secretory pathway as a viroporin. Previous studies have also shown that FMDV 2B plays a role in the modulation of host type-I interferon (IFN) responses through the inhibition of expression of RIG-I and MDA5, key cytosolic sensors of the type-I IFN signaling. However, the exact molecular mechanism is poorly understood. Here, we demonstrated that FMDV 2B modulates host IFN signal pathway by the degradation of RIG-I and MDA5. FMDV 2B targeted the RIG-I for ubiquitination and proteasomal degradation by recruiting E3 ubiquitin ligase ring finger protein 125 (RNF125) and also targeted MDA5 for apoptosis-induced caspase-3- and caspase-8-dependent degradation. Ultimately, FMDV 2B significantly inhibited RNA virus-induced IFN-β production. Importantly, we identified that the C-terminal amino acids 126-154 of FMDV 2B are essential for 2B-mediated degradation of the RIG-I and MDA5. Collectively, these results provide a clearer understanding of the specific molecular mechanisms used by FMDV 2B to inhibit the IFN responses and a rational approach to virus attenuation for future vaccine development.

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

Foot-and-Mouth Disease Virus Inhibits RIP2 Protein Expression to Promote Viral Replication.

Receptors interaction protein 2 (RIP2) is a specific adaptor molecule in the downstream of NOD2. The role of RIP2 during foot-and-mouth disease virus (FMDV) infection remains unknown. Here, our results showed that RIP2 inhibited FMDV replication and played an important role in the activation of IFN-β and NF-ĸB signal pathways during FMDV infection. FMDV infection triggered RIP2 transcription, while it reduced the expression of RIP2 protein. Detailed analysis showed that FMDV 2B, 2C, 3Cpro, and Lpro proteins were responsible for inducing the reduction of RIP2 protein. 3Cpro and Lpro are viral proteinases that can induce the cleavage or reduction of many host proteins and block host protein synthesis. The carboxyl terminal 105-114 and 135-144 regions of 2B were essential for reduction of RIP2. Our results also showed that the N terminal 1-61 region of 2C were essential for the reduction of RIP2. The 2C-induced reduction of RIP2 was dependent on inducing the reduction of poly(A)-binding protein 1 (PABPC1). The interaction between RIP2 and 2C was observed in the context of viral infection, and the residues 1-61 were required for the interaction. These data clarify novel mechanisms of reduction of RIP2 mediated by FMDV.

DDX56 cooperates with FMDV 3A to enhance FMDV replication by inhibiting the phosphorylation of IRF3

The components of foot-and-mouth disease virus (FMDV) interact with host cellular proteins to promote self-replication and evade the host immune response. Previous studies have shown that FMDV 3A, 2C and 2B proteins interact with host cellular proteins involved in FMDV replication. However, whether the other host proteins have an impact on FMDV replication is less understood. In this study, we identified DDX56 as a positive regulator of FMDV replication. DDX56 overexpression increased FMDV replication, whereas DDX56 knockdown had the opposite effect. DDX56 interacted and cooperated with FMDV 3A to inhibit the type I interferon by reducing the phosphorylation of IRF3. Moreover, the D166 site of DDX56 played a role in increasing FMDV replication and cooperating with FMDV 3A to inhibit the phosphorylation of IRF3. Additionally, knockdown of DDX56 or FMDV 3A results also showed that DDX56 cooperated with FMDV 3A to inhibit the phosphorylation of IRF3. These results suggest that the interaction between FMDV 3A protein and the host protein DDX56 is critical for FMDV replication.

New approaches for post-vaccination surveillance of foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a contagious and important transboundary disease of cloven-hoofed animals and ruminants. In ruminants, an animal is considered as a foot-and-mouth disease virus (FMDV) carrier if a live FMDV/FMDV RNA is obtained from the oro-pharyngeal fluid (OPF) beyond 28 days after infection. These carrier animals may pose a risk for causing outbreaks in healthy animals. Moreover, it is important to conduct serosurveillance to know the virus circulation. In the present study, an ELISA was developed using field samples to detect FMDV specific secretory IgA antibodies. These samples were also tested for the presence of FMDV RNA using quantitative real-time PCR (qRT-PCR). It was found that more carrier animals were detected by IgA ELISA in comparison to qRT-PCR. Thus, IgA ELISA is an important tool to detect FMD carriers. An ELISA based on detection of antibodies against FMDV 2B non-structural protein (NSP) was also used to confirm the results obtained from screening of 3AB3 NSP ELISA. These two new approaches (IgA ELISA and 2B ELISA) form important tools for detection of carriers and virus circulation, respectively, during FMD eradication program. Keywords: foot-and-mouth disease virus; carriers; IgA; 2B non-structural protein; 3AB3 non-structural protein.

