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 that has caused significant economic loss to global livestock industry (1). The causative agent of the disease is foot-and-mouth disease virus (FMDV), which belongs to the genus of Aphthovirus in family of Picornaviridae. FMDV includes a positive single-strand RNA genome that encodes a large polyprotein. The polyprotein is processed into several intermediate proteins and finally yields four mature structural proteins and 10 nonstructural proteins (2). Both the intermediate and mature viral proteins perform functions in the viral life cycle (3). A number of interactions between the viral proteins and host proteins have been reported for understanding the functions of FMDV proteins and clarifying how FMDV manipulates the host machinery (4).

Innate immune system is the first line of defense against invading pathogens, and the pathogen recognition receptors (PRRs) are responsible for detection of the pathogens and induction of host immune responses (5). PRRs mainly contain TLRs, RIG-I–like receptors (RLRs), NOD-like receptors, and C-type lectin receptors. These PRRs sense different pathogen signatures and trigger the activation of different pathways (6, 7). RLRs, as one of the major subsets of PRRs, play critical roles in the host defense against numerous viral infections (8). The activation of the downstream transcription factors of RLRs pathway drives type I IFN and proinflammatory cytokines production that induce host antiviral signaling cascades (9).

Many picornaviruses suppress host innate immune system by a variety of mechanisms to evade clearance by the host and facilitate viral replication. For example, hepatitis A virus and FMDV proteolytically cleave host NF-κB essential modulator (NEMO) by viral proteinase 3C^pro to counteract host innate immune response (10, 11). NEMO is a bridging adaptor that plays a critical role in activation of both IFN-β and NF-κB signaling pathways (12). The cleavage of NEMO significantly impairs IFN-β and proinflammatory cytokines production. The L proteinase (L^pro) and 2A protein of most of picornaviruses cleave or decrease host translation initiation factor eIF4G to shut off host protein synthesis and promote viral replication (13–15). Moreover, the antagonistic role of other viral proteins in innate immune suppression is being recently unveiled, indicating sophisticated immune suppression mechanisms and strategies of picornaviruses (4, 16–19).

FMDV 3B (also known as VPg) is present in three similar but nonidentical copies (3B1, 3B2, and 3B3) (20), and there are no reports of naturally occurring FMDV strains with fewer than three copies of 3B (21). The three nonidentical copies are highly conserved and all contain the position 3 Tyr (Y) residue (Fig. 2D) which is involved in the phosphodiester linkage to the viral genome RNA. 3B is covalently attached to the 5′ end of the viral genome RNA via the conserved Y residue by the viral RNA polymerase (3D^pol), acting as a primer for the synthesis of the RNA during viral replication (22, 23). Although not all of the three copies are essential for FMDV replication, the copy number of 3B is critically associated with the host range and the virulence of FMDV. This may help to explain why all naturally isolated FMDVs have retained three copies of 3B (2).

FMDV has developed significant ability to suppress host innate immune response by diverse strategies. The viral proteinases L^pro and 3C^pro are the most well-known factors that present antagonistic role on host innate immune response (4, 24). Both L^pro and 3C^pro cleave eIF4G to shut off host protein synthesis (13, 25). In addition, they suppress IFN production by impairing the activation of RLRs signaling pathway (26). In the current study, we identified FMDV nonstructural protein 3B as a new antagonistic factor of host innate immune response. 3B suppressed the expression of IFN-β and antiviral genes during FMDV infection and efficiently promoted viral replication. We found that 3B interacted with RIG-I and impaired the interaction between RIG-I and the adaptor protein mitochondrial antiviral signaling protein (MAVS), which is critical for IFN-β production and antiviral genes expression. The lysine 63 (K63)-linked ubiquitination of RIG-I is crucial for the interaction between RIG-I and MAVS. Our results showed that the K63-linked ubiquitination of RIG-I was significantly inhibited by 3B. The interaction of 3B with RIG-I blocked the RIG-I–TRIM25 interaction, preventing the TRIM25-mediated K63-linked ubiquitination and activation of RIG-I. Therefore, we determined that 3B targeted the viral sensor RIG-I to disrupt its activation, leading to a blockade of RIG-I–mediated immune signaling and host antiviral response.

FMDV strain O/BY/CHA/2010 (GenBank number: JN998085), which was isolated from a pig in China in 2010 (27), was used in this study. Sendai virus (SeV), a model RNA virus routinely used to activate type I IFN pathway in cell culture, was obtained from H. Shu’s Laboratory in Wuhan University (16, 28). Human embryonic kidney HEK293T cells and porcine kidney PK-15 cells were cultured in in DMEM (Invitrogen), supplemented with 10% heated-inactivated FBS at 37°C in a humidified 5% CO₂ incubator.

