Foot-and-mouth disease virus capsid protein VP1 interacts with host ribosomal protein SA to maintain the activation of MAPKs signal pathway and promote virus replication.

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.

four structural proteins and eight nonstructural proteins (NSPs). The structural proteins VP1, VP2, VP3, and VP4 form the viral capsid, and the other NSPs are mainly involved in viral replication and the pathogenesis process (2). Recent studies have demonstrated that some NSPs antagonize host antiviral responses by modulating immunity (3,4). The VP1 protein, as the main immunogenic component of FMDV particle, has also been determined to play additional roles in regulating host responses (5,6). The interaction between VP1 and host proteins may determine the outcome of disease and pathogenesis as well. However, the host proteins that were targeted by VP1 in the viral replication cycle and the involved mechanisms are not yet fully understood.
To better understand the potential pathogenic consequences of VP1-host protein interactions, we performed a yeast two-hybrid (YTH) assay as previously described and reported that VP1 interacted with host DnaJ heat shock protein family member A3 (DNAJA3) and showed that DNAJA3 played an essential role for suppression of FMDV replication (7). In the present study, we report that FMDV VP1 interacted with host ribosomal protein SA (RPSA), a component of the ribosome (8,9). RPSA, also known as laminin receptor 1, is ubiquitously expressed and plays multifunctional roles in various cellular responses. RPSA is involved in RNA processing (10), cell migration (11), angiogenesis (12), and human spleen development (8). It is also suggested that RPSA is involved in regulation of the mitogen-activated protein kinase (MAPK) signaling pathway correlating with tumor dissemination (13). However, whether it includes a regulatory function during viral replication remains unknown. The MAPK signaling cascade regulates a wide variety of physiological processes, such as cell differentiation, survival, growth, cancer-immune evasion, and apoptotic cell death (14,15). Both extracellular and intracellular stimuli, such as cytokines, hormones, oxidative stress, and virus infection, can activate MAPK pathways (16)(17)(18).
Deviation from the well-balanced control of MAPK signaling cascade has been implicated in many virus infections. For example, Ebola virus persistently infects and escapes from cells through activation of the Ras/MAPK pathway (19). Influenza A virus-induced MAPK signaling cascade results in a large amount of cytokines production and airway inflammation (20). Infectious salmon anemia virus activates MAPK pathway to favor the synthesis of new viral progeny by promoting cellular apoptosis (21). RPSA is involved in regulation of MAPK signaling pathway. Here, VP1 interacted with RPSA, which implied a potential role of FMDV in manipulating the MAPK signal pathway.
The VP1-RPSA interaction initially identified by the YTH assay was confirmed by a coimmunoprecipitation assay. We further showed that the overexpression of RPSA decreased FMDV replication, and knockdown of RPSA enhanced FMDV replication within FMDV-infected cells. FMDV infection induced the activation of MAPK signaling cascade. Suppression of the MAPK signal pathway by the MAPK pathway-specific inhibitor U1026 significantly impaired FMDV replication. RPSA played an essential role in suppression of the MAPK signal pathway activation during FMDV infection. Overexpression of FMDV VP1 impaired the RPSA-mediated suppressive effect on the MAPK signal pathway activation. Overexpression of VP1 truncated mutant with abolished ability to bind with RPSA showed no effect on MAPK signal pathway activation, supporting the possibility that the interaction between VP1 and RPSA contributed to the increased MAPK signaling and enhanced viral replication within FMDV-infected cells.