Foot-and-mouth disease virus non-structural protein 2B negatively regulates the RLR-mediated IFN-β induction

Foot-and-mouth disease virus (FMDV) is the causative agent of Foot-and-mouth disease (FMD), which is an acute and highly contagious disease affecting pigs, cattle and other cloven-hoofed animals. Several studies have shown that FMDV has evolved multiple strategies to evade the host innate immune response, but the underlying mechanisms for immune evasion are still not fully understood. In the current research, we have demonstrated that FMDV utilizes its non-structural protein 2B to sabotage the host immune response. Over-expression of the FMDV 2B inhibited Poly(I:C)-induced or SeV-triggered up-regulation of IFN-β, IL-6 as well as ISG15. When HEK293T cells were transfected with FMDV 2B, the phosphorylation of TBK1 and IRF3 was inhibited. Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-I and MDA5. Moreover, FMDV 2B also inhibited the expression of the RIG-I and MDA5. Thus, FMDV 2B negatively regulates the RLR-mediated IFN-β induction by targeting RIG-I and MDA5.

Foot-and-mouth disease virus induces lysosomal degradation of NME1 to impair p53-regulated interferon-inducible antiviral genes expression

Nucleoside diphosphate kinase 1 (NME1) is well-known as a tumor suppressor that regulates p53 function to prevent cancer metastasis and progression. However, the role of NME1 in virus-infected cells remains unknown. Here, we showed that NME1 suppresses viral replication in foot-and-mouth disease virus (FMDV)-infected cells. NME1-enhanced p53-mediated transcriptional activity and induction of interferon-inducible antiviral genes expression. FMDV infection decreased NME1 protein expression. The 2B and VP4 proteins were identified as the viral factors that induced reduction of NME1. FMDV 2B protein has a suppressive effect on host protein expression. We measured, for the first time, VP4-induced lysosomal degradation of host protein; VP4-induced degradation of NME1 through the macroautophagy pathway, and impaired p53-mediated signaling. p53 plays significant roles in antiviral innate immunity by inducing several interferon-inducible antiviral genes expression, such as, ISG20, IRF9, RIG-I, and ISG15. VP4 promoted interaction of p53 with murine double minute 2 (MDM2) through downregulation of NME1 resulting in destabilization of p53. Therefore, 5-flurouracil-induced upregulation of ISG20, IRF9, RIG-I, and ISG15 were suppressed by VP4. VP4-induced reduction of NME1 was not related to the well-characterized blocking effect of FMDV on cellular translation, and no direct interaction was detected between NME1 and VP4. The 15–30 and 75–85 regions of VP4 were determined to be crucial for VP4-induced reduction of NME1. Deletion of these VP4 regions also inhibited the suppressive effect of VP4 on NME1-enhanced p53 signaling. In conclusion, these data suggest an antiviral role of NME1 by regulation of p53-mediated antiviral innate immunity in virus-infected cells, and reveal an antagonistic mechanism of FMDV that is mediated by VP4 to block host innate immune antiviral response.

Foot-and-mouth disease virus nonstructural protein 2B interacts with cyclophilin A, modulating virus replication.

Foot-and-mouth disease (FMD) is a highly contagious virus that affects cloven-hoofed animals. To understand better the role of nonstructural protein 2B of the causative agent FMD virus (FMDV) in the process of virus replication, we identified a porcine host protein, cyclophilin A (CypA), which interacts with FMDV 2B. The 2B-CypA interaction was confirmed by coimmunoprecipitation and GST pull-down assays. CypA showed antiviral functions during FMDV infection. Overexpression of CypA decreased FMDV leader protein (Lpro) and 3A at protein levels. CypA-induced reduction of Lpro enhanced the synthesis of host proteins and increased the integrality of host eukaryotic translation initiation factor (eIF)-4γ (eIF4G). The reduction of Lpro and 3A was dependent on the proteasome pathway. No interaction was identified between CypA and Lpro or 3A. However, CypA-induced reduction of Lpro and 3A was suppressed by 2B, and disruption of 2B-CypA interaction impaired this inhibitive effect induced by 2B. In summary, our findings identify the antiviral role of CypA against FMDV and provide key insights into how FMDV antagonizes host antiviral response by 2B protein.-Liu, H., Xue, Q., Cao, W., Yang, F., Ma, L., Liu, W., Zhang, K., Liu, X., Zhu, Z., Zheng, H. Foot-and-mouth disease virus nonstructural protein 2B interacts with cyclophilin A, modulating virus replication.

Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression.

ABSTRACTThe role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein.IMPORTANCE This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca2+ concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

Effects of Foot-and-Mouth Disease Virus Nonstructural Proteins on the Structure and Function of the Early Secretory Pathway: 2BC but Not 3A Blocks Endoplasmic Reticulum-to-Golgi Transport

Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.