Porcine Sapelovirus 3Cpro Inhibits the Production of Type I Interferon

Porcine sapelovirus (PSV) is the causative pathogen of reproductive obstacles, acute diarrhea, respiratory distress, or severe polioencephalomyelitis in swine. Nevertheless, the pathogenicity and pathogenic mechanism of PSV infection are not fully understood, which hinders disease prevention and control. In this study, we found that PSV was sensitive to type I interferon (IFN-β). However, PSV could not activate the IFN-β promoter and induce IFN-β mRNA expression, indicating that PSV has evolved an effective mechanism to block IFN-β production. Further study showed that PSV inhibited the production of IFN-β by cleaving mitochondrial antiviral signaling (MAVS) and degrading melanoma differentiation-associated gene 5 (MDA5) and TANK-binding kinase 1 (TBK1) through viral 3Cpro. In addition, our study demonstrated that PSV 3Cpro degrades MDA5 and TBK1 through its protease activity and cleaves MAVS through the caspase pathway. Collectively, our results revealed that PSV inhibits the production of type I interferon to escape host antiviral immunity through cleaving and degrading the adaptor molecules.


INTRODUCTION
Porcine sapelovirus (PSV) is a non-enveloped positive single-stranded RNA virus, belonging to the Sapelovirus genus within the Picornaviridae family (Adams et al., 2015). The genome of PSV is similar to other picornaviruses: 5′-UTR-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3′-UTR (Krumbholz et al., 2002). Since 1960, PSV infection was reported in the United Kingdom, and it has been reported in other countries around the world, including China, South Korea, and Brazil (Donin et al., 2014;Schock et al., 2014;Son et al., 2014;Li et al., 2019). PSV infection can cause acute diarrhea, poliomyelitis, pneumonia, reproductive disorders, and other clinical symptoms (Lan et al., 2011). The infection of PSV has broad cell tropism in vitro (Li et al., 2019). The molecular mechanism of PSV evading host antiviral innate immunity is still unclear, and the related research contributes to revealing the pathogenicity of PSV infection and the formulation of PSVpreventive measures.
Picornaviruses have evolved strategies to counteract the host innate immune systems. Most members of the Picornaviridae family can evade the host innate immune response by inhibiting the production of type I interferon, thus successfully proliferating and causing infection and pathogenicity. As a member of the Picornaviridae family, the molecular mechanism of PSV evading the host antiviral innate immunity is still unclear. In this study, we took the clinical isolation of PSV strains in our laboratory as the research object, and the association between PSV and the host IFN antiviral responses was investigated. Our study indicates that PSV infection cannot induce the production of type I interferon and it is an interferonsensitive virus. In addition, PSV 3C pro inhibits IFN production by degrading TBK1/MDA5 and cleaving MAVS/TANK.

Western Blotting
Total cellular proteins were prepared using lysis buffer (1.19% HEPES, 0.88% NaCl, 0.04% EDTA, 1% NP-40, and a protease inhibitor) with occasional vortexing. Lysates were then collected by centrifugation at 12,000 rpm for 10 min at 4°C, and protein concentrations were determined by the bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA, USA). The equal amounts of protein for each sample were loaded and separated by 8% to 12% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Welwyn Garden City, UK). Membranes were blocked with 5% skim milk in PBST with 5% Tween 20 (DGBio, Beijing, China) for 2 h at room temperature. The membranes were then washed two times with PBST and incubated with primary antibodies at room temperature for 2 h. Afterward, the membranes were washed five times with PBST and incubated with anti-rabbit or antimouse IgG antibodies conjugated to horseradish peroxidase (HRP) at room temperature for 1 h. The membranes were developed using enhanced chemiluminescence detection reagents (Thermo Scientific, USA).

Luciferase Reporter Gene Assay
HEK293T cells were seeded in 24-well plates, and the monolayer cells were co-transfected with 100 ng/well of pGL3-IFNb-Luc plasmid, 5 ng/well of pRL-TK plasmid (as an internal control), and the indicated expression plasmids or an empty control plasmid. The cells were infected PSV or Sev 20 h after the initial co-transfection. The cells were then collected and lysed, and firefly luciferase and Renilla luciferase activities were determined using the dual-luciferase reporter assay system (Beyotime, Shanghai, China) according to the manufacturer's protocol. Three independent experiments were performed in duplicate. Data are presented as means ± standard deviations(SD).