RESULTS
FMDV structural VP1 protein interacted with porcine host protein RPSA. Cellular RPSA was identified as a potential target of FMDV VP1 by YTH assay in our previous study (7). To confirm the VP1-RPSA interaction, we performed the coimmunoprecipitation assay. Another VP1-targeted candidate host protein, vimentin (VIM), screened by YTH assay and two VP1-nonbinding proteins, esterase D (ESD) and tropomyosin 4 (TPM4), were also analyzed in the experiment. Human embryonic 293T (HEK-293T) cells were cotransfected with Myc-vector-, Myc-VIM-, Myc-RPSA-, Myc-ESD-, or Myc-TPM4-, and Flag-VP1-expressing plasmids. The cell lysates were immunoprecipitated with a mouse monoclonal anti-Myc antibody, and the coprecipitated proteins were analyzed by immunoblotting with a mouse monoclonal anti-Flag antibody. We found that RPSA efficiently pulled down VP1; however, VIM, ESD, and TPM4 did not pull down VP1 (Fig.  1A). A reverse immunoprecipitation using a rabbit polyclonal anti-Flag antibody was also performed. Similarly, VP1 pulled down RPSA, and VP1 did not pull down VIM, ESD, and TPM4 (Fig. 1B). These results suggested that VP1 interacted with RPSA. To confirm that the VP1-RPSA interaction occurs in FMDV-infected cells, the subcellular localization of VP1 and RPSA in FMDV-infected cells was evaluated. The colocalization of VP1 and RPSA was clearly observed in the cytoplasm of FMDV-infected PK-15 cells (Fig. 1C). The coimmunoprecipitation assay was also performed using the lysates from FMDVinfected cells. RPSA clearly pulled down FMDV VP1 protein in the context of viral infection (Fig. 1D). The reverse immunoprecipitation assay was similarly performed using the anti-VP1 antibody, and the results showed that VP1 also immunoprecipitated RPSA (Fig. 1E). Together, these results suggested that FMDV VP1 interacted with host RPSA protein.
RPSA was not a host receptor for FMDV infection. RSPA is ubiquitously distributed in host cells, and it is also expressed on the cell surface. RPSA is a viral receptor for dengue virus and classical swine fever virus (22,23). Here, we showed that FMDV VP1 interacted with host RPSA in the context of viral infection. To investigate whether RPSA is a potential receptor for FMDV, we performed an antibody inhibition assay. RPSA spans the plasma membrane once with its C terminus exposed to the extracellular space (24). We used two antibodies against the N terminus or C terminus of RPSA for the inhibition assay. Baby hamster kidney (BHK-21) or porcine kidney (PK-15) cells were preincubated with the antibody against the N terminus or C terminus of RPSA, and the cells were then infected with FMDV. The results indicated that neither antibody had an inhibitory effect on FMDV infection at a concentration of 1 or 10 g/ml ( Fig. 2A and B). Whether FMDV used some other alternative receptors during the blocking process remained unknown. The present data suggested that blocking of RPSA had no significant interference on FMDV entry. The expression of RPSA in BHK-21 and PK-15 cells was confirmed by immunoblotting with both the N-terminus and C-terminus RPSA anti- bodies, which showed that RPSA could be detected from both BHK-21 and PK-15 cells using the two antibodies (Fig. 2C). The cellular membrane fractions of BHK-21 and PK-15 cells were extracted, and the expression of RPSA in the membrane fractions was also confirmed (Fig. 2D). We further overexpressed porcine RPSA in HEK-293T cells, which are not susceptible to FMDV infection. HEK-293T cells were transfected with increasing amounts of Myc-RPSA plasmids for 24 h and then infected with FMDV for 12 h. The cells were subjected to Western blotting to evaluate the viral infection status in the cells. The overexpression of RPSA did not cause the virus replication in HEK-293T cells (Fig. 2E). We also determined that overexpression of RPSA did not promote the virus attachment to HEK-293T cells (data not shown). The expression of Myc-RPSA in the cellular membrane fractions of HEK-293T cells was also confirmed by Western blot analysis (Fig. 2F). These results indicated that RPSA was not a receptor for FMDV infection.

RPSA inhibited FMDV replication during virus infection.