The commercial Abs used in this study include anti-FLAG mouse Ab (catalog no. F1804; Sigma-Aldrich), anti-FLAG rabbit Ab (catalog no. 701629; Invitrogen), anti-HA mouse Ab (catalog no. 26183; Invitrogen), anti-HA rabbit Ab (catalog no. 3724; Cell Signaling Technology), anti-Myc mouse Ab (catalog no. sc-40; Santa Cruz Biotechnology), anti–RIG-I rabbit Ab (catalog no. ab180675; Abcam), anti–RIG-I mouse Ab (catalog no. sc-376845; Santa Cruz Biotechnology), anti-MAVS rabbit Ab (catalog no. 24930; Cell Signaling Technology), anti-ubiquitin (linkage-specific K63) rabbit Ab (catalog no. ab179434; Abcam), anti-GST rabbit Ab (catalog no. 2625; Cell Signaling Technology), and anti–β-actin mouse Ab (catalog no. sc-8432; Santa Cruz Biotechnology). Anti-3B rabbit polyclonal Ab was prepared by our laboratory.

The FLAG-tagged 3B plasmid was constructed by our laboratory previously, and the cDNA sequence of FMDV 3B was inserted into pCAGGS vector with a C-terminal FLAG tag (29). For GST-3B and GST-3B single-copy–deletion mutants expressing plasmids (including GST-△3B1, GST-△3B2, and GST-△3B3), the GST tag was inserted into pCDNA3.1 at the C terminus of the multiple cloning sites, and FMDV 3B sequence was constructed into the pCDNA3.1-GST plasmid with the GST located at the C terminus. The deletion of the 3B1, 3B2, or 3B3 was introduced into GST-3B through the PCR-based mutagenesis as described previously (30). The GST-3B1–, GST-3B2–, and GST-3B3–expressing plasmids were also constructed through the PCR-based mutagenesis. A series of FLAG-tagged 3B mutants constructs were generated by inserting the synthesized fragment into the FLAG–CMV-7.1 plasmid. IFN-stimulated response element (ISRE), IFN-β, and NF-κB promoter luciferase reporter plasmids and pRL-TK internal control plasmids were described previously (16, 31). HA-tagged RIG-I, HA-tagged RIG-I (CARD) and various HA-tagged components including MAVS, TBK1, IRF3, and IRF7 expressing plasmids used in this study were kind gifts from Professor H. Shu in Wuhan University (16, 31). Myc-tagged RIG-I and HA-tagged K63 ubiquitin plasmids were described previously (32, 33). All the plasmids used in this study were verified by DNA sequencing analysis. The Lipofectamine 2000 reagent (Invitrogen) was used for plasmid transfection per the manufacturer’s instructions.

Total RNA was extracted from the cell culture using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. The first-strand cDNA was synthesized using the M-MLV reverse transcriptase (Promega) and random hexamer primers (TaKaRa Bio). The synthesized cDNAs were subjected to quantitative real-time PCR (qPCR) analysis. The relative amounts of cDNAs were determined using the SYBR Premix Ex Taq reagents (TaKaRa Bio) and a QuantStudio 5 Real-Time PCR instrument (Applied Biosystems) following the manufacturer’s protocol. Relative mRNA levels were calculated using 2^−ΔΔCT method (CT indicates threshold cycle) as described previously (32). The house keeping gene GAPDH was used as an internal control in qPCR analysis. The qPCR primers used in this study were shown in Table I.

HEK293T cells were cultured on 24-well plates, and the monolayer cells were transfected with the indicated host protein expressing plasmids, empty vector, or viral protein expressing plasmids, in the presence of 100 ng per well reporter plasmid and 10 ng per well internal control Renilla luciferase reporter plasmid pRL-TK (normalization of the transfection efficiency). The Lipofectamine 2000 reagent was used for plasmid transfection. The empty vector plasmids were used in the transfection experiments to ensure the cells receive the same amounts of total plasmids. Where indicated, the transfected cells were mock-infected or infected with SeV (100 hemagglutinating activity units per milliliter) for 16 h at 24 h posttransfection (hpt). The dual-luciferase activities were measured using the Promega Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. As for the critical components of RIG-I pathway induced luciferase reporter assay, HEK293T cells were cotransfected with Flag vector or Flag-3B–encoding plasmids and the plasmids expressing a set of components of RIG-I pathway, together with the ISRE-driven luciferase reporter plasmid and the internal control pRL-TK reporter plasmid. Luciferase activity was detected and analyzed at 24 hpt. The dual-luciferase activities were then measured using the Promega Dual-Luciferase Reporter Assay System.

Briefly, the cells were harvested at the indicated time and lysed in the lysis buffer supplemented with various proteinase inhibitors. The cell lysates were collected in the precold tubes. Equal amounts of samples were subjected to 10% SDS-PAGE and analyzed for the expression of different proteins. The nitrocellulose membranes (Pall) were used to retain proteins, and the Ab–Ag complexes were generated by incubation of various Abs. The generated Ab–Ag complexes were detected by ECL detection reagents (Thermo Fisher Scientific). β-actin was used as a loading control to demonstrate equal protein sample loading. For the coimmunoprecipitation assays, the cell lysates were immunoprecipitated with indicated Abs, and the immunoprecipitated samples were subjected to immunoblotting analysis as described previously (32).

