Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the  IFN response through inhibitng TRIM25 oligomerization and functional activation of RIG-I/TRIM25

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibitng TRIM25 oligomerization and functional activation of RIG-I/TRIM25 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibitng TRIM25 oligomerization and functional activation of RIG-I/TRIM25 Jiyu Zhang, Hongyan Shi, Liaoyuan Zhang, Tingshuai Feng, Jianfei Chen, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3814773/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Apr, 2024 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus , brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed IFN production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I tandem caspase activation and recruitment domain and TRIM25 CCD domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-I production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses. Swine acute diarrhea syndrome coronavirus Nucleocapsid Interferon RIG-I TRIM25 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coronaviruses (CoVs) infect various animal species and pose a significant threat to both public health and economies [ 1 ]. Over the last two decades, severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2, have caused severe respiratory disease in humans [ 2 ]. As an important livestock species, pigs have been particularly susceptible to severe CoV diseases, thus resulting in substantial economic effects [ 3 ]. Porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (PHEV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine respiratory virus (PRCV) are five distinct swine CoVs. The sixth identified porcine coronavirus, swine acute diarrhea syndrome coronavirus (SADS-CoV), was responsible for two significant outbreaks in China [ 4 ]. SADS-CoV, classified as a swine enteric alpha-CoV, induces swine acute diarrhea syndrome in piglets and leads to vomiting, severe diarrhea and weight loss. The mortality rates of 5-day-old piglets affected by SADS-CoV can reach 90% [ 5 ]. SADS-CoV (family Coronaviridae , genus alphacoronavirus ) is an enveloped, single-stranded positive-sense RNA virus. The genome of SADS-CoV has a typical CoV structure, seven open reading frames encoding four structural proteins, 16 non-structural proteins and an accessory protein [ 6 ]. Nucleocapsid (N) proteins have important functions in the transcription, replication, and assembly phases of viruses [ 7 ]. N proteins can also act as IFN antagonists. For example, the N protein of PDCoV, PEDV, SARS-CoV-1, SARS-CoV-2 and Mouse hepatitis virus (MHV) suppress IFN production through different mechanisms [ 8 – 11 ]. The innate immune response, as the initial barrier against pathogens, encompasses the type I interferon (IFN-I) signaling pathway, which plays an essential role in defending against viral infections [ 12 ]. After the identification of viral RNA within the cytoplasm, retinoic acid-inducible gene I (RIG-I)-like receptors initiate the antiviral response through the activation of signaling cascades [ 13 , 14 ]. CoVs replication produce 5´-ppp RNA intermediates and dsRNA, and RIG-I specifically recognized these non-self-RNA signature in the cytoplasm [ 15 ]. RIG-I detects viral 5´-ppp RNA species and subsequently initiates signaling pathways via mitochondrial antiviral signaling (MAVS) protein, thereby inducing the interferon regulatory factors (IRF3 and IRF7) translocation, as well as the IFN and inflammatory cytokines expression [ 16 , 17 ]. Tripartite motif (TRIM) proteins have three domains: an N-terminal Really Interesting New Gene (RING) domain, one or two B-boxes, and a coiled coil domain (CCD) [ 18 ]. The RING domain of TRIM proteins acts as an ubiquitin E3 ligase to catalyze the target proteins ubiquitination [ 19 ]. Gack and colleagues first demonstrated that RIG-I caspase activation and recruitment domains (CARDs) ubiquitination involves tripartite motif-containing protein 25 (TRIM25) [ 20 ]. RIG-I exhibits a specific interaction with TRIM25 via its CARD, which facilitates the transfer of the K63-linked ubiquitin moiety to another CARD and leads to the interaction with MAVS. Furthermore, the RIG-I splice variant (amino acids 36–80 deletion in the first CARD) is unable to bind TRIM25 and activate downstream effectors [ 21 ]. Viruses evade host immune attack by antagonizing antiviral defenses. Specifically, virus-encoded protein impede the innate antiviral responses of the host by selectively targeting the expression of IFN genes or the effector molecules induced by IFN [ 22 ]. The N protein, which is encoded by CoVs, is the main IFN antagonist. For example, MERS-CoV N interacts with TRIM25 to suppress both IFN-I and IFN-Ⅲ production [ 23 ]. Similarly, SARS-CoV-1 N protein binds to protein activator of protein kinase R (PACT) and TRIM25 to restrain RIG-I activation [ 8 ]. SARS-CoV-2 N protein plays a role in TRIM25 and RIG-I complex by suppressing TRIM25 E3 ligase activity toward RIG-I [ 9 ]. PRRSV N protein competitively interferes with