Foot-and-mouth disease virus non-structural protein 3A inhibits the interferon-β signaling pathway

Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects cloven-hoofed animals. The pathophysiology of FMDV has not been fully understood and the evasion of host innate immune system is still unclear. Here, the FMDV non-structural protein 3A was identified as a negative regulator of virus-triggered IFN-β signaling pathway. Overexpression of the FMDV 3A inhibited Sendai virus-triggered activation of IRF3 and the expressions of RIG-I/MDA5. Transient transfection and co-immunoprecipitation experiments suggested that FMDV 3A interacts with RIG-I, MDA5 and VISA, which is dependent on the N-terminal 51 amino acids of 3A. Furthermore, 3A also inhibited the expressions of RIG-I, MDA5, and VISA by disrupting their mRNA levels. These results demonstrated that 3A inhibits the RLR-mediated IFN-β induction and uncovered a novel mechanism by which the FMDV 3A protein evades the host innate immune system.

Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease (FMD) that is highly contagious for affect domestic and wild cloven-hoofed animals worldwide. There are seven known serotypes of FMDV (A, O, Asia1, C, SAT1, SAT2, and SAT3), which consists of numerous subtypes 1,2 . The FMDV genome is an 8.5 kb, positive sense, single-stranded RNA molecule containing one open reading frame (ORF). The ORF encodes a single polyprotein that is post-translationally processed into four structural proteins (VP1-4) and eight non-structural proteins (L pro , 2A, 2B, 2C, 3A, 3B, 3C pro , and 3D) 3 . Similar to most viruses, FMDV has evolved to inhibit or evade the innate immune system. Innate immunity plays an important role in defending the host from invading pathogens. Type I interferons (IFNs), such as IFN-α and IFN-β , are recognized as an essential component of the innate immune response, especially against viral infections. The recognition of pathogen-associated molecular patterns (PAMPs) is conducted by a series of pattern recognition receptors, including toll-like receptor (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) [4][5][6] . TLRs are mostly expressed by macrophages, dendritic cells, and other immune cells, where they detect PAMPs at the cell surface or within endosomes 7 . In contrast, RIG-I and melanoma differentiation-associated gene 5 (MDA-5) are localized in the cytosol and mainly function in detecting RNA virus. RIG-I like receptors (RLRs) compose two copies of a caspase recruitment domains (CARD) at the N terminus, a regulatory/repression domain at the C terminus, and a central DExD/H box ATPase/helicase. Following activation after viral infection, the CARDs of RIG-I or MDA-5 are released and interact with the CARD motif of VISA, which subsequently lead to the activations of IFN regulatory factor 3 (IRF3) and nuclear factor-κ B (NF-κ B) [8][9][10] . Upon activation, IRF3 and NF-κ B translocate to the nucleus and collaborate in inducing expression of a series of genes, such as type I IFNs, interferon-stimulated genes (ISG) and inflammatory cytokines 11,12 .
In order to replicate and spread in the hosts, many pathogens subvert the host cellular defense machinery to combat innate immune system, such as FMDV has evolved to actively suppress the production of host interferon in vitro [13][14][15] . Furthermore, FMDV or some proteins of FMDV could antagonize the host innate response in mammalian cells by preventing the translational 16 or transcriptional level of host proteins 17 . Such as, the FMDV leader proteinase (L pro) plays a key role in preventing IFN-α /β protein synthesis, by reducing the level of immediate-early induction of IFN-β mRNA and IFN stimulated gene products 16 , degrading p65/RelA 18 or IRF3/7 19 , cleaving ubiquitin moieties from critical signaling proteins of the type I IFN signaling pathway, and/or abrogating the deubiquitinating activity of enzymes 20 .
In addition, FMDV 3C pro inhibits the virus-triggered expression of IFN-α and -β expression by cleaving NEMO (NF-κ B essential modulator) 21 . It was recently reported that 3C pro antagonizes the IFN signaling pathway by blocking the nuclear translocation of STAT1 and STAT2 17 . FMDV VP1 suppresses type I IFN induction by interacting with sorcin, which appears to regulate the cellular response to viral infections 22 . It remains unclear whether other FMDV nonstructural proteins are involved in inhibition of type I IFN signaling. In the present study, the results demonstrated that FMDV 3A inhibited virus-triggered the IFN-β signaling pathway. It also inhibited the expression of RIG-I, MDA5 and VISA proteins by disrupting their mRNA levels. It uncovered the complicated mechanisms underlying the evasion of the host immune response by FMDV.