Immunofluorescence
Cells were fixed in 4% paraformaldehyde at 4°C for 10 min and permeabilized with 0.1% Triton X-100 at room temperature for 10 min; the cells were incubated with 2% BSA in PBS for 1 h at room temperature. The cells were then washed three times with PBS and incubated with anti-IRF3 for 2 h. After four washes with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies at room temperature for 1 h. The cells were washed again four times with PBS and then incubated with DAPI (Sigma, St. Louis, MO, USA) for 10 min, followed by five washes. Fluorescent images were examined using inverted fluorescence.

Real-Time RT-PCR
Total RNA was extracted from the targeted cells using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. 1 µg total RNA was reverse transcribed to cDNA using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme). The relative mRNA level of targeted genes was measured with qPCR using SYBR Green real-time PCR master mix (Applied Biological Materials Inc) with specific prime. All reactions were performed in triplicate. The mRNA level of housekeeping gene GAPDH was used as an internal control. The relative gene fold was determined with the comparative cycle threshold (2 -DDCT ) method.

Statistical Analysis
Statistical analysis was conducted using GraphPad Prism software, version 5. All results were determined by at least three times independent experiments. The various treatments were compared using an unpaired, two-tailed Student t-test with an assumption of unequal variance. P value < 0.05 was considered statistically significant.

RESULT PSV Infection Does Not Induce Type I Interferon Production
Previous studies have reported that IFN-b can inhibit several picornavirus replication, such as FMDV, EMCV, and SVV (Chinsangaram et al., 2001;Wang et al., 2011;Qian et al., 2017). To evaluate whether IFN-b could inhibit PSV proliferation, HEK293T cells were pretreated with different doses of IFN-b for 12 h and then infected with PSV (0.01 MOI) for 24 h. We found that IFN-b inhibited PSV-induced cytopathic effects (CPE) and PSV proliferation in a dosedependent manner ( Figure 1A). Taken together, our results demonstrate that PSV is an interferon-sensitive virus. The impact of PSV infection on the host innate immune response is unknown. The effects of PSV infection on IFN-b promoter activity were analyzed by dual-luciferase assays. We found that IFN-b promoter activity was hardly activated in PSV-infected cells ( Figure 1B). HEK293T cells were infected with PSV (0.1 MOI) or Sev (2 5 HA) for the indicated time, and the expression level of IFN-b mRNA was detected through quantitative realtime RT-PCR assay. Interestingly, the mRNA level of IFN-b hardly increased in PSV-infected cells as the infected time increased ( Figure 1C). Similarly, we observed that the IFN-b promoter activity and the mRNA level of IFN-b hardly increased in PSV-infected PK15 cells ( Figures 1D, E). We also measured the phosphorylation level of IRF3 via Western blotting. HEK293T cells were infected with PSV (0.1 MOI) or Sev (2 5 HA), and then the cells were collected at indicated times. The phosphorylation of IRF3 could not be detected ( Figure 1F). In addition, we also analyzed whether PSV could influence Sevinduced IFN-b production. The quantitative real-time PCR results showed that PSV infection inhibited the expression level of Sev-induced IFN-b mRNA ( Figure 1G). Collectively, these results indicate that PSV infection cannot induce type I interferon production.