To access the role of RPSA during FMDV infection, we evaluated FMDV replication level in cells overexpressing RPSA. Porcine kidney (PK-15) cells were transfected with different amounts of RPSA-expressing plasmids for 24 h; empty vector plasmids were used in the transfection process to ensure the cells received same amounts of total plasmids. The transfected cells were then infected with FMDV for another 12 h. The expression of viral proteins was then detected by Western blotting. A considerable reduction of viral proteins expression was observed in the RPSA-overexpressing cells compared to cells transfected with vector plasmids. The total expression of RPSA in the virus-infected cells was measured, and this showed that RPSA inhibited the viral replication in a dosedependent manner (Fig. 3A). The viral RNA in the cells and the viruses in the cell culture supernatant were also measured. As expected, both the viral RNA and virus yields significantly decreased in the RPSA-overexpressing cells compared to vector- transfected cells (Fig. 3B). Furthermore, the viral RNA, viral protein, and virus yields in the RPSA knockdown cells were measured. The knockdown of RPSA was performed by transfection of small interference RNA (siRNA) that targets RPSA. PK-15 cells were transfected with the negative-control siRNA (NC siRNA) or RPSA siRNA for 36 h, and the expression of endogenous RPSA was assessed by Western blotting to validate the silencing efficiency of the siRNAs used in the present study. Transfection of NC siRNA did not affect the basal expression of RPSA protein. The synthesized siRNA-1 and siRNA-2 efficiently silenced both mRNA and protein expression of RPSA (Fig. 3C). PK-15 cells were transfected with siRNA-1 or siRNA-2 for 36 h and then infected with equal amounts of FMDV for 12 h. The expression of RPSA was detected, and the viral RNA, viral protein, and virus yields were measured, respectively. Knockdown of RPSA caused remarkable increase in viral RNA, viral protein expression, and virus yields (Fig. 3D). These results indicated that RPSA played an antiviral role during FMDV infection.
FMDV infection activated MAPK signal pathway to promote virus replication. As mentioned previously, RPSA is a multifunctional protein. How RPSA inhibits FMDV replication remains unknown. To uncover the involved mechanism of RPSA-mediated anti-FMDV effect, we first explored the expression of RPSA in FMDV-infected cells. PK-15 cells were infected with FMDV and collected at 0, 2, 4, 8, 12, or 16 hpi, and then the cells were subjected to Western blotting. The basal expression level of RPSA in the mockinfected cells was also evaluated. No remarkable change of RPSA protein abundance was observed in the mock-infected cells at different times. A gradual increase in RPSA protein abundance was observed as infection progressed at the early infection period (from 2 to 8 h postinfection [hpi]), and the abundance remained almost unchanged after 8 hpi (from 8 to 16 hpi) (Fig. 4A). This indicated that the expression of RPSA was upregulated at the early stage during FMDV infection. The status of MAPK signal pathway during FMDV infection was also investigated. The degrees of total and phosphorylated ERK, JNK, and p38 kinases at 0, 2, 4, 8, 12, and 16 hpi were examined by Western blotting. We found that MAPK signaling was activated in FMDV-infected cells. As shown in Fig. 4B, FMDV infection remarkably triggered the phosphorylation of JNK1/2, ERK1/2, and p38, and the phosphorylation of JNK1/2, ERK1/2, and p38 started at 2 hpi, indicating that all of these three MAPKs were activated in response to FMDV infection.
It has been reported that MAPKs are phosphorylated during various virus infection and that the activation of MAPK pathway promotes the replication of many viruses (17,25,26). The role of MAPK pathway during FMDV infection remains unknown. To explore the effect of MAPK activation on FMDV replication, a chemically synthesized organic compound U0126 that specifically inhibits the activation of MAPK (ERK1/2) was used, as previously described (27). An MTT assay was performed to evaluate the compound on cell viability which showed that there was no significant effect on the viability of PK-15 cells treated with 20 M U0126 (data not shown). PK-15 cells were pretreated with U0126 and then infected with FMDV. Pretreatment of U0126 significantly suppressed ERK1/2 phosphorylation and resulted in dramatically decreased replication of FMDV (Fig. 4C). A time course analysis further confirmed that FMDV infection activated MAPK signaling and pretreatment of U0126 considerably blocked FMDV replication (Fig. 4D). MAPK activation plays a critical role in mediating proinflammatory cytokine production (28). To evaluate the expression of proinflammatory cytokines in FMDV-infected cells pretreated with U0126, the total RNA was extracted from infected cells pretreated with dimethyl sulfoxide (DMSO) control or U0126. The transcripts of CCL2, CCL5, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-␣), and FMDV were measured by qPCR. We found that the expression of CCL2, CCL5, IL-6, and TNF-␣ was inhibited through the course of infection in U0126-pretreated cells and that viral RNA abundance was also decreased in U0126-pretreated cells (Fig. 4E). This suggested that MAPK signaling played a key role on FMDV replication in PK-15 cells and that the upregulation of proinflammatory cytokines was involved in this process.