Results
FMDV 3A is a negative regulator of virus-triggered IFN-β expression. To address the potential roles of FMDV nonstructural proteins in regulating cellular antiviral response, the effects of FMDV nonstructural proteins on virus-triggered activation of IFN-β were examined by reporter assays. The data showed that 3A could markedly inhibited Sendai virus (SeV)-triggered activations of the IFN-β promoters (Fig. 1a) and ISRE (an IRF3binding motif) (Fig. 1b), but not affect IFNγ -induced activation of IRF1 promoter (Fig. 1c). Overexpression of FMDV 3A significantly inhibited SeV-triggered activation of the IFN-β promoter and ISRE in a dose-dependent manner in human embryonic kidney 293T (HEK293T) cells (Fig. 1d,e), while IFNγ -induced activation of IRF1 promoter was not affected (Fig. 1f). In real-time PCR experiments, it was observed that SeV-triggered transcriptions of the Ifnb1, Cxcl-10, Isg56 and Rantes genes were decreased in FMDV 3A-transfected cells in comparison with the control transfected cells (Fig. 1g). To investigate the relevance of the activation of the IFN-β signaling pathway in FMDV replication, the infectious progeny virus of the type O FMDV in 3A-transfected PK-15 cells was examined. The transcriptional level of IFN-β was reduced in the 3A-transfected PK-15 cells (Fig. 1h), indicating that the activation of the IFN-β signaling pathway was inhibited by FMDV 3A. Consistent with the decrease of IFN-β mRNA level, the FMDV genome copies were increased in the 3A-transfected PK-15 cells when compared with non-transfected cells (Fig. 1i). Furthermore, it was found that overexpression of the FMDV 3A inhibited IRF3 phosphorylation and dimerization, which are hallmarks for IRF3 activation (Fig. 1j), in addition, overexpression of FMDV 3A decreased the expressions of RIG-I/MDA5 after SeV infection (Fig. 1k). Collectively, these results suggest that FMDV 3A is a negatively regulator of virus-triggered IFN-β induction.
FMDV 3A negatively regulates virus-triggered signaling at or upstream of the VISA level. There are various components involved in IFN-β signaling pathways. In order to determine the molecular mechanisms of FMDV 3A in regulating RLR-mediated antiviral pathways, we co-transfected FMDV 3A with RIG-I (CARD)/MDA5 or their downstream signaling proteins and examined the activations by dual-luciferase assays. Overexpression of FMDV 3A inhibited activation of the IFN-β promoter induced by VISA as well as its upstream components RIG-I (CARD) and MDA5, but did not affect IFN-β promoter activation induced by VISA downstream components TBK1, IRF3-5D and IRF7 (Fig. 2a). Consistently, overexpression of FMDV 3A inhibited activation of the ISRE induced by RIG-I (CARD), MDA5, and VISA, but not TBK1, IRF3-5D and IRF7 (Fig. 2b). These data suggest that FMDV 3A targets at or upstream of VISA to regulate RLR-mediated signaling pathway. FMDV 3A interacts with RIG-I, MDA5, and VISA. To further evaluate the complicated regulatory mechanisms of FMDV 3A in virus-triggered signaling pathway, we examined the ability of FMDV 3A to associate with RIG-I, MDA5, and VISA which were shown to be inhibited in report assays. In transient transfection and co-immunoprecipitations experiments, FMDV 3A was found to specifically interacts with VISA and its upstream sensor RIG-I and MDA5, but not with TRAF3, TRAF6, TBK1, IRF3, IRF7, IKKα , IKKβ or P65 (Fig. 3a). Furthermore, to determine whether FMDV 3A interacts with RIG-I/MDA5/VISA in physiological conditions, we did co-immunoprecipitation experiments with antibodies recognizing endogenous RIG-I, MDA5, and VISA. The results showed FDMV 3A could interact with endogenous RIG-I, MDA5, and VISA ( Fig. 3b-d). Taken together, these results suggest that FMDV 3A interacts with RIG-I, MDA5 and VISA.