HEK293T cells were cultured into Nunc glass-bottom dishes, and transfected with 2 μg of FLAG-3B and 2 μg of HA–RIG-I plasmids for 24 h. The cells were collected, washed with PBS, and then fixed with 4% paraformaldehyde in PBS at room temperature for 0.5 h. Then the fixed cells were washed with ice-cold PBS three times. The 0.2% Triton X-100 was used to permeabilize the cells for 10 min. Five percent BSA in PBS was used to block the cells for 1 h at 37°C. The cells were then incubated with proper Abs as described previously (32). The stained cells were analyzed and imagined with a Nikon eclipse 80i fluorescence microscope and NIS Elements F 2.30 software.

The samples were collected at the indicated hours postinfection (hpi). The 50% tissue culture infective dose (TCID₅₀) assay was carried out as described previously (34). Briefly, the monolayer cells were grown in the 96-well plates. The virus suspension was diluted with 10⁻¹–10⁻⁹ serial dilutions and 50-μl portion of the diluent was added to the cell cultures. Each dilution was repeated in eight wells. The cells were maintained until cytopathic effects were clearly observed. The TCID₅₀ values were then calculated by the Reed-Muench method.

The construction strategy for the r3B-FMDV and r3B-A/E-FMDV infectious clones was based on the reverse-genetics system that has been developed by our laboratory previously (35). A synonymous substitution led to no changes in amino acids in the 2C was introduced into the viral genome of O/BY/CHA/2010 (mutation of the 691–696 region in 2C from 5′-ATTGAC-3′→5′-ATCGAT-3′) to generate a unique ClaI restriction enzyme site. A unique SbfI restriction enzyme site was present in the C terminus after the viral poly (A) in our reverse-genetics system. A ∼3120 bp of fragment was obtained by digestion of the r3B-FMDV infectious clone using the ClaI and SbfI restriction enzymes. The obtained fragment was then inserted into the POK12 plasmid, and the aa A17E mutation in each copy of 3B was then introduced into the fragment at the indicated position by the PCR-based mutagenesis as described previously (30). After confirmation of the A→E mutation in all of the three nonidentical copies of 3B, the ClaI and SbfI restriction enzymes were used to generate the fragment bearing the 3B-A/E mutation. The collected fragment was then inserted into the r3B-FMDV infectious clone instead of the original region. All of the constructed plasmids were sequenced. The viral rescue and identification was then performed as described previously (35).

All the data are represented as the mean with SE from three independent experiments. A Student t test was used for a comparison of three independent experiments. All tests were two-sided. A p value < 0.05 was considered to be statistically significant (designated by a single asterisk). A p value < 0.01 was considered to be statistically highly significant (designated by a double asterisk). n.s. indicated not significant.

FMDV 3B protein plays important roles in viral replication (21). Our previous study found that FMDV 3A suppressed SeV-triggered IFN-β promoter activation (29). To identify the suppressive role of 3B on host innate immune pathways signaling, the SeV that is routinely used to induce type I IFNs was used as an agonist of innate immune pathway, and SeV-induced activation of IFN-β promoter, ISRE, and NF-κB promoter was evaluated in 3B-overexpressing cells. HEK293T cells were transfected with vector plasmids or increasing amounts of FLAG-3B–expressing plasmids together with ISRE-, IFN-β promoter–, or NF-κB promoter–driven reporter plasmids and the internal control plasmid pRL-TK. The transfected cells were infected with SeV to activate type I IFN pathway or NF-κB pathway, and the luciferase activity was measured. Overexpression of 3B protein significantly suppressed SeV-induced ISRE (Fig. 1A), IFN-β promoter (Fig. 1B), and NF-κB promoter (Fig. 1C) activation, showing a dose-dependent manner. The expression of FMDV 3B protein was detected by immunoblotting using an anti-FLAG Ab. We also evaluated the effect of FMDV VP2 on SeV-induced ISRE, IFN-β promoter, and NF-κB promoter activation, which showed that a high amount of VP2 weakly but statistically significantly inhibited ISRE activation (Fig. 1D), although it did not undermine SeV-induced IFN-β promoter (Fig. 1E) and NF-κB promoter activation (Fig. 1F). This indicated that FMDV 3B significantly suppressed RLR pathway signaling.