RPSA inhibits MAPK signaling in FMDV-infected cells to suppress viral replication. RPSA is involved in regulation of MAPK signaling pathway correlating with tumor dissemination (13). However, little is known about the association of RPSA with MAPK signaling in virus-infected cells. To investigate whether RPSA regulates MAPK signaling during FMDV infection, PK-15 cells were transfected with RPSA-expressing plasmids or empty vector for 24 h and then infected with FMDV. The expression of total and phosphorylated MAPKs was examined by Western blotting. The upregulation of RPSA significantly suppressed the phosphorylation of JNK1/2, ERK1/2, and p38 induced by FMDV, and the viral replication was also decreased (Fig. 5A). This suggested that RPSA suppressed the activation of MAPKs signal pathway to inhibit FMDV replication. The regulative role of RPSA on MAPK signaling in FMDV-infected cells was also investigated in the RPSA knockdown cells. PK-15 cells were transfected with RPSA siRNA to silence the expression of RPSA, and the NC siRNA was used as a control. The transfected cells were infected with FMDV at 36 h posttransfection for 0, 4, or 8 h, and the state of MAPKs signaling was evaluated. Knockdown of RPSA remarkably promoted the phosphorylation of ERK1/2, JNK1/2, and p38 during FMDV infection. As expected, FMDV replication was considerably enhanced in the RPSA knockdown cells (Fig. 5B).
Knockdown of RPSA resulted in highly enhanced MAPK phosphorylation and viral replication in FMDV-infected cells. We further investigated whether this enhancive effect could be blocked by incubation of U0126. The results showed that incubation of U0126 clearly blocked MAPKs phosphorylation in RPSA knockdown cells during FMDV infection, and the viral replication was also strikingly decreased (Fig. 5C). The silence of RPSA enhanced MAPK signaling and FMDV replication, and this enhancive effect can be blocked by treatment of U0126. This indicated that the activation of MAPKs signal pathway was essential for FMDV replication, and RPSA performed a suppressive role on the activation of the MAPK signal pathway to inhibit FMDV replication.
The activation of the MAPK signal pathway by virus infection results in the expression of a large amount of proinflammatory cytokines. To confirm the regulative role of RPSA on the MAPK signal pathway activation during FMDV infection, the expression of proinflammatory cytokines in FMDV-infected RPSA-overexpressing cells and RPSA knockdown cells was subsequently evaluated. The results showed that the expression of CCL5, IL-6, and TNF-␣ was impaired by the upregulation of RPSA, accompanied by a decrease in FMDV replication and yields (Fig. 5D). In contrast, the expression of CCL2, CCL5, IL-6, and TNF-␣ were considerably enhanced in RPSA knockdown cells, with an increased FMDV replication and yields (Fig. 5E). Together, these results indicated that RPSA inhibited MAPK signal pathway activation in FMDV-infected cells to suppress viral replication.
FMDV VP1 interacted with RPSA to repress the RPSA-mediated effect on the regulation of MAPK signaling. As described above, FMDV VP1 interacted with RPSA.  To evaluate the role of the VP1-RPSA interaction on MAPK signaling during FMDV replication, the expression of total and phosphorylated MAPKs in RPSA-overexpressing cells was evaluated in the absence or presence of Flag-VP1 protein. We found that the overexpression of VP1 restored the phosphorylation of ERK1/2, JNK1/2, and p38 which had been suppressed by RPSA. As shown in Fig. 6A, the overexpression of RPSA significantly suppressed ERK1/2, JNK1/2, and p38 phosphorylation at 4 and 8 hpi. However, this inhibitive effect was considerably impaired by the overexpression of VP1. The restored phosphorylation of MAPKs in turn resulted in increased FMDV replication (Fig. 6A).
To identify the region of FMDV VP1 that was responsible for the VP1-RPSA interaction, a series of Flag-tagged truncated VP1 constructs were constructed (Fig. 6B). Myc-RPSA and Flag-VP1 or Flag-VP1 truncated mutants were cotransfected into HEK-293T cells. The interaction status was investigated by a coimmunoprecipitation assay. The results showed that all of the truncated mutants could bind to RPSA, suggesting that multiple regions might be involved in VP1-RPSA interaction (Fig. 6C). We further constructed three Flag-tagged truncated VP1 constructs that included longer regions of VP1 (Fig. 6D). The interaction between the VP1 mutants and RPSA was further investigated. The N-terminal region of VP1 from amino acids 1 to 115 did not bind to RPSA, and the regions of VP1 from amino acids 37 to 188 and amino acids 115 to 214 bound to RPSA (Fig. 6E). The truncated mutant 1-180 (VP1-Δ180-214) could bind to RPSA  (Fig. 6C). These results suggested that there was no binding site in the 1-115 region of VP1 and that there was at least one binding site or region located in the 115-180 region of VP1.