Domain mapping of the FMDV 3A interaction. To further determine the structure domains of FMDV 3A that responsible for the interactions with RIG-I, MDA5 and VISA, a series of truncated mutants of FMDV 3A, RIG-I, MDA5, and VISA were generated (Fig. 4a-c). The results suggested that the N-terminal 51 amino acids of 3A was required for its interaction with RIG-I, MDA5, and VISA ( Fig. 4d-f). We next examined which domains of RIG-I/MDA5/VISA are required for its interaction with FMDV 3A. The results also showed that CARD and helicase domains of RIG-I/MDA5, and C-terminal amino acids (360-540) of VISA interacted with FMDV 3A (Fig. 4g,h). In reporter assays, it was found that the full-length and N-terminal 102 aminol acids of FMDV 3A inhibited SeV-, RIG-I-, MDA5-, and VISA-triggered IFN-β and ISRE activations (Fig. 5a,b), while C-terminal 51 aminol acids (103-153) of the protein had minor effects (Fig. 5c,d). These results suggest that the N-terminal 51 amino acids of FMDV 3A interacted with the VISA/RIG-I/MDA5 and the N-terminal 102 amino acids of FMDV 3A inhibited the IFN-β signaling pathway.  is intriguing to find out if FMDV 3A inhibits the expressions of RIG-I, MDA5 and VISA at translational levels. Treatment with a proteasome inhibitor MG-132, autophagy inhibitor 3-MA, and lysosome inhibitor NH4Cl were not significantly blocked the protein reduction (Fig. 7a-c). In real time PCR experiments, it was found that FMDV 3A inhibited the mRNA abundance of RIG-I, MDA5 and VISA, but not TBK1, TRAF3 and IRF3 (Fig. 7d). These data demonstrate that that FMDV 3A reduced the expression of the RIG-I, MDA5 and VISA by inhibiting their mRNA level, not at translational levels.

Discussion
Activation of the innate immune system in mammals upon in response to viral infections, leads to the expression of hundreds of antiviral genes that control the spread of infections 23 . The IFN signaling pathway is a crucial component of the innate antiviral response.
FMDV is a member of the Aphthovirus genus within the Picornaviridae family. Its pathophysiology is likely related to its ability to evade the innate immune system of the host. Some studies have shown that FMDV proteins inhibit the IFN pathway, and therefore evade host innate immunity 18,21,24 . The mechanisms that FMDV use to manipulate the host for its replication and to evade the host immune response, are not fully understood. Although there are known FMDV-mediated inhibitory mechanisms that affect the initiation and effector phases of the innate immune response, little is known about the effects of FMDV on the signaling transductions. This pathway involves RIG-I-, MDA5-, and VISA-mediated type I IFN production, and is regulated by a sophisticated interplay between host and viral proteins. In the current study, we revealed that the expressions of RIG-I, MDA5, and VISA are reduced by FMDV 3A, which results in inhibition of type I IFN production. The findings could help to explain the influence of FMDV on RIG-I-and MDA-5 signaling transduction pathways, and further improve our understandings of the mechanisms by which FMDV evades the host innate immune system of the host.