PSV 3C pro Negatively Regulates the Production of Type I Interferon
Based on above findings, we evaluated which viral proteins would likely be involved in the inhibition of type I production. HEK293T cells were transfected with plasmids expressing L, VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3C pro , or 3D in combination with IFN-b-Luc and pRL-TK. The results indicated that viral proteins L, 3C pro , and 3D could significantly inhibit IFN-b promoter activity ( Figure 2A). Previous studies revealed that picornaviruses evolved various strategies to inhibit IFN-b production by certain viral proteins, such as L pro , VP2, VP3, 2A pro , 2B, 2C, 3A, 3B, and 3C pro (Dang et al., 2011;Mukherjee et al., 2011; HEK293T cells were seeded in 12-well plates, and the monolayer cells were infected with PSV (0.5 MOI) or not for 3 h, then infected or not with Sev (25 HA) for another 6 and 9 h. The mRNA expression level of IFN-b was evaluated with qPCR assays, and housekeeping gene GAPDH was used as the control. Data are represented. Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.  Bei et al., 2013;Dang et al., 2014;Zixiang et al., 2016;Wen et al., 2019;Xiangle et al., 2020). Our data showed that overexpression of L, 3C pro , and 3D reduced Sevinduced IFN-b production. Moreover, the results of quantitative real-time PCR and Western blotting demonstrated that 3C pro exhibited an extreme inhibitory effect on IFN-b production ( Figures 2B, C). This implies that 3C pro is an antagonist protein to inhibit IFN-b production. HEK293T cells were transfected with an increasing dose of HA-3C for 24 h. As shown in Figure 2D, PSV 3C pro could significantly inhibit Sevinduced IFN-b promoter activity. In addition, PSV 3C pro decreased Sev-induced endogenous transcription of IFN-b in a dose-dependent manner ( Figure 2E). Furthermore, we also found that the phosphorylation of IRF3 gradually reduced when the protein level of PSV 3C pro was increased ( Figure 2F). Taken together, these data confirm that PSV 3C pro inhibits type I IFN production in a dosedependent manner.

PSV 3C pro Suppresses Sev-Induced Type I IFN Production Through Its Protease Activity
Similar to other picornaviruses 3C pro , PSV 3C pro contains the conserved catalytic box with histidine (His) and cysteine (Cys) residues ( Figure 3A). Therefore, we constructed a series of PSV 3C pro mutation expression plasmids, including single-site mutations H40A and C146A and double-site mutation H40A-C146A (3CDM) ( Figure 3B). The result showed that wild-type 3C pro (WT) suppressed Sev-induced phosphorylation of IRF3. In contrast, all 3C pro mutants [3C-H40A, 3C-C146A, and H40A-C146A (3C-DM)] lost the ability to inhibit the phosphorylation of IRF3 ( Figure 3C). This result suggested that 3C pro inhibited Sev-induced type I interferon production via its protease activity.

PSV 3C pro Inhibits IFN-b Expression by Targeting the Adaptor MAVS, MDA5, and TBK1
Previous studies demonstrated that several picornavirus 3C proteases cleave or degrade innate immune adaptors to evade host innate immune responses. SVV 3C pro cleaves MAVS, TRIF, and TANK to weaken type I IFN production (Qian et al., 2017). CVB3 and EV71 cleave RIG-I via viral 3C pro (Feng et al., 2014). SVV 3C pro can target RIG-I, IRF3, and IRF7 for degradation to downregulate IFN-b production (Qiao et al., 2018;Wen et al., 2019). To investigate which innate immune adaptors are cleaved or degraded by PSV 3C pro , HEK293T cells were transfected with plasmids expressing FLAG-MAVS, -RIG-I, -MDA5, -IRF3, -TBK1, or -IKKx in combination with an empty vector or plasmids expressing HA-3C. As shown in Figure 4A, overexpression of PSV 3C pro could cleave MAVS, and we observed that TBK1 and MDA5 were degraded by PSV 3C pro . To confirm whether PSV 3C pro degraded TBK1 and MDA5 in a dose-dependent manner, HEK293T cells were co-transfected with increasing amounts of PSV 3C pro and MDA5 or TBK1 for 24 h. Western blotting results showed that the abundance of MDA5 and TBK1 was gradually reduced when the protein level of PSV 3C pro was increased ( Figure 4B). Meanwhile, HEK293T cells were co-transfected with FLAG-MAVS and an increasing dose of PSV 3C pro . We observed that the cleavage products of MAVS were increased by PSV 3C pro in a dose-dependent manner ( Figure 4C). In addition, we also assessed the effects of PSV 3C pro on endogenous MDA5 and TBK1 protein expression and endogenous MAVS cleavage. HEK293T cells were transfected with increasing amounts of HA-3C for 24 h. The result showed that endogenous MDA5 and TBK1 were degraded by 3C pro and 3C pro cleaved endogenous MAVS ( Figure 4D). We also analyzed the expression of MDA5 and TBK1 and the cleavage of MAVS during PSV infection. HEK293T cells were infected with 0.5 MOI PSV for the indicated time. We found that the expression of MDA5 and TBK1 was gradually reduced during PSV infection, and PSV infection induced the cleavage of MAVS ( Figure 4E). Collectively, our data indicated that PSV 3C pro targets MAVS, MDA5, and TBK1 to impair type I IFN production.

PSV 3C pro Reduces the mRNA Level of TBK1 and MDA5
A previous study demonstrated that SVV 3C pro degraded RIG-I through the caspase pathway (Wen et al., 2019). To investigate whether PSV 3C pro -mediated MDA5 and TBK1 is dependent on apoptosis-related caspase activity, proteasome, or lysosome signaling, HEK293T cells expressing MDA5 or TBK1 and PSV 3C pro were treated with DMSO, proteasome inhibitor MG132 (10 mM), lysosomal inhibitor NH4Cl (10 mM), or pan-caspase inhibitor Z-VAD-FMK (50 mM). As shown in Figures 5A, B, we found that none of these inhibitors rescued MDA5 or TBK1 expression. We next examined whether 3C pro -induced reduction of MDA5 and TBK1 depended on its protease activity; HEK293T cells were co-transfected with FLAG-MDA5 or -TBK1 and all 3C pro mutants (H40A, C146A, and H40A-C146A (3C-DM)) for 24 h. Western blot results showed that 3C pro degraded MDA5 and TBK1 through its protease activity ( Figures 5C, D). Next, we tested whether PSV 3C pro downregulated the level of TBK1 and MDA5 mRNA. The result of qRT-PCR suggested that PSV 3C pro could significantly reduce the mRNA level of TBK1 and MDA5 ( Figures 5E, F).

PSV 3C pro Cleaves MAVS Through the Apoptosis Pathway
Previous reports have clearly shown that MAVS could be cleaved by virus proteases (NS3/4A and 3ABC) or caspases (Yang et al., 2007;Yu et al., 2010;Anggakusuma et al., 2016). To determine whether cellular proteases participate in 3C pro -mediated cleavage of MAVS, the effect of proteasome inhibitor MG132, lysosomal inhibitor NH4Cl, and pan-caspase Z-VAD-FMK on the cleavage of MAVS were detected by Western blot. Flag-MAVS and PSV 3C pro were cotransfected into HEK293T cells for 12 h, and then the cells were treated with DMSO, MG132 (10 mM), NH4Cl (10 mM), and Z-VAD-FMK (50 mM) for another 12 h. The results showed that PSV 3C pro lost the ability to mediate the cleavage of MAVS in the presence of Z-VAD-FMK, indicating that caspases were involved in 3C pro -mediated MAVS cleavage ( Figure 6A). To investigate whether 3C protease activity also participated in 3C pro -mediated cleavage of MAVS, FLAG-MAVS and 3C mutants [3C-H40A, 3C-C146A, and 3C-H40A-C146A (3C-DM)] were co-transfected into HEK293T cells for 24 h. Compared to the wild-type PSV 3C pro , all 3C pro mutants could not cleave MAVS ( Figure 6B). To confirm whether PSV 3C pro could induce cell apoptosis, HEK293T cells were transfected with an increased amount of HA-3C. As shown in Figure 6C, PSV 3C pro cleaved PARP1 in a dose-dependent manner. This result revealed that 3C pro could induce apoptosis. Interestingly, we found that PSV could also induce cell apoptosis ( Figure 6D). To determine whether 3C pro induced apoptosis through its protease activity, HEK293T cells were transfected with the HA-3C or 3C pro mutant. PSV 3C pro lost the ability to cleave PARP1 in the presence of Z-VAD-FMK, and PSV 3C pro without protease activity could not cleave PARP1 ( Figure 6E). These data showed that the protease activity was essential for PSV 3C pro -mediated apoptosis.

PSV 3C pro -Mediated MAVS Cleavage Products Lose the Ability to Induce Type I Interferon Production
To investigate the 3C pro -mediated MAVS cleavage site, we constructed three MAVS mutants in which aspartic (D) was replaced with alanine (A) ( Figure 7A). HEK293T cells were transfected with MAVS or MAVS mutants and HA-3C. As shown in Figure 7B, we observed that the cleavage fragment entirely disappeared in MAVS-D429A. Taken together, these results demonstrated that PSV 3C pro cleaved MAVS at D429. MAVS is the key adaptor molecule in RLR signaling pathways.
To investigate whether PSV 3C pro could disrupt MAVSmediated type I interferon production, a dual-luciferase activity assay was performed to detect the IFN-b promoter activity. The result showed that MAVS-mediated IFN-b promoter activity was significantly inhibited by 3C-WT, but not 3C-DM ( Figure 7C). In addition, MAVS-D429A-mediated IFN-b promoter activity was not affected by 3C-WT or 3C-DM, suggesting that MAVS-D429A could resist 3C pro -mediated cleavage and retain the ability of IFN-b promoter activity ( Figure 7C). To confirm the results, HEK293T cells were transfected with MAVS, MAVS-D429A, and 3C-WT or 3C-DM for 24 h, and the cells were collected for qRT-PCR analysis. As expected, MAVS-D429A could induce IFN-b production and 3C-WT did not inhibit MAVS-D429A-mediated IFN-b production ( Figure 7D). We next assessed whether MAVS cleavage fragments maintain the activity of MAVS-mediated IFN-b production; the truncated plasmids expressing the N-terminal (residues 1 to 429; N429) and C-terminal (residues 430 to 540; C430) fragments of MAVS were constructed, and the effect was evaluated by dual-luciferase activity assay and qRT-PCR. The cleavage products cannot activate IFN-b promotor activity and increase the IFN-b mRNA level, indicating that N429 and C430 lost the ability to induce IFN-b production ( Figures 7E, F). Taken together, PSV 3C pro -mediated MAVS cleavage products lose their ability to induce type I interferon production.