The VP1-1-115 mutant did not interact with RPSA. To investigate whether this region also lost the ability to regulate the MAPK signal pathway during FMDV infection, the effect of VP1-1-115 on MAPK signaling in FMDV-infected RPSA-overexpressing cells was evaluated. The results showed that overexpressing Flag-VP1-1-115 did not restore the phosphorylation of ERK1/2, JNK1/2, and p38 that had been suppressed by RPSA (Fig.  6F). Meanwhile, the transcripts of IL-6, TNF-␣, and FMDV were investigated in FMDVinfected RPSA-overexpressing cells in the presence or absence of Flag-VP1 or Flag-VP1-1-115. The wild-type VP1 protein enhanced IL-6 and TNF-␣ expression, leading to an increased replication of FMDV. However, the VP1-1-115 mutant did not enhance the expression IL-6 and TNF-␣ and also failed to promote FMDV replication (Fig. 6G). The effect of VP1-37-188 and VP1-115-214 mutants on MAPK signaling in FMDV-infected RPSA-overexpressing cells was further evaluated. The results showed that overexpression of either VP1-37-188 or VP1-115-214 mutants restored the phosphorylation of ERK1/2 and abolished Myc-RPSA-mediated antiviral activity against FMDV (Fig. 6H). These results suggested that VP1 interacted with RPSA to restore the activation of MAPKs signaling and promote FMDV replication.

DISCUSSION
FMDV utilizes various strategies to counteract host antiviral responses for its replication (4). However, the involved mechanisms are not fully understood. One potential strategy that FMDV may use to counteract host antiviral response involves interaction with host proteins, enabling the virus to impair the function of host protein and subverting natural signal transduction in favor of viral replication. In our previous work, we determined that FMDV 2B protein interacted with cellular RIG-I and LGP2 to suppress host antiviral responses, promoting viral replication (3,29). Here, we identified that the FMDV structural protein VP1 interacted with the host RPSA protein, a multifunctional and ubiquitously expressed host protein that regulates various physiological processes.
VP1 is an important structural protein of picornavirus that reveals the ability to bind to various host proteins. For example, VP1 of enterovirus 71 (EV71) interacts with host annexin II on the cell surface to enhance viral entry and infectivity (30). FMDV VP1 interacts with host soluble resistance-related calcium-binding protein to inhibit the type I interferon response in favor of viral replication (5). FMDV VP1 is extensively involved in the regulation of FMDV infection and replication. The amino acid sequence RGD at positions 145 to 147 and the C-terminal region of VP1-203-213 are involved in cell attachment by binding to host integrins (31,32). Moreover, VP1 inhibits IRF3 phosphorylation to impair type I interferon production and promote virus replication. The host DNAJA3 protein also interacts with the C-terminal 188 -211 region of VP1 to induce the lysosomal degradation of VP1 and attenuate VP1-mediated suppressive effect on interferon production (7). The 135-144 region of FMDV VP1 contains both Band T-cell epitopes and has been used as an immunogen in hosts (33). RPSA is identified as a receptor for several viruses, including adeno-associated virus serotypes 2, 3, 8, and 9 and dengue virus (23,34). However, in the present study, we found that incubation of the cells using the anti-RPSA antibodies did not block the infection of FMDV. The 115-180 region of VP1 was involved in RPSA-VP1 interaction. Although this region also includes the RGD sequence that is responsible for cell attachment by FMDV, deletion of the 120 -155 region of VP1 that contains the RGD sequence did not affect RPSA-VP1 interaction. We suggest that RPSA is not a cellular receptor for FMDV entry. In contrast, overexpression of RPSA resulted in a decreased replication of FMDV, and knockdown of RPSA increased FMDV replication. RPSA-VP1 interaction did not affect the viral entry, and it played an antiviral function during FMDV infection. The 115-180 region of VP1 did not overlap the binding region for DNAJA3, indicating that RPSA was not involved in DNAJA3-induced degradation of VP1.