RIG-I and MDA-5 play important roles in innate immunity. Recently, several viruses have been reported to use various strategies to disrupt the functions of RIG-I and MDA-5. The influenza virus NS1 protein can antagonize the RIG-1-VISA pathway by interacting with RIG-1 and VISA 25 . The V proteins of the Paramyxoviruses, including simian virus 5, human parainfluenza virus 2, mumps virus, SeV, and Hendra virus, were shown to disrupt the MDA5-IFN-β pathway via direct interactions with MDA5 26,27 . Other studies showed that several viruses disrupt the function of VISA to evade host innate immunity. The NS3-4A protease of hepatitis C virus inhibits the IFN signaling pathway by cleaving the C-terminal end of VISA to disrupt its interaction with the outer mitochondrial membrane [28][29][30] . The HBx protein of hepatitis B virus binds with VISA and promotes its degradation to inhibit IFN-β production 31,32 . Viruses of the Picornaviridae family disrupt the function of VISA through various mechanisms. The 3ABC protein of Hepatitis A virus co-localizes with VISA at the mitochondria and degrades VISA 33 . The rhinovirus 2A pro and 3C pro proteases, and 3C pro of coxsackievirus B3 inhibit the IFN signaling pathway by cleaving VISA 34 . The 2A pro of enterovirus 71 inhibits type I IFN production by cleaving VISA at Gly209, Gly251, and Gly265 35 . All of the above studies were focused on the degradations of the proteins of the IFN signaling pathway by virus-encoded proteases. Therefore, our finding that FMDV 3A inhibited the expression of the RIG-I, MDA5, and VISA provides a new insight into how non-protease FMDV-derived proteins can affect the IFN signaling pathway. FMDV 3A has no sequence homology to any other known proteases. We postulated that other proteins mediates the inhibition of the expressions of RIG-I, MDA5, and VISA proteins by FMDV 3A. Many viruses have developed mechanisms to subvert the IFN pathway. The nonstructural proteins of bovine respiratory syncytial virus (BRSV) block the induction of genes encoding IFN-α /β , which results in the increased virulence of BRSV 36 . The replication and virulence of West Nile virus is enhanced via resistance to IFN-α /β 37 . The PB2 subunit of influenza virus RNA polymerase inhibits the expression of IFN-β , thereby affecting its virulence 38 . Previous studies have suggested that there is a correlation between IFN expression and the virulence of a virus, but some studies have focused on FMDV pathogenicity and virulence. FMDV 3A consists of 153 amino acids that are partially conserved. The first half of the 3A coding region, which is highly conserved among all FMDVs, encodes an N-terminal hydrophilic domain and a hydrophobic domain capable of binding to membranes 39 . The 3A protein is multifunctional and plays important roles in virus replication, virulence, and determination of host-range 40,41 . Previous studies have shown that deletion of amino acids 93-102 of 3A protein in a Taiwanese strain (O/TAW/97) of FMDV is associated with an inability to cause disease in cows. Further research suggested that this deletion does not affect FMDV replication efficiency in BHK-21 (hamster), PK-15 (porcine), and FPK (fetal porcine) cell lines. However, virus replication efficiency was reduced in the FBK (fetal bovine) cell line, although this alone cannot account for the inability of FMDV to replicate in bovine cells. Another Asian strain of FMDV with a second deletion within 3A (amino acids 133-143) or a series of substitutions, revealed that changes in the genome in addition to the original deletion are responsible for the porcinophilic properties of current Asian viruses in this lineage 39 . In FMDV, deletions of 19 to 20 amino acids between 3A residues 87-106     (32,28,24,20,16,8 and 0 μ g) for 24 h. A small volume of the protein lysate was collected to detected VISA, TBK1, TRAF3, and IRF3 by western blotting. Other protein lysate was used for immunoprecipitation with protein G-beads coupled to polyclonal antibodies directed against RIG-I or MDA5. The immunoprecipitation was analyzed by immunoblots with the polyclonal antibodies against RIG-I and MDA5.
Scientific RepoRts | 6:21888 | DOI: 10.1038/srep21888 results in reduced virulence in cattle 42 . A recombinant O1 Campos virus harboring a 20 amino acid deletion in 3A exhibited reduced virulence in cattle 43 . It remain unclear that there is a relationship between the amino acid composition of 3A and the IFN signaling pathway, which affects the virulence of FMDV. We demonstrated that amino acids 1-102 of FMDV 3A inhibits the IFN-β signaling pathway, which provides new information regarding the influence of IFN signaling on FMDV virulence. Further studies are required to determine whether the roles of the IFN response reported here culminate in FMDV pathophysiology and which key amino acids of FMDV 3A inhibit the IFN-β signaling pathway.