TRIM25-RIG-I interaction to inhibit RIG-I ubiquitination. However, the detailed roles of SADS-CoV N protein in suppressing type I interferon production and antiviral gene expression have not been directly investigated. Here, our findings suggested that TRIM25 is upregulated after SADS-CoV infection and significantly inhibits SADS-CoV infection. We identified that SADS-CoV N protein interacts with TRIM25 CCD domain and RIG-I CARDs, which inhibits TRIM25 multimerization and TRIM25-RIG-I interaction, thereby suppresses RIG-I signaling and IFN production. The study offered valuable mechanism that may facilitate therapeutic drugs development against Coronaviridae family members infection. Materials and methods Cell culture and virus Porcine intestinal epithelial cells (IPI-2I) and human embryonic kidney cells (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, USA) containing 10% fetal bovine serum (Invitrogen, USA) and 1% antibiotic-antimycotic (Invitrogen, USA). SADS-CoV, SeV and vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) were stored at our lab. Plasmids and antibodies Pig full-length TRIM25 (GenBank accession number XM_005656971.3) was cloned from IPI-2I cell cDNA and reconstructed with pCMV-Flag and pCAGGS-HA vectors. The deletion mutants of TRIM25 (RING_1–90 aa, B-boxes_91–200 aa, CCD_180–450 aa, and SPRY_451–630 aa) were constructed on the basis of the full-length TRIM25 plasmid and inserted into the pAcGFP-C1 vector to generate green fluorescent protein (GFP) fusion proteins. TRIM25 DelCCD (lacking the CCD domain) was cloned into the pCAGGS-HA vector, and we named the recombinant plasmid pHA-TRIM25 DelCCD. GFP-SADS-CoV N and N truncated plasmids were stored in our laboratory as previously described [ 24 ]. The plasmids for expression of RIG-I, RIG-IN and RIG-IC with Flag-tag, RIG-I 2CARD with GST-tag, and IFN-β-Luc were preserved at our laboratory. The primer sequences are listed in Table 1 . Monoclonal antibodies specific for SADS-CoV N protein were stocked in our laboratory [ 24 ]. We purchased antibody against DDDDK (ab205606), HA (ab9110), TBK1 (ab40676), p-TBK1 (ab109272), IRF3 (ab68481), p-IRF3 (ab76493), GFP (ab290), GST (ab138491) or TRIM25 (ab167154) from Abcam (Cambridge, MA). We purchased antibody against Myc (M4439) or GAPDH (G9545) from Sigma-Aldrich (St. Louis, MO, USA). RNase A, Alexa Fluor 488 goat anti-mouse IgG (H + L) and Alexa Fluor 594 goat anti-rabbit IgG (H + L) secondary antibodies were purchased from Thermo Fisher Scientific (Carlsbad, CA). Poly(I:C) was purchased from InvivoGen (Hong Kong, China). Table 1 PCR primer sequences used in this study Names Sequences(5’-3’) HA-TRIM25 F AGATTACGCTGAATTAATGGGCGGAACTGTGCCC HA-TRIM25 R GATCTGCTAGCTCGACTACCTGGTGGAGCAGATGGAGA Flag-TRIM25 F AGATTACGCTGAATTAATGGCGGAACTGTGCCC Flag-TRIM25 R GATCTGCTAGCTCGACTACCTGGTGGAGCAGATGGAGA GFP-RING F GGACTCAGATCTCGAATGGCAGAGCTGTGCCC GFP-RING R GATCCCGGGCCCGCGTTACCAGACGTCGGCGGGT GFP-B-boxes F GGACTCAGATCTCGAACGCCGCCCGCC GFP-B-boxes R GATCCCGGGCCCGCGTTAGGCCTCCAGGTCGGCG GFP-CCD F GGACTCAGATCTCGACTGGTGGAGCATAAGACCTGC GFP-CCD R GATCCCGGGCCCGCGTTAAGGTCTGGACTTGGCCAGGAAG GFP-SPRY F GGACTCAGATCTCGAGAGCTCCTGGAGTATTACATTAAAGTCATCC GFP-SPRY R GATCCCGGGCCCGCGTTACTTGGGGGAGCAGATGGAGAG Myc-SADS-CoV N F ATGGAGGCCCGAATTATGGCCACTGTTAATTGGGGTGACGC Myc-SADS-CoV N R GCCGCGGTACCTCGACTAATTAATAATCTCATCCACCATCTCAACCT GFP-N2a F TCTCGAGCTCAAGCTAGAAGTGCTTCACGTTCACAGTCT GFP-N2a R TAGATCCGGTGGATCAATGTCAACAGACTGTGACGGC GFP-N2b F GGACTCAGATCTCGAGTTGCTGCAGTTAAACAAGCTTTGG GFP-N2b R GATCCCGGGCCCGCGGACAGCTCTGCTTCTTGGTTTGG GFP-N2c F GGACTCAGATCTCGATCACCTGCACCTGCCC GFP-N2c R GATCCCGGGCCCGCGGCGAGGACCAAAGCATTTACG Flag-RIG-I F TGACGATGACAAGCTTATGACCACCGAGCAGCG Flag-RIG-I R CTCTAGAGTCGACTGTCATTTGGACATTTCTGCTGGATCAAATGG GST-RIG-I 2CARD F CTCCAAAAATCGATGGTATGACCACCGAGCAGCG GST-RIG-I 2CARD R GATCTGCTAGCTCGATTAAGATCTTCTGTTTCAACATCTTTTATACCTTT Flag-RIG-I-IN F TGACGATGACAAGCTTATGACCACCGAGCAGCG Flag-RIG-I-IN R CTCTAGAGTCGACTGTTATTTAAGATGATGTTCACATATAAGCAGTGAA Flag-RIG-I-IC F TGACGATGACAAGCTTCCAGAATGCCAGAATCTTAGTGAGAATTCA Flag-RIG-I-IC R CTCTAGAGTCGACTGTCATTTGGACATTTCTGCTGGATCAAATGG RNA interference Three siRNAs targeting TRIM25 were designed by Shanghai GenePharma (Shanghai, China); the target sequences were as follows: siTRIM25-1 (sense, 5'-GGCTCACATTGATGCTTAT-3'), siTRIM25-2 (sense, 5'-GCTGAGGCATAAACTGACT-3'), and siTRIM25-3 (sense,5'-GCGATCACGGCTTTGTCAT-3'). SiTRIM25 and siNC negative control were transfected into IPI-2I cells with Lipofectamine RNAiMAX reagent (13778150, Thermo Fisher Scientific, USA). After 48 h transfection, we infected the cells with SADS-CoV at a multiplicity of infection (MOI) of 0.1. The levels of SADS-CoV N mRNA and protein in the infected cells were detected at 24 h postinfection (hpi). Dual-luciferase reporter assays We seeded HEK293T cells in 24-well plates, and transfected with luciferase reporter plasmids (IFN-β-Luc) and the indicated plasmid alone or together with SADS-CoV N plasmid for 24 h (pRL-TK Renilla luciferase reporter plasmid as an internal control). After 24 h transfection, we test the luciferase activity