DISCUSSION
The innate immune response is the key of host against invading pathogens, and it is the first line of host defense against pathogen invasion (Schoggins and Rice, 2011;Qiao et al., 2018). The type I interferon (IFN) family, including IFN-a and IFN-b, is an essential component of the host innate immune response and the first line of host response against invading pathogens (Sadler and Williams, 2008). Pathogen ligands interact with pattern recognition receptors (PRRs), and thereby the IFN regulators (IRFs) induce interferon (IFN) production. Subsequently, the IFN molecules bind to IFN receptors and activate numerous IFN-stimulated genes (ISGs), which directly or indirectly exert antiviral functions (Schoggins and Rice, 2011;Schneider et al., 2014). However, the host cells have evolved highly specialized mechanisms to detect and resist virus invasion. However, many viruses have developed countermeasures to disrupt these signaling pathways (Dang et al., 2012). Picornavirus mainly destroys the RLR signaling pathway to inhibit IFN production. SVV 3C pro cleaves MAVS, TRIF, and TANK to abrogate host innate immune responses (Qian et al., 2017). The cleavage of TANK by EMCV 3C pro impairs the ability of TANK to inhibit TRAF6-mediated NF-kB signaling, which helps it evade the host innate immune responses (Li et al., 2015). HAV cleaves MAVS to ablate RLR-mediated type I IFN response (Yang et al., 2007). Enterovirus infection induces the cleavage or degradation of host factors, including RIG-I, MDA5, MAVS, TRIF, and IRF7/9 (Xiaobo et al., 2016). FMDV infection restricts the expression of RIG-I, MDA5, and MAVS Zixiang et al., 2016). (E, F) The effects of cleavage fragments of MAVS by PSV 3C pro on MAVS-mediated IFN-b production were assessed via luciferase activity assay and qPCR assay. The expression of MAVS and its mutants was detected by Western blotting using the FLAG antibody. Data are represented as means ± SD. Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001.
Previous studies have reported that some other picornavirus 3C pro impairs type I IFN production (Dang et al., 2012;Xiaobo et al., 2016;Qian et al., 2017). In this study, we found that PSV infection could inhibit type I interferon production. PSV 3C pro inhibited IFN production dependent on its protease activity. PSV 3C pro suppressed type I interferon production by degrading TBK1/MDA5 and cleaving MAVS, which depends on 3C protease activity. This degradation is not associated with cellular proteasome-, lysosome-, or apoptosis-mediated degradation. Surprisingly, our study demonstrated that PSV 3C pro could not interact with TBK1 and MDA5 (data not shown). Previous studies have suggested that FMDV 3A inhibited the expression of MDA5 by disrupting their mRNA level . Similarly, PSV 3C pro could significantly reduce the mRNA levels of TBK1 and MDA5, indicating that PSV 3C pro degrades TBK1 and MDA5 by targeting transcription levels. Meanwhile, we detected the endogenous degradation of TBK1/MDA5 in PSV-infected or PSV 3C pro -overexpressed cells. SVV 3C pro cleaved MAVS depending on its protease activity (Qian et al., 2017). Interestingly, our data suggested that PSV infection and 3C pro expression could induce apoptosis, which is the key to 3C pro -mediated MAVS cleavage. Apoptosis is a unique and important way of programmed cell death, involving genetically determined cell elimination (Fomigli et al., 2000;Sperandio et al., 2000;Martinvalet et al., 2005). Apoptosis usually occurs in the process of cell development and senescence, which maintains the stable number of cells in the tissue. In addition, apoptosis also occurs as a defense mechanism, such as in immune response or when cells are destroyed by diseases or toxic substances (Norbury and Hickson, 2001). Many viruses have developed different strategies to mediate apoptosis to ensure their continued reproduction and/or spread (Cuconati and White, 2002). SVV impacts the extrinsic and intrinsic pathway and then mediates apoptosis (Tingting et al., 2019). Enterovirus 71 (EV71) infection induces apoptosis in many cell lines through impacting the mitochondrial apoptotic pathway (Liang et al., 2004;Shih-Cheng et al., 2004). SARS-CoV ORF7a and 7b induce apoptosis by activating caspase 3 (SS et al., 2007). HIV-1 Vpr destroys mitochondrial transmembrane potential and activates both caspases-8 and 9 to induce apoptosis (Jacotot et al., 2000;Nie et al., 2002). HCV NS4A changes the distribution of mitochondria in cells and causes mitochondrial damage, which finally induces apoptosis by activating caspase-3 (Nomura- Takigawa et al., 2006). Previous studies have reported that SVV 3C pro induced apoptosis depending on its protease activity, which did not directly cleave or interact with PARP1 (Tingting et al., 2019). Similarly, our study showed that PSV 3C pro -induced PARP1 cleavage disappeared in the presence of Z-VAD-FMK.
Previous studies have shown that some picornaviruses could cleave MAVS at different sites. CVB3 and SVV 3C pro induce the cleavage of MAVS at Gly148 (Mukherjee et al., 2011;Qian et al., 2017). EV71 2A pro cleaves MAVS at multiple distinct sites (Bei et al., 2013). MAVS is cleaved during HRV1a infection by viral proteinases 2A pro , 3C pro , and activated caspase-3 (Drahos and Racaniello, 2009). Caspase-3 cleaves MAVS at D429/490 during virus infection and cell apoptosis (Ning et al., 2019); a series of MAVS mutants were constructed in which potential D residues were replaced with A residues. Similar to the previously reported study (Ning et al., 2019), we found that the key site of 3C promediated MAVS cleavage was D429, and the cleavage fragments lost the ability to induce the production of type I IFN. Moreover, our data demonstrated that MAVS D429A still induced IFNb production.
In summary, our results identify that PSV blocks host antiviral innate immunity by cleaving MAVS and degrading MDA5 and TBK1. The comprehensive mechanism of PSV suppressing the host innate immune response needs to be further explored.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
WW drafted the main manuscript and performed the data analysis. WW, QZ and MY planned and performed the experiments. HW, XL, HC, QZ, and PQ were responsible for the experimental design. All authors contributed to the article and approved the submitted version.