In summary, FMDV 3A adversely affects RIG-I, MDA5, and VISA-mediated type I IFN production by inhibiting the mRNA levels of these genes. Furthermore, we have identified a key domain (amino acids 1-102) of FMDV 3A that inhibits the IFN-β signaling pathway. Taken together, the findings reveal a novel mechanism of FMDV 3A-mediated evasion of host innate immunity.   was purchased from Amersham Biosciences and M-MLV reverse transcriptase was from Invitrogen. The Sendai virus (SeV) used in the experiments and rabbit antibodies against VISA, MDA5, TRAF3, and RIG-I were previously described [44][45][46] . The type O FMDV was propagated in PK-15 cells, and the supernatants of infected cells were clarified and stored at − 80 °C. The generation of luciferase reporter plasmids encoding the promoters for ISRE, IRF1, and IFN-β ,   along with mammalian expression plasmids for RIG-I (CARD), RIG-I (helicase), RIG-I, MDA5, VISA, TBK1,  TRAF3, TRAF6, IRF3, IKKα , IKKβ , P65 and VISA mutants have been described previously 7,44,[46][47][48][49] . The genes of FMDV proteins were amplified from cDNA of the type O FMDV, strain Tibet/CHA/99 (GenBank accession no. AJ539138). FMDV proteins cDNA were cloned into the pCAGGS vector using EcoRI and XhoI restriction enzymes. A series of FMDV 3A mutants were cloned into the pRK-GFP or pMSCV vectors using EcoRI and XbaI restriction enzymes generated from pCAGGS-3A by conventional PCR techniques with the mutagenesis primers listed in Table 1. All plasmids were verified by sequencing.

Plasmids.
Transfection and reporter assays. HEK293T cells (5 × 10 4 ) in 48-well plates were transfected with 100 ng of reporter plasmid, 10 ng of pRL-TK (Promega) (as an internal control), and 100 ng indicate plasmids. At 24 hours post transfection, the cells were treated with SeV (moi: 1.0) or IFNγ (100 ng/ml) or left untreated for 12 h, and the whole-cell extracts were prepared for the analysis of dual-luciferase activities. The activities of the reporter genes, including firefly luciferase and renilla luciferase, were determined using a dual-luciferase reporter 1000 assay system (Promega) according to the manufacturer's instructions. The data represented the firefly luciferase activity normalized to the renilla luciferase activity. Three independent experiments were carried out in duplicate.  Table 1. Relative fold changes in gene expression were determined by the threshold cycle (2 −ΔΔCt ) method. The viral genome copies in FMDV-infected PK-15 cells were quantified by a quantitative real-time RT-PCR. Total RNA of the FMDV-infected cells was extracted using TRIzol as described above. Quantification of genome copies of FMDV was performed as described previously 50 .

Real-time RT-PCR.
Co-immunoprecipitation and western immunoblotting. Transfected HEK293T cells from 10-cm dishes were lysed in l ml of lysis buffer (20 mM Tris-HCl pH 7.4-7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 μ g/mL aprotinin, 10 μ g/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). For each sample, 0.8 mL of cell lysate was incubated with 0.5 μ g of appropriate antibody and 25 μ l of 50% (v/v) slurry of protein G agarose beads (Amersham Biosciences) at 4 °C for 2 h. Agarose beads were washed three times with 1 mL of lysis buffer containing 500 mM NaCl. Precipitates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then western immunoblotting analysis.
Virus infection and treatment. PK-15 cells were transfected with plasmids for 12 h then treated with puromycin (1 mg/ml) for 24 h followed by infection with the type O FMDV (MOI: 0.1). After 2 h, the viral inoculum was removed and the infected cells were washed twice with phosphate-buffered saline (PBS) (pH 7.4) and re-fed with DMEM containing 2% FBS. IFN-β mRNA abundance or FMDV genome copies in PK-15 cells were assessed using a relative quantitative RT-PCR assay or using a quantitative RT-PCR assay. To examine effect of FMDV 3A on the expressions of RIG-I, MDA5 and VISA, HEK293T cells co-transfected with RIG-I, MDA5 or VISA and 3A or empty vector for 18 h followed by treatment with DMSO, MG132 (20 μ M), 3-MA (0.5 mg/ml), NH4Cl (20 mM) for 6 h. The samples were harvested and analyzed using western immunoblotting. Statistical analysis. Statistical analysis was performed using the SPSS17.0 software. The student's t test was used for a comparison of three independent treatments. For the test, a P value < 0.05 was considered significant ( * ) while P value < 0.01, very significant ( ** ).