using a dual luciferase reporter assay kit (E1901, Promega, USA). Confocal fluorescence microscopy The plasmid-transfected HEK293T cells were fixed with 4% paraformaldehyde (16005, Sigma-Aldrich, USA) at 4°C for 30 min, and permeabilized with 1% Triton X-100 (T8787, Sigma-Aldrich) at room temperature for 15 min. After 5% skim milk blocking at 37°C for 2 h, cells were incubated with different primary antibodies at 4°C overnight. After PBS-Tween-20 (PBS containing 0.05% Tween-20; P1379, Sigma-Aldrich) washing, cells were incubated with Alexa Fluor 594/488 conjugated secondary antibody at room temperature for 1 h. After PBST washing, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 15 min, fluorescence images were directly captured under a LSM880-ZEISS confocal laser scanning microscope equipped with Fast Airyscan (Carl Zeiss AG, Oberkochen, Germany). RNA extraction and real-time PCR Total RNA was extracted with an RNeasy Mini Kit (52906, Qiagen, Germany), and reverse transcription was performed with PrimeScript™ IV 1st strand cDNA Synthesis Mix (6215A, Takara, Japan). Real-time PCR was conducted with TB Green Premix Ex Taq™ II (RR820A, Takara, Japan) on a QuantStudio 5 real-time PCR system (Applied Biosystems, Carlsbad, USA). The fold change in gene expression levels was calculated with the comparative CT (ΔΔC T ) method as described previously [ 25 ]. All experiments were performed in at least triplicate. The primers used in the qRT-PCR assays are listed in Table 2 . Table 2 qRT-PCR primer sequences used in this study Names Sequences(5’-3’) IFN-β F TGGGAGGCTTGAATACTGCCTCAA IFN-β R TCCTTGGCCTTCAGGTAATGCAGA CXCL10 F GTGGCATTCAAGGAGTACCTC CXCL10 R TGATGGCCTTCGATTCTGGATT ISG56 F CATACATTTCCACTATGG ISG56 R TACTCCAGGGCTTCATTCA hGAPDH F TCATGACCACAGTCCATGCC hGAPDH R GGATGACCTTGCCCACAGCC sGAPDH F ACCTCCACTACATGGTCTACA sGAPDH R ATGACAAGCTTCCCGTTCTC hTRIM25 F GCAGGATGTGCGGATGACTG hTRIM25 R GCGTCCAAGAGAGCCTTCAT sTRIM25 F AGCACCGACCTGGAGAACAA sTRIM25 R CCTGCTGTTTAGCTCTCACG SADS-CoV N F CCCCTAAACCGGCTCGTAA SADS-CoV N R CAGAATTAGGAACACGCTTCCA Western blotting analyses Whole cell lysates of IPI-2I cells and HEK293T cells were prepared after SADS-CoV infection or transfection with the indicated plasmids in six-well plates. The contents of each well were lysed with RIPA lysis buffer (R0278, Sigma-Aldrich, USA) with 1 mM PMSF (ST506-2, Beyotime) on ice for 30 min. After centrifuging at 12,000× g at 4°C for 20 min, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to the supernatant and boiled for 10 min. Proteins were separated with 12.5% SDS-PAGE, then transferred to nitrocellulose membranes at 300 mA for 120 min. After 5% skim milk blocking for 2 h, the membranes were incubated with different primary antibodies at 4°C for 6–8 h, and then incubated with IRDye 800CW goat anti-mouse lgG (H + L) (1:10,000) (926-32210, LiCor BioSciences) or IRDye 680RD goat anti-rabbit lgG (H + L) (1:10,000) (926-68071, LiCor BioSciences) for 1 h. The membranes were then visualized with Odyssey infrared imaging system (LiCor BioSciences). Immunoprecipitation We performed the immunoprecipitation assays as described previously [ 26 ]. Plasmid-transfected HEK293T cells were lysed with IP lysis buffer (87788, Thermo Fisher Scientific), which contained 1 mM PMSF and 1 mg/mL protease inhibitor cocktail (04693132001, Roche) on ice for 30 min. Five percent of the cell lysate was collected as input, and the remainder was incubated with the indicated primary antibody at 4°C overnight, then precipitated with Protein A/G agarose beads (78609, Thermo Fisher Scientific) for 6 h. After five times washing, the beads were collected and analyzed with western blotting. Flow cytometry analysis After 24 h transfection with SADS-CoV N plasmids, HEK293T cells were infected with VSV-GFP for 12 h. Subsequently, we harvested and resuspended the cells in PBS. Basing on the background signal emitted by uninfected cells, cells were subjected to gating for GFP signals. Fluorescence intensity was tested through BD FACSCalibur instrument. The data analysis was performed in FlowJo software. Viral titration IPI-2I cells were infected with SADS-CoV after HA-TRIM25 or siTRIM25 transfection. The culture supernatants were collected at 24 hpi. Vero E6 cells were infected with 10-fold serial dilutions of each supernatant. At 4–6 days postinfection, cytopathic effects in cells were observed through microscopy. We used the Spearman-Kärber method to calculate the median tissue culture infective dose (TCID 50 ). Experimental infection of piglets We performed the piglets infection experiment as described previously [ 27 ]. We randomly separated six 3 days old specific pathogen free (SPF) piglets into the challenge and control group. The challenge group was orally infected with 5×10 4 TCID 50 of SADS-CoV, whereas the control group was orally infected with the same volume DMEM. We recorded the clinical symptoms (vomiting and diarrhea), and euthanized all piglets at 36 hpi. Intestinal tissues were collected for qRT-PCR analyses. Statistical analysis The figures display the mean and standard deviation (SD) of results obtained from three independent experiments. Data were analyzed in Graph Pad Prism 8.0. Error bars represent the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant. Results Upregulation of TRIM25 by SADS-CoV infection in vitro and in vivo Previous studies have indicated that multiple virus infection affect the expression of TRIM25 [ 28 , 29 ]. To determine the expression level and potential role of TRIM25 in SADS-CoV infection, we infected IPI-2I cells with 0.1 MOI SADS-CoV. After qRT-PCR and western blotting analysis, we observed marked upregulation of both TRIM25 mRNA and protein at 24 and 48 hpi (Fig. 1 A, B). In addition, SADS-CoV significantly upregulated the expression of TRIM25 with increasing SADS-CoV MOI values (Fig. 1 C, D). Subsequently, we assessed the expression of TRIM25 in the ileum in piglets infected with SADS-CoV. Compared with the control group, TRIM25 mRNA levels were higher in infected group (Fig. 1 E). Together, these results suggested that TRIM25 was upregulated by SADS-CoV infection in vitro and in vivo . TRIM25 affects SADS-CoV replication To determine the role of TRIM25 in SADS-CoV infection, IPI-2I cells were transfected with TRIM25 expression plasmid and infecte with SADS-CoV. Western blotting showed that the N protein levels of SADS-CoV significantly decreased in a dose-dependent manner by TRIM25 overexpression (Fig. 2 A). In agreement with the observed changes in protein levels, increased TRIM25 expression was associated with decreases in mRNA levels of N protein ranging from 34.2–76.43% in IPI-2I cells, according to qRT-PCR (Fig. 2 B, C). Furthermore, TCID 50 assays indicated that the titer of released virus decreased after TRIM25 overexpression (Fig. 2 D). Together, these results indicated that TRIM25 upregulation inhibited SADS-CoV replication. To further ascertain the effect of TRIM25 on SADS-CoV replication, three siRNAs targeting TRIM25 were synthesized. The qRT-PCR results showed that TRIM25 mRNA levels decreased significantly after transfection of IPI-2I cells with siTRIM25-1 (Fig. 2 E). In contrast, the mRNA level of the viral N gene exhibited an increase (Fig. 2 F). IPI-2I cells was transfected with siTRIM25-1 at different concentrations, and subsequently infected with 0.1 MOI SADS-CoV for 36 h. As shown in Fig. 2 G, siTRIM25-1 promoted SADS-CoV propagation. The qRT-PCR results indicated lower TRIM25 mRNA levels (Fig. 2 H) and higher SADS-CoV N mRNA levels (Fig. 2 I) than observed in the siNC group. Furthermore, TCID 50 assays indicated that the viral titer was greater after TRIM25 knockdown than control siNC transfection (Fig. 2 J). These results indicated that TRIM25 silencing promoted SADS-CoV replication. Together, our results suggested that TRIM25 acts as an antiviral factor inhibiting SADS-CoV infection. TRIM25 interacts with SADS-CoV N CoVs N protein is highly abundant in infected cells and has a crucial function in viral transcription and assembly [ 30 , 31 ]. SARS-CoV-1 and PRRSV N protein inhibit IFN-I production by competitively interfering with TRIM25-RIG-I interaction and suppressing RIG-I ubiquitination [ 32 , 33 ]. SADS-CoV, SARS-CoV-1, and PRRSV belong to the Nidovirales order, and TRIM25 also has important roles in the cellular anti-SADS-CoV response, we next investigated whether SADS-CoV N protein might interact with TRIM25 and antagonize its antiviral effects. To test this possibility, we assessed the interaction between TRIM25 and SADS-CoV N protein. We found that Myc-tagged SADS-CoV N interacted with HA-tagged TRIM25 protein (Fig. 3 A). In addition, we immunoprecipitated virus-infected IPI-2I cells with mAb 3E9, then performed western blotting. As shown in Fig. 3 B, endogenous TRIM25 co-immunoprecipitated with SADS-CoV N protein. To eliminate the effects of RNA interference on the interaction between TRIM25 and the N protein of SADS-CoV, we used RNase A to remove any RNA molecules present in the experimental system. The SADS-CoV N-TRIM25 interaction was not impeded in the presence of RNase A, suggesting that the interaction between TRIM25 and SADS-CoV N protein did not rely on RNA (Fig. 3 C). Additionally, the co-localization of SADS-CoV N and TRIM25 was observed through indirect immunofluorescence (Fig. 3 D). These data indicated that TRIM25 interacted with the SADS-CoV N protein. Amino acids 215–249 of SADS-CoV N protein interact with TRIM25 CCD domain and inhibit CCD-dependent TRIM25 oligomerization To clarify the specific region of SADS-CoV N protein required for SADS-CoV N-TRIM25 interaction, we cotransfected HEK293T cells with GFP-tagged truncated fragments of SADS-CoV N protein (aa 1–146, 147–249, and 250–376) with HA-TRIM25. Co-IP assays revealed that the N2 domain of SADS-CoV N (aa 147–249) was the essential region for SADS-CoV N-TRIM25 interaction (Fig. 4 A, B). In contrast, the other truncated regions of SADS-CoV N protein were not responsible for this association. To further determine the necessary amino acid residues in SADS-CoV N2 domain (aa 147–249) directing the SADS-CoV N-TRIM25 interaction, we constructed a series of overlapping recombinant N2 truncated mutant proteins and determined their interaction with TRIM25 through Co-IP assays (Fig. 4 C). As shown in Fig. 4 D, GFP-N2c and GFP-N2b + N2c were responsible for the interaction with TRIM25. These results further suggested that S 215 PAPAPKPARKQMDKPEWKRVPNSEEDVRKCFGPR 249 of N protein is the key region for SADS-CoV N-TRIM25 interaction. TRIM25 consists of a N-terminal RING domain, two B-boxes, a central CCD and a C-terminal SPRY domain [ 34 ]. To enhance understanding of the SADS-CoV N-TRIM25 interaction, we comprehensively analyzed the structural domains necessary for TRIM25 binding to SADS-CoV N proteins, by using a series of GFP-tagged deletion mutants (Fig. 4 E). As depicted in Fig. 4 F, the CCD domain of TRIM25 interacted with SADS-CoV N, whereas the other domains did not show any interaction. Furthermore, the CCD deletion TRIM25 mutant (TRIM25 Del CCD) was unable to bind SADS-CoV N protein (Fig. 4 G). The coiled coil region of the TRIM family proteins primarily participated in homo-oligomeric interactions, and promoted the formation of a hypersecondary structure with multiple a helices [ 35 ]. Mutants lacking the CCD region of TRIM25 cannot multimerize [ 36 ]. Given that SADS-CoV N interacted with the CCD domain of TRIM25, we next tested whether SADS-CoV N might interfere with TRIM25 multimerization. Indeed, SADS-CoV N expression effectively inhibited CCD-mediated TRIM25 multimerization (Fig. 4 H). SADS-CoV N protein interferes with TRIM25-RIG-I interaction Given that TRIM25 is a RIG-I regulatory partner, our subsequent studies were designed to determine SADS-CoV N-RIG-I interaction. To test this possibility, we investigated the SADS-CoV N-RIG-I interaction through Co-IP. On the basis of precipitation with antibodies against Flag or Myc tag, we determined that RIG-I interacted with SADS-CoV N protein (Fig. 5 A). Further Co-IP assays indicated that only full-length SARS-CoV N interacted with RIG-I (Fig. 5 B). We also generated Flag-tagged N-terminal (Flag-RIG-IN) and C-terminal (Flag-RIG-IC) constructs and assessed them in Co-IP experiments. Co-IP assays revealed that the SADS-CoV N protein interacted with Flag-RIG-I and Flag-RIG-IN, but not Flag-RIG-IC (Fig. 5 C). To further establish that the RIG-I 2CARD was sufficient for interaction with SADS-CoV N, GST-RIG-I-2CARD and Myc-SADS-CoV N were co-transfected in HEK293T cells. The results indicated that the N-terminal CARD of RIG-I was crucial for SADS-CoV N-RIG-I interaction (Fig. 5 D). To explore the localization of the N/RIG-I, and TRIM25/RIG-I, and N/TIRM25/RIG-I complexes, we performed confocal microscopy. Imaging showed that RIG-I was colocalized with SADS-CoV N and TRIM25 in the cytoplasm (Fig. 5 E). These results further suggested that SADS-CoV N interacts with the RIG-I. Study showed that TRIM25 SPRY domain interacted with RIG-I, which induced K63-linked ubiquitination of the RIG-I N-terminal CARDs, thereby markedly activating RIG-I downstream signaling [ 20 ]. Given the association of the RIG-I N-terminal CARDs with SADS-CoV N protein, we speculated that the binding of RIG-I to TRIM25 might be affected by SADS-CoV N protein. To examine this possibility, we coexpressed HA-TRIM25 and Flag-RIG-I in HEK293T cells, both with and without Myc-SADS-CoV N. Co-IP assays revealed that overexpression of SADS-CoV N protein attenuated TRIM25-RIG-I interaction, which was a dose-dependent manner (Fig. 5 F). Thus, SADS-CoV N protein competitively binds RIG-I N-terminal CARDs and interferes with the TRIM25-RIG-I interaction. SADS-CoV N protein suppresses IFN-I production To explore whether the presence of SADS-CoV N protein in the RIG-I/TRIM25 complex might inhibit RIG-I downstream signaling and IFN-β production, HEK293T were transfected with Myc-N or Myc expression plasmid for 24 h, and then infected with Sendai virus (SeV) or stimulated with poly(I:C) for another 12 h. SADS-CoV N protein inhibited the IFN-β promoter activity inducing by SeV and poly(I:C) (Fig. 6 A, B). qPCR-RT analysis indicated that the expression of SADS-CoV N protein decreased the transcription of IFN-β, CXCL10, and ISG56 inducing by SeV and poly(I:C) (Fig. 6 C, D). Consistently, ectopic expression of SADS-CoV N inhibited the phosphorylation of IRF3 and TBK1 stimulated by SeV and poly(I:C) (Fig. 6 E, F). These data indicated that SADS-CoV N protein suppresses IFN-β production. To examine whether SADS-CoV N protein regulated RIG-I mediated IFN-β activation, HEK293T cells were transfected with firefly luciferase reporter plasmid, empty vector, or SADS-CoV N and RIG-I expression plasmid. The RIG-I induced IFN-β promoter activation was significantly suppressed in a dose-dependent manner by SARS-CoV N protein (Fig. 6 G). Similarly, N protein of SADS-CoV inhibited RIG-I mediated IFN-β transcription (Fig. 6 H). Furthermore, qRT-PCR indicated that TRIM25 further enhanced IFN-β transcription levels by acting on RIG-I; however, SADS-CoV N decreased the TRIM25-mediated RIG-I signaling enhancement (Fig. 6 I). We next investigated whether SADS-CoV N protein regulated VSV-GFP infection and proliferation. Fluorescence microscopy, flow cytometry, and western blotting showed that Myc-N plasmid-transfected HEK293T cells, compared with vector-treated cells, facilitated the replication of VSV-GFP, as measured by GFP signaling (Fig. 6 J-L), thus suggesting that SADS-CoV N inhibits the secretion of antiviral factors. Collectively, these data indicated that SADS-CoV N inhibits the TRIM25-mediated enhancement of RIG-I signaling. Discussion SADS-CoV infection entails a multifaceted interplay between virus and host. Understanding the SADS-CoV and host protein interaction can not only elucidate the mechanism of viral infection but also identify antiviral factors. In this study, we found TRIM25 was a new host restriction factor in SADS-CoV infection. The experiments provided clear evidence that SADS-CoV N interacted with TRIM25 and RIG-I protein. This interaction inhibits RIG-I signaling via TRIM25, which is essential for full RIG-I activation [20]. A multitude of TRIM family proteins have significant functions in the inhibition of viral replication. After viral infection, the mRNA and protein level of several TRIM protein are upregulated; these proteins subsequently act as host factors promoting the production of IFNs and inhibiting viral replication. TRIM14 is upregulated after HSV-1 infection and positively regulates type I IFN signaling [37]. TRIM22, a restriction factor, is upregulated at both the mRNA and protein levels in influenza A virus infected human alveolar epithelial A549 cells [38]. In the current study, SADS-CoV infection upregulated the levels of TRIM25 mRNA and protein in vitro and in vivo . The most prominent role of TRIMs is inhibition of viral replication. TRIM56 impairs hepatitis B virus, PEDV, yellow fever virus, and human coronavirus OC43 infection [39-41]. TRIM5α inhibits human immunodeficiency virus type 1 (HIV-1) infection [42]. We found that upregulation of TRIM25 restricts SADS-CoV infection, whereas downregulation of this protein enhances SADS-CoV infection (Figure 2). Therefore, TRIM25 is a novel host factor that exerts inhibitory effects on SADS-CoV replication. The N proteins of different coronaviruses are highly conservated and perform multiple functions during viral infection [6]. TRIM25 is targeted by MERS-CoV N [23] and SARS-CoV N protein [32] for immune evasion. Similarly, SADS-CoV N protein also interacts with TRIM25 (Figure 3). The N S215-R249 domain of SADS-CoV N was critical for binding TRIM25 in vitro . Despite variations in length and primary sequence among nucleoproteins from different coronaviruses, a conserved three-domain organization has been identified. Notably, domain Ⅱ of the N protein is highly conserved, as compared with domains I and Ⅲ [43]. The N S215-R249 domain of SADS-CoV N protein is in domain Ⅱ. TRIM25 consists of a RING-finger domain, two B-box domains, a central CCD, and a C-terminal SPRY domain [18]. SADS-CoV N protein directly binds TRIM25 and targets the TRIM25 CCD, thereby disrupting TRIM25 multimer formation (Figure 4H). The formation of TRIM25 multimers acts a pivotal role in RIG-I CARDs ubiquitination—a crucial modification necessary for the optimal IFN production in response to viral infection [20]. SADS-CoV N directly binds TRIM25, and inhibits the signal transduction of RIG-I and IFN-β production. The similarity of the observed mechanism to those used by paramyxovirus V [44] and influenza A virus NS1 [36] demonstrates that TRIM25 may be a common viral target for RIG-I antagonism. Interestingly, despite SADS-CoV N and paramyxovirus V both targeting TRIM25 , our data indicated that SADS-CoV N utilized a different mechanism to inhibit TRIM25 and RIG-I compared with paramyxovirus V. Paramyxovirus V targets TRIM25 SPRY domain, whereas SADS-CoV N protein interacts with TRIM25 CCD domain. Despite these differences, SADS-CoV N, paramyxovirus V, and influenza A virus NS1 proteins ultimately similarly suppress RIG-I signaling ubiquitination/activation by precluding TRIM25 binding to RIG-I. Viruses evolving to inhibit antiviral activity induced by IFN is well-documented. For instance, viral protein specifically impedes RIG-I- mediated the signal transduction. Influenza A virus NS1 inhibits the IFN-I induction via the interaction with RIG-I [45]. We found SADS-CoV N binds two N-terminal CARDs of RIG-I, thus suppressing the TRIM25-RIG-I interaction (Figure 5). TRIM25-SADS-CoV N-RIG-I interaction supports the hypothesis that N protein may potentially impede the progression of TRIM25-RIG-I downstream signaling pathway. Different coronavirus N proteins have been indicated to inhibit IFN production. PDCoV N protein suppresses Riplet-induced ubiquitination of RIG-I through interaction with porcine RIG-I and TRAF3 protein [46]. SARS-CoV-1 and MHV N proteins interaction with PACT protein attenuates the activation of RIG-I and MDA5 [8]. PEDV N protein interferes with TBK1-mediated IRF3 phosphorylation and thus inhibits IFN-β expression [11]. Our current results indicated that ectopic expression of SADS-CoV N protein significantly inhibited mRNA expression of IFN-β, CXCL10 and ISG56 induced by SeV or poly I:C, and RIG-I activity. Overexpression of SADS-CoV N protein in HEK293T cells enhanced the replication of VSV-GFP (Figure 6). Thus, the SADS-CoV N protein facilitates viral replication by inhibiting the host interferon response. In conclusion, our results suggested that TRIM25 impeded SADS-CoV replication. Furthermore, SADS-CoV N protein inhibits TRIM25 multimerization and TRIM25-RIG-I interaction to antagonize antiviral activity. In addition, SADS-CoV N suppressed IFN-I production and facilitated VSV-GFP replication. Our findings elucidate a significant molecular mechanism through which SADS-CoV utilizes its N protein to evade innate immune response mediated by TRIM25, thus providing an explanation of the natural viral defense mechanism and potentially facilitating the development of more effective strategies for controlling SADS-CoV infection. Declarations Authors’ contributions Conceptualization: JZ, DS, and LF. Methodology: JZ, ZJ, XZ, DL, XY, MZ, DS, and LF. Experiment operation: JZ, LZ, ZJ, and TF. Supervision: DS and LF. Data analysis: JZ, HS, JC, XZ, DS, and LF. Writing—original draft: JZ. Writing—review and editing: JZ, DS, and LF. All authors read and approved the final manuscript. Funding This research was funded by the Regional Innovation and Development Joint Fund of National Natural Science Foundation of China, grant number U23A20236; the National Key R&D Program of China, grant number 2021YFD1801105; and the Central Public-interest Scientific Institution Basal Research Found, grant number 1610302022015. Availability of data and materials The data that support the findings of this study are available from the authors on reasonable request. Ethics approval and consent to participate The animal experiments were approved by Harbin Veterinary Research Institute. The animal ethics committee approval number was 231108-05-GR. Competing interests The authors declare that they have no competing interests. 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05:00:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":961390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSADS-CoV infection upregulates TRIM25 expression\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e Relative expression levels of TRIM25 were quantified by qRT-PCR. IPI-2I cells were either mock infected or infected with SADS-CoV 0.1 MOI at different times (A) or different MOIs at 24 hpi (C). Total RNA was extracted, and TRIM25 mRNA levels were evaluated with qRT-PCR. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Relative expression levels of TRIM25 were quantified by western blotting. IPI-2I cells were either mock infected or infected with SADS-CoV 0.1 MOI at different times (B) or different MOIs at 24 hpi (D). The cell lysates were collected, and the expression of TRIM25 was tested by western blotting. \u003cstrong\u003eE\u003c/strong\u003e SADS-CoV infection upregulates TRIM25 expression \u003cem\u003ein vivo\u003c/em\u003e. qRT-PCR analysis of the mRNA expression levels of TRIM25 in ileal samples from SADS-CoV -non-infected and -infected piglets at 36 hpi.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/99451b65a7be894c72f345de.png"},{"id":50281953,"identity":"d725b8f5-8ad0-4ab3-b2ed-04f96ffa3a7c","added_by":"auto","created_at":"2024-01-29 04:52:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1911639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM25 affects the replication of SADS-CoV.\u003c/strong\u003e \u003cstrong\u003eA-D\u003c/strong\u003e Overexpression of TRIM25 inhibits SADS-CoV replication. IPI-2I cells were transfected with HA-tagged TRIM25 (0, 1, 2, or 4 μg) for 24 h and then infected with SADS-CoV at an MOI of 0.1 for 24 h.\u003cstrong\u003e A\u003c/strong\u003e The protein expression of TRIM25 and SADS-CoV N was detected by western blotting. \u003cstrong\u003eB\u003c/strong\u003e Overexpression of TRIM25 was verified by qRT-PCR. \u003cstrong\u003eC\u003c/strong\u003e The mRNA levels of SADS-CoV N were determined by qRT-PCR. \u003cstrong\u003eD\u003c/strong\u003e The SADS-CoV TCID\u003csub\u003e50\u003c/sub\u003e in the supernatants was titrated on Vero E6 cells. \u003cstrong\u003eE-J\u003c/strong\u003e Knockdown of TRIM25 enhanced SADS-CoV replication. \u003cstrong\u003eE\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e IPI-2I cells were transfected with siTRIM25-1, siTRIM25-2, siTRIM25-3, or siNC (negative control) at 50 nM for 36 h and subsequently infected with SADS-CoV at an MOI of 0.1 for 24 h. The mRNA levels of TRIM25 (E) and SADS-CoV N (F) in TRIM25 knockdown cells were determined by qRT-PCR.\u003cstrong\u003e G-J\u003c/strong\u003e IPI-2I cells were transfected with siNC or siTRIM25-1 (50 nM or 100 nM) for 36 h and subsequently infected with SADS-CoV at an MOI of 0.1 for 24 h. \u003cstrong\u003eG\u003c/strong\u003e The protein expression of TRIM25 and SADS-CoV N was detected by western blotting. \u003cstrong\u003eH\u003c/strong\u003e The mRNA levels of TRIM25 were verified by qRT-PCR. \u003cstrong\u003eI\u003c/strong\u003e The mRNA levels of SADS-CoV N were determined by qRT-PCR. \u003cstrong\u003eJ \u003c/strong\u003eThe SADS-CoV TCID\u003csub\u003e50\u003c/sub\u003e in the supernatants was titrated on Vero E6 cells.