To examine whether FMDV 3B protein antagonizes IFN-β and IFN-stimulated genes (ISGs) expression (Table I), HEK293T cells were transfected with empty vector or 3B-expressing plasmids and then infected with SeV. The cell supernatants and cells were collected separately for the ELISA and qPCR detection of IFN-β expression as previously described (36). The expression levels three ISGs (ISG56, ISG54, and MX1) were also measured. Overexpression of 3B protein considerably inhibited IFN-β and ISGs expression (Fig. 2A, 2B). The activation of NF-κB pathway induces the expression of a set of proinflammatory cytokines that are involved in innate immunity (37). 3B had been shown to inhibit SeV-induced NF-κB promoter activation (Fig. 1C). To confirm 3B-mediated suppressive role on NF-κB pathway, the expression levels of several cytokines, including TNF-α, IL-6, and IL-1β were measured in the cells transfected with vector or 3B-expressing plasmids and infected by SeV. SeV infection induced weakly but statistically significantly TNF-α, IL-6, and IL-1β expression in the vector-transfected cells, and the expression of TNF-α, IL-6, and IL-1β was considerably decreased in the 3B-overexpressing cells (Fig. 2C). FMDV 3B includes three nonidentical copies (3B1, 3B2, and 3B3) (Fig. 2D). The effect of 3B1, 3B2, and 3B3 on IFN-β production was also investigated in both HEK293T and PK-15 cells. The results showed that deletion of a single copy of 3B did not subvert 3B-mediated suppressive effect on IFN-β production (Fig. 2E, 2F). It showed a similar outcome by using three deletion mutants. We further generated the constructs expressing GST-tagged 3B1, 3B2, or 3B3 and evaluated whether a single copy of 3B could suppress IFN-β production. The results showed that the expression of any copy of 3B inhibited SeV-induced IFN-β production (Fig. 2G). These results further confirmed that FMDV 3B protein antagonized host innate immune signaling and inhibit the expression of downstream antiviral genes.

3B suppressed SeV-induced innate immune signal transduction. To investigate the viral replication status in 3B-overexpressing cells, PK-15 cells were transfected with empty vector or FLAG-3B–expressing plasmids and then mock-infected or infected with FMDV for 12 h. The viral RNA levels and virus yields were detected by qPCR and TCID₅₀ assay respectively as described previously (17, 29). The results showed that overexpression of 3B significantly promoted FMDV replication (Fig. 3A). The expression levels of IFN-β and ISGs (ISG56, ISG15, and MX1) were also determined by qPCR analysis, which showed that 3B decreased the expression of IFN-β and ISGs (Fig. 3B). FMDV infection in PK-15 cells led to very low expression of ISGs. This implied that FMDV infection blocked the production of IFNs and subsequent IFN signaling in the PK-15 cells, therefore resulted in low expression of ISGs. An ∼50-fold increase in viral titers was observed on the basis of an 8-fold increase in intracellular FMDV RNA in Fig. 3A. FMDV 3B inhibited expression of ISGs. These ISGs performed antiviral functions through multiple mechanisms. Some of the ISGs possibly have affected the assembly/release of the virus. We also investigated the effect of 3B on the replication level of another picornavirus Senecavirus A (SVA), which showed that overexpression of 3B also promoted SVA replication (Fig. 3C). These results indicated that 3B suppressed IFN-β and ISGs expression which promoted viral replication.

FMDV 3B suppressed both SeV-induced IFN-β and NF-κB promoter activation (Fig. 1). This suggested that 3B targeted the RIG-I signaling pathway at or upstream of MAVS. To confirm this inhibitory effect and the potential components targeted by FMDV 3B protein, HEK293T cells were cotransfected with empty vector or FLAG-3B–expressing plasmids and a series of plasmids expressing the components of type I IFN pathway, including RIG-I (CARD) (the CARD domain of RIG-I), MAVS, RIG-I/MDA5 (full-length RIG-I together with MDA5), TBK1, IRF3, or IRF7, together with ISRE luciferase reporter plasmid and the internal control plasmid pRL-TK. The component protein-induced activation of ISRE luciferase activity was evaluated at 24 h after transfection. Overexpression of these components significantly activated ISRE reporter system (Fig. 4). However, 3B inhibited the activation of ISRE reporter system driven by MAVS, RIG-I(CARD), and RIG-I/MDA5 (Fig. 4A). In contrast, TBK1, IRF3, or IRF7-induced activation of ISRE reporter system was not affected by 3B (Fig. 4B). Consequently, this confirmed that FMDV 3B targeted MAVS or its upstream molecule RIG-I.

To investigate whether FMDV 3B suppressed the expression of MAVS or RIG-I, HEK293T cells were transfected with empty vector or increasing amounts of FLAG-3B–expressing plasmids. The expression levels of MAVS or RIG-I mRNA and endogenous MAVS or RIG-I proteins were evaluated at 36 hpt. Both the MAVS mRNA (Fig. 5A) and MAVS protein levels (Fig. 5B) were not changed in the 3B-overexpressing cells compared with the vector-transfected cells. Similarly, we did not find the decrease of RIG-I mRNA (Fig. 5C) or RIG-I protein levels (Fig. 5D) in 3B-overexpressing cells. The effect of 3B on endogenous expression of MAVS or RIG-I in PK-15 cells was also examined. PK-15 cells were transfected with increasing amounts of FLAG-3B–expressing plasmids. The expression levels of MAVS or RIG-I mRNA and endogenous MAVS or RIG-I proteins were examined at 36 hpt. The results were consistent with those noted above. Therefore, 3B did not affect the expression of MAVS (Fig. 5E) and RIG-I mRNA (Fig. 5F) and endogenous MAVS (Fig. 5G) and RIG-I proteins (Fig. 5H). These results suggested that 3B might affect the function of MAVS or RIG-I by other manner rather than decreasing their expression.

To investigate the possible interaction between 3B protein and the components of RIG-I–mediated immune signal pathway, HEK293T cells were cotransfected with FLAG-3B and the empty vector or the plasmids expressing the HA-tagged components of RIG-I–mediated immune signal pathway. The results showed that RIG-I but not MAVS was coprecipitated with FLAG-3B (Supplemental Fig. 1), suggesting that there was an interaction between FMDV 3B and RIG-I protein. To evaluate whether 3B protein and RIG-I share similar subcellular locations, the indirect immunofluorescence microscopy (IFA) assay was carried out. PK-15 cells were transfected with Myc–RIG-I–expressing plasmids for 24 h, and then followed by mock-infection or FMDV infection for 12 h. The subcellular localization of 3B and Myc–RIG-I was investigated. FMDV 3B and Myc–RIG-I showed remarkable colocalization in the cytoplasm (Fig. 6A). The colocalization of FMDV 3B and endogenous RIG-I in PK-15 cells was further investigated, which also showed that FMDV 3B colocalized with RIG-I (Fig. 6B). The interaction of 3B protein with endogenous RIG-I was further evaluated. PK-15 cells were transfected with vector or FLAG-3B–expressing plasmids for 36 h, and the coimmunoprecipitation assay was performed. Both the immunoprecipitation experiment and the reverse immunoprecipitation experiment showed that FMDV 3B protein interacted with RIG-I protein (Fig. 6C, 6D). The interaction between 3B and endogenous RIG-I in the context of viral infection was subsequently examined. PK-15 cells were infected by FMDV for 12 h, and the coimmunoprecipitation assay was performed. The results showed that FMDV 3B also efficiently interacted with endogenous RIG-I in FMDV-infected cells (Fig. 6E). This confirmed that FMDV 3B protein interacted with host RIG-I protein during viral infection. The PK-15 cells were also cotransfected with vector or FLAG-3B–expressing plasmids and HA-tagged porcine RIG-I (HA-pRIG-I)– or HA-tagged porcine MDA5 (HA-pMDA5)–expressing plasmids for 36 h, and the coimmunoprecipitation assay was performed. The results showed that FMDV 3B protein interacted with porcine RIG-I protein but not MDA5 (Fig. 6F, 6G). The expression level of IFN-β and viral replication status in FMDV-infected RIG-I knockout (RIG-I KO) PK-15 cells and wild-type PK-15 cells were determined and compared with reveal the essential antiviral role of RIG-I against FMDV. Knockout of RIG-I considerably decreased IFN-β expression and promoted FMDV replication as well as viral yields (Fig. 6H). Furthermore, RIG-I KO PK-15 cells were transfected with vector plasmids or increasing amounts of FLAG-3B–expressing plasmids for 24 h, and followed FMDV infection for 12 h. The replication of FMDV was then evaluated. Overexpression of 3B did not enhance FMDV replication in the RIG-I KO PK-15 cells (Fig. 6I). As expected, we also found that overexpression of 3B also did not promote FMDV replication in BHK-21 cells (Fig. 6J), which have a deficient IFN signal transduction system (38, 39). These results indicated that RIG-I was critical for suppressing FMDV replication during FMDV infection and 3B-mediated antagonistic effect against RIG-I was favorable for FMDV replication.

The interaction between RIG-I and MAVS is essential for signal transduction of RIG-I–mediated immune signal pathway (40). 3B was determined to interact with RIG-I. Therefore, to investigate whether 3B protein impaired the interaction between RIG-I and MAVS, HEK293T cells were transfected with increasing amounts of FLAG-3B expressing plasmids together with Myc–RIG-I– and HA-MAVS–expressing plasmids and then infected with SeV. The transfectants were immunoprecipitated with anti-Myc Ab and subjected to Western blotting analysis. RIG-I efficiently pulled down MAVS. However, the amount of MAVS that bound to RIG-I gradually decreased as the increasing expression of 3B protein (Fig. 7A). These results suggested that FMDV 3B protein sequestered the RIG-I–MAVS interaction in a dose-dependent manner.

The interaction of RIG-I with MAVS is dependent upon K63-linked ubiquitination of RIG-I, which is essential for RIG-I–mediated immune signaling (41). The ubiquitin ligase TRIM25 positively mediates K63-linked ubiquitination of RIG-I (42). To examine whether 3B protein blocked the K63-linked ubiquitination of RIG-I, HEK293T cells were transfected with Myc–RIG-I–, FLAG-TRIM25–, HA–Ub-K63 (HA-tagged K63 ubiquitin)–expressing plasmids together with different amounts of FLAG-3B–expressing plasmids and then infected with SeV. The transfectants were immunoprecipitated with anti-Myc Ab and subjected to Western blotting analysis. Overexpression of 3B protein significantly suppressed the K63-linked ubiquitination of RIG-I in a concentration-dependent manner (Fig. 7B). The effect of FMDV infection on poly(I:C)–induced K63-linked ubiquitination of RIG-I was examined as well. PK-15 cells were transfected with poly(I:C) for 12 h and then infected by FMDV for 12 h. The K63-linked ubiquitination of RIG-I was then detected. FMDV infection also inhibited poly(I:C)–induced K63-linked ubiquitination of RIG-I (Fig. 7C). These results indicated that FMDV 3B prevented the K63-linked ubiquitination of RIG-I, resulting in the suppression of RIG-I–MAVS complex formation.

The interaction of TRIM25 with RIG-I is essential for TRIM25-mediated K63-linked ubiquitination of RIG-I. 3B inhibited the K63-linked ubiquitination of RIG-I. To decipher the mechanism by which 3B interaction with RIG-I leads to the suppression of K63-linked ubiquitination of RIG-I, we evaluated whether 3B interfered with the interaction between RIG-I and TRIM25. HEK293T cells were cotransfected with Myc–RIG-I, HA-TRIM25, and vector or FLAG-3B–expressing plasmids. The transfectants were immunoprecipitated with anti-Myc Ab and subjected to Western blotting analysis. Coimmunoprecipitation revealed that RIG-I interacted with TRIM25. However, the amount of TRIM25 that bound to RIG-I clearly decreased in the presence of 3B protein (Fig. 8A). Consistent with its ability to suppress the K63-linked ubiquitination of RIG-I, 3B inhibited the RIG-I–TRIM25 interaction in a dose-dependent manner (Fig. 8B). We also investigated whether 3B interacted with TRIM25, and no interaction was observed between 3B and TRIM25 (Fig. 8C). These results collectively indicated that the interaction of 3B with RIG-I blocked the RIG-I–TRIM25 interaction, inhibiting K63-linked ubiquitination of RIG-I.

CARD domain is involved in RIG-I–MAVS complex formation and the K63-linked ubiquitination of RIG-I. To investigate the binding domain of RIG-I that is responsible for its interaction with 3B, a series of constructs expressing the CARD, DEAD Helicase, or C-terminal domain (CTD) of RIG-I were generated (Fig. 9A). The interaction between these mutants and 3B was evaluated. HEK293T cells were cotransfected with HA vector, HA–RIG-I, HA–RIG-I–CARD, HA–RIG-I–DEAD Helicase, or HA–RIG-I–CTD– and FLAG-3B–expressing plasmids. The transfectants were immunoprecipitated with anti-FLAG Ab and subjected to Western blotting analysis. Coimmunoprecipitation revealed that both the CARD and DEAD Helicase of RIG-I interacted with 3B (Fig. 9B). The previous data showed 3B inhibited the RIG-I (CARD)–triggered IFN-β promoter activation (Fig. 4A). These results suggested that the interaction of 3B with RIG-I–CARD domain blocked the K63-linked ubiquitination of RIG-I and suppressed RIG-I–mediated immune signaling.

FMDV 3B binds to the 5′ end of viral genomic RNA via the conserved Y residue, acting as a primer for the synthesis of the RNA during viral replication (22, 23). To investigate whether the binding activity of 3B prevented viral RNA recognition by RIG-I, we generated a construct expressing mutated 3B (3B-Y3A) bearing the Y to A mutations at the conserved sites (Fig. 10A). To evaluate the suppressive role of 3B-Y3A on host innate immune pathway signaling, SeV-induced activation of IFN-β promoter and IFN-β mRNA expression were analyzed in the presence or absence 3B-Y3A. The results showed that 3B-Y3A retained the activity to inhibit SeV-induced RLR pathway signaling and IFN-β expression (Fig. 10B). We also found that FMDV VP2 did not affect SeV-induced IFN-β expression (Fig. 10C). To confirm the suppressive role of 3B-Y3A on IFN-β production during FMDV infection, PK-15 cells were transfected with empty vector or FLAG–3B-Y3A–expressing plasmids and then mock-infected or infected with FMDV for 12 h. The mRNA expression levels of IFN-β and ISGs (ISG56 and MX1) were then measured. Overexpression of 3B-Y3A inhibited the expression of IFN-β and ISGs in FMDV-infected cells as well (Fig. 10D). These data suggested that the RNA-binding activity of 3B was independent for 3B to suppress RIG-I–mediated signal transduction.

The expression of any of the single copy of 3B inhibited SeV-induced IFN-β production (Fig. 2G). This indicated that all of the three copies of 3B possessed the activity to inhibit IFN-β production. Therefore, we aligned the amino acid sequences of 3B1, 3B2, as well as 3B3 and identified five conserved regions/sites among the three copies (Fig. 10E). These regions or sites were then mutated respectively, and their activity to suppress SeV-induced IFN-β production was subsequently evaluated. Mutation of the GP-1 (the first two residues), GP-2 (the fifth to sixth residues), LKV (the 13–15th residues), or E (the last one residue) of the three copies did not abolish the activity of 3B to inhibit IFN-β production. However, mutation of the 17th alanine to glutamicacid (A/E) completely abolished the activity of 3B to inhibit IFN-β production (Fig. 10F). The interaction between single 3B and RIG-I was investigated as well, all of the three copies of 3B interacted with RIG-I (Fig. 10G). The interaction between the other 3B mutants and RIG-I was further investigated, which showed that all of the mutants interacted with RIG-I (Fig. 10H). These results indicated multiple sites in 3B were involved in 3B–RIG-I interaction. The 3B-A/E mutant that lost the potential to inhibit IFN-β transcription also bound to RIG-I. It suggested that the A–E mutation in 3B possibly just disrupted the activity of 3B to inhibit RIG-I–mediated signal transduction. RIG-I, as an ISG, also has a direct antiviral activity, other mechanisms might also be involved in 3B–RIG-I interaction.

To confirm the function of the residues of 17A in 3B, a recombinant virus bearing the 3B-A/E instead of 3B was generated, and the parental wild-type virus was named as r3B-FMDV and the recombinant virus was named r3B-A/E-FMDV (Fig. 11A). The two viruses showed similar viral titers in the BHK-21 cells (Fig. 11B). We further evaluated the replication status of the two viruses in PK-15 cells. PK-15 cells were infected with equal amount of r3B-FMDV and r3B-A/E-FMDV, respectively, the expression of viral RNA, IFN-β, and ISGs were measured at 12 hpi. r3B-A/E-FMDV showed a decreased replication ability compared with r3B-FMDV. However, it induced increased IFN-β and ISGs expression (Fig. 11C). These results confirmed that the 17A in each copy of FMDV 3B was involved in suppression of IFN-β production.

It is well known that type I IFNs initiate a series of signaling cascades and then induce the expression of hundreds of ISGs, which play important roles not only in host innate immune responses but also in adaptive immune responses (43, 44). Various viruses have been found to subvert the host innate immune response by disruption of the function of the PRRs, adaptor factors, and critical components of the antiviral pathway for IFN-β signaling (11, 19, 24, 43). Modifications in viral 3B protein of FMDV decrease virus yield in different cell lines, suggesting the important role of 3B in FMDV replication (45). Our previous study found that FMDV 3B inhibited SeV-triggered IFN-β promoter activation (29). In this study, we extensively probed the link between the capacity of FMDV 3B protein to inhibit the function of targets in the host innate immune signaling pathway and its capacity to suppress antiviral response. We found that FMDV 3B significantly suppressed SeV-induced IFN-β and NF-κB pathways signaling, thus, considerably decreasing the expression of IFN-β, ISGs, and proinflammatory cytokines. Further investigation identified that 3B blocked the K63-linked ubiquitination of RIG-I, in turn, leading to the decreased interaction of RIG-I and MAVS. Therefore, RIG-I is the target of FMDV 3B to attenuate host antiviral response.

RIG-I, as a critical cytosolic sensor for IFN-β pathway, plays a critical role in host antiviral response. Upon viral infection, RIG-I is redistributed and polyubiquitinated to bind the adaptor protein MAVS, which initiates the host antiviral immune signaling (41). Given the importance of RIG-I in antiviral immune signaling, it is not surprising that viruses have evolved a variety of strategies that target RIG-I to antagonize IFN response. Many studies have reported that viral proteins target RIG-I by different mechanisms to block IFN pathway activation. For example, poliovirus 3C proteinase cleaves RIG-I during viral replication to attenuate host antiviral response (46). Ebola virus VP35 protein binds dsRNA to prevent the sensing of viral RNA by RIG-I (47). Human respiratory syncytial virus NS2 protein binds to RIG-I to inhibit the RIG-I–MAVS interaction (48). This suggests that RIG-I–mediated activation process can be disrupted by viral proteins in various ways.

The N-terminal CARD domain of RIG-I is critical for MAVS binding and downstream signaling (9). In this study, we identified that FMDV 3B protein impaired the K63-linked ubiquitination of RIG-I to interfere with RIG-I–MAVS complex formation and antagonize downstream signaling. TRIM25 interacts with the CARD domain of RIG-I, and this interaction effectively induces the K63-linked ubiquitination of RIG-I, leading to its interaction with MAVS (42). FMDV 3B interacted with the CARD domain of RIG-I and blocked the interaction of the E3 ubiquitin ligase TRIM25 with RIG-I, then resulting in the decreased K63-linked ubiquitination of RIG-I and RIG-I–MAVS complex formation to suppress IFN production and prevent an efficient host immune. Influenza virus NS1 suppresses the K63-linked ubiquitination of RIG-I by interacting with the E3 ubiquitin ligase TRIM25 and blocking TRIM25 multimerization (49). This suggests that the disruption of RIG-I–TRIM25 interaction is a target for different viruses to impair host antiviral response. The DEAD helicase domain is involved in RIG-I filament formation and pathway activation. The interaction of 3B with DEAD helicase domain might also interfere with the function of RIG-I by other unknown mechanisms. The very recently prominent papers have proposed that the E3 ligase RIPLET promotes RIG-I–mediated immune signaling through both Ub-dependent and -independent manners, which may be more important than TRIM25 (50, 51). In this study, we confirmed that 3B inhibited the K63-linked ubiquitination of RIG-I. Overexpression of TRIM25 promoted the K63-linked ubiquitination of RIG-I, and 3B blocked TRIM25-induced K63-linked ubiquitination of RIG-I. In addition, 3B interacted with both the CARD and DEAD helicase domains. There might be an alternative model that 3B binds RIG-I and keeps it in a “locked” state, which will make RIG-I inaccessible for any of E3 ligases including the RIPLET.

A recent study reported that the host zinc-finger protein ZCCHC3 promotes the K63-linked ubiquitination of RIG-I (52). Although it remains unknown whether FMDV 3B protein affects the ZCCHC3-mediated K63-linked ubiquitination of RIG-I, the current study identified a new strategy for FMDV to suppress RIG-I–mediated immune signaling by inhibiting the activation of RIG-I. Apart from sensing of viral RNA, RIG-I also serves as an ISG and functions as a direct antiviral effector (53, 54). For example, RIG-I promotes the disassembly of the viral polymerase complex to suppress influenza virus replication (55). RIG-I counteracts the interaction between hepatitis B virus polymerase and pregenomic RNA to suppress viral replication (56). The direct antiviral function of RIG-I in FMDV-infected cells should be further investigated. Whether 3B protein is also involved in attenuation of the antiviral function of RIG-I should be exploited. In this study, we also found that 3B suppressed the adaptor protein MAVS-mediated ISRE activation. However, 3B did not affect the transcripts and protein levels of MAVS, and we did not observe the interaction between 3B and MAVS. The involved mechanism will be further investigated. Besides, our data showed that FMDV 3B could not completely abolish the SeV-induced RLRs pathway activation (Figs. 1, 2). However, 3B, indeed, efficiently inhibited RIG-I function and promoted FMDV replication (Figs. 3, 4). The IFN-β expression could also be detected in the FMDV-infected RIG-I KO cells. Therefore, we speculated that other PRRs and pathways might have also been involved in the initiation of host innate immune response during FMDV infection. RIG-I KO decreased IFN-β production almost by 50%, and this still left many of IFNs in the system, which may induce substantial expression of ISGs. Multiple antagonistic mechanisms are used by different viral proteins during FMDV infection. Some of the viral proteins target the components of RLRs pathway, and some of the viral proteins target the JAK-STAT pathway or even directly interfere with the functions of several ISGs. There might be a lot of synergy among different viral proteins and a viral replication cascade effect. Therefore, the effect of the RIG-I KO on FMDV replication is very significant. 3B effectively blocked RIG-I–mediated immune signaling. Whether 3B could also target other PRRs or other antiviral proteins remains unknown.

FMDV is distinguished from other members of the Picornaviridae by the presence of three copies of 3B (2). Therefore, the role of 3B in different picornaviruses might not be completely same. FMDV 3B protein plays important roles in viral replication. Deletion of the 3B3 sequence within the full-length infectious cDNA resulted in the production of a noninfectious RNA transcript (21). Modifications in FMDV 3B protein could decrease virus yield in porcine and bovine cells. However, it does not significantly affect the ability of FMDV to grow in highly susceptible BHK cells (45). In the current study, we also determined that overexpression of 3B did not affect FMDV replication in BHK-21 cells (Fig. 6J), and mutation of the 17A in each copy of 3B resulted in a decreased replication of FMDV in PK-15 cells but not in BHK-21 cells (Fig. 11). This implied the multiple roles of 3B protein during viral replication. In the current study, we determined the antagonistic role of 3B on host innate immune signaling, and showed that 3B1, 3B2, and 3B3 all inhibited IFN-β production (Fig. 2G). This suggested that the three nonidentical copies are all involved in suppression of host antiviral response. 3B may exist as part of a larger precursor of FMDV. This suggested that the precursor of 3B might be involved in FMDV-mediated immune evasion process as well. 3B-mediated antagonistic role was critically involved in the virulence and pathogenesis of FMDV. BHK-21 cells do not have a normal innate immune signaling system. This might be one of the reason that why modification of 3B does not affect the viral replication. In contrast, it has been reported that picornavirus 3B possesses a regulatory effect on 3A function (57). Modifications in viral 3B protein might potentially affect the function of 3A and results in the decreased virus yield. These studies imply the multifunctional role of 3B in viral replication.

In conclusion, we identified the antagonistic role of FMDV 3B on RIG-I–mediated immune signaling. Furthermore, we revealed that FMDV 3B interacted with the CARD domain of RIG-I to block the RIG-I–TRIM25 interaction, preventing TRIM25-mediated, K63-linked ubiquitination and activation of RIG-I. These findings could improve our understandings of the pathogenesis and immune evasion mechanisms of FMDV.

We thank Dr. Xin Mu (Tianjin University) for discussion and suggestions and Prof. Hongbing Shu for the gift of SeV.

The authors have no financial conflicts of interest.