RPSA induces the dephosphorylation of kinases ERK, JNK, and p38 and decreases the activity of the three MAPK cascades in tumor cell lines (13). The MAPK pathway is activated and manipulated by diverse group of viruses during viral infection (35). However, little is known about the MAPK signal pathway in FMDV-infected cells. Excessive inflammation is becoming accepted as a critical factor in many viral infection diseases. MAPKs are important regulators of inflammatory cytokine and chemokine expression, and many viruses have been shown to exploit MAPK signaling by causing excessive inflammation for their own replication (20,28,35). Excessive inflammatory response has been associated with FMDV-induced diseases (36)(37)(38). A recent study reported that in evolved BHK-21 cell lines that were insensitive to FMDV infection, the MAPK signaling was weaker than in wild-type BHK-21 cells, suggesting that the decreased MAPK signaling may contribute to the resistance of cells to FMDV infection (39). In the present study, we demonstrated that FMDV infection activated the MAPK signal pathway, and we further showed that the MAPK signal pathway positively regulated FMDV replication. Treatment of cells with the specific inhibitors of MAPK signal pathway U0126 significantly blocked MAPK signaling and suppressed FMDV replication. This confirmed that the MAPK pathway is manipulated by FMDV and that the MAPK signal pathway might be a potential target for the suppression of FMDV infection.
RPSA repressed MAPK signaling in PK-15 cells; therefore, it is easily understood that the overexpression of RPSA suppressed FMDV replication and the knockdown of RPSA enhanced FMDV replication. RPSA performed its antiviral function against FMDV by the suppression of MAPK signal pathway activation. The MAPK signal pathway is also required for EV71 replication (40). Whether RPSA also suppresses EV71 and other picornaviruses remains unknown. Moreover, the mechanism by which RPSA suppresses MAPK signaling should further be investigated.
It is widely known that FMDV becomes an effective pathogen by evading the host immune response. Various FMDV proteins have been shown to interact with host proteins to suppress host antiviral response, including VP1 protein (4). Here, we identified that VP1 interacted with RPSA to impair the inhibitive effect of RPSA on MAPK signal pathway activation. VP1-RPSA interaction enhanced MAPK signaling and promoted FMDV replication. FMDV infection causes excessive inflammation in the oral cavity and the coronary bands (38); the unbalanced activation of MAPK signaling might be involved in the excessive inflammation, and VP1 protein might be one of the viral factors responsible for induction of the disease.
In conclusion, the present study determined the positive regulatory role of the MAPK signal pathway in FMDV replication and demonstrated the antiviral role of RPSA by repressing MAPK signaling during FMDV infection. It also showed a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and counteract the RPSA-mediated antiviral effect.

MATERIALS AND METHODS
Cell lines and reagents. PK-15, BHK-21, and HEK-293T cells were maintained in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin and then cultured at 37°C under 5% CO 2 . PK-15 and BHK-21 cells are susceptible to FMDV infection and were used for viral infection experiments in the present study. HEK-293T cells are easily transfected and have been extensively used as an expression tool for host or viral proteins and used to investigate protein-protein interactions. We also used HEK-293T cells to perform the coimmunoprecipitation assays and identify the protein-protein interactions. The commercial antibodies used in this study were as follows: anti-Flag M2 antibody produced in mice (Sigma, St. Louis, MO), anti-Flag antibody produced in rabbits (Sigma), anti-␤-actin mouse antibody (Thermo Scientific, Waltham, MA), MAPK family antibody sampler kit (Cell Signaling Technology, Beverly, MA), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit antibody (Cell Signaling Technology), anti-phospho-p38 MAPK (Thr180/Tyr182) rabbit antibody (Cell Signaling Technology), anti-phospho-SAPK/JNK (Thr183/ Tyr185) rabbit antibody (Cell Signaling Technology), anti-RPSA rabbit antibodies (ab245561 and ab133645; Abcam, Cambridge, MA), anti-RPSA mouse antibody (MyBioSource, San Diego, CA), and anti-Myc mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-VP1 antibody was provided by OIE FMD reference laboratory of China (Lanzhou, Gansu, People's Republic of China). The MAPK signal pathway inhibitor U0126 was purchased from Cell Signaling Technology.