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/efdbe63d351aefbc2f0dbdf7.png"},{"id":50281956,"identity":"bc5c3b91-1d34-463a-a48b-e9f7985f7c36","added_by":"auto","created_at":"2024-01-29 04:52:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1811457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRIM25 interacts with SADS-CoV N protein.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Co-IP analysis of the interaction between SADS-CoV N protein and TRIM25 in HEK293T cells. HEK293T cells were transfected with HA-TRIM25, Myc-SADS-CoV N, or empty vector. At 24 h post-transfection, cell lysates were subjected to immunoprecipitation with anti-Myc or anti-HA antibodies, and subsequent immunoblotting with anti-Myc or anti-HA antibodies. \u003cstrong\u003eB\u003c/strong\u003eThe interaction of endogenous TRIM25 and SADS-CoV N protein was detected with Co-IP assays. IPI-2I cells were infected with SADS-CoV at an MOI of 0.1 for 24 h. Cell lysates were collected and then incubated with anti-mouse IgG or anti-SADS-CoV N mAb (3E9), and detected with the indicated antibodies by western blotting. \u003cstrong\u003eC\u003c/strong\u003e SADS-CoV N protein binds TRIM25 in a manner independent of RNA. HA-TRIM25, Myc-SADS-CoV N, or empty vector were coexpressed in HEK293T cells. At 24 h post-transfection, the cell lysates were either immunoprecipitated with antibodies against the HA-tag or treated with 100 mg/ml RNase A on ice for 2 h before IP. SADS-CoV N and TRIM25 were detected by western blotting with anti-Myc or anti-HA antibodies, respectively. \u003cstrong\u003eD\u003c/strong\u003eCo-localization of TRIM25 and SADS-CoV N. HEK293T cells were co-transfected with HA-TRIM25, GFP-SADS-CoV N, or GFP empty vector plasmids. The GFP and GFP-SADS-CoV N are in green, and the TRIM25 fusion protein is in red. Merged images are also presented, and the positions of the nuclei are indicated by DAPI (blue) staining in the merged images.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/3b334869bf6923905f66f6ed.png"},{"id":50282318,"identity":"acf27f66-473b-44e7-8ea1-86a9d1742ee2","added_by":"auto","created_at":"2024-01-29 05:00:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2484939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSADS-CoV N protein inhibits CCD-dependent TRIM25 oligomerization.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e Schematics of SADS-CoV N and the SADS-CoV N2 domain truncations. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eD \u003c/strong\u003eThe domain of SADS-CoV N responsible for interaction with TRIM25 was identified at 24 h post-transfection with HA-TRIM25 together with GFP empty vector, GFP-SADS-CoV N, or SADS-CoV N truncations. Whole cell lysates were collected and subjected to IP with anti-HA antibodies, followed by immunoblotting with anti-GFP or anti-HA antibodies. \u003cstrong\u003eE\u003c/strong\u003e Schematics of TRIM25 truncations. \u003cstrong\u003eF\u003c/strong\u003e The domain of TRIM25 responsible for interaction with SADS-CoV N was identified. Myc-SADS-CoV N, GFP empty vector, or TRIM25 truncations with GFP were coexpressed in HEK293T cells. At 24 h post-transfection, the cell lysates were immunoprecipitated with antibodies against GFP. SADS-CoV N and TRIM25 truncations were detected by western blotting with anti-Myc or anti-GFP antibodies, respectively. \u003cstrong\u003eG\u003c/strong\u003e SADS-CoV N did not interact with mutants of TRIM25 lacking the CCD domain. HEK293T cells were co-transfected with Myc-SADS-CoV N together with empty vector, HA-TRIM25, or HA-TRIM25 DelCCD. At 24 h post-transfection, whole cell lysates were collected and used for IP with anti-HA antibodies, followed by immunoblotting with anti-Myc or anti-HA antibodies. \u003cstrong\u003eH\u003c/strong\u003e SADS-CoV N protein inhibited the interaction of differently labeled TRIM25. HEK293T cells were transfected with Myc-SADS-CoV N together with empty vector, Flag-TRIM25, or HA-TRIM25. At 24 h post-transfection, whole cell lysates were collected and used for IP with anti-Flag antibodies, followed by immunoblotting with anti-Myc, anti-HA, and anti-Flag antibodies.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/c0b2c7b5c626a79f9c02f356.png"},{"id":50282319,"identity":"17298b66-ffde-47a2-a780-860e93534058","added_by":"auto","created_at":"2024-01-29 05:00:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2836528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSADS-CoV N protein interferes with the interaction between TRIM25 and RIG-I.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e RIG-I interacts with SADS-CoV N protein. HEK293T cells were transfected with Flag-RIG-I, Myc-SADS-CoV N, or empty vector. At 24 h post-transfection, cell lysates were subjected to immunoprecipitation with anti-Myc or anti-Flag antibodies, and subsequent immunoblotting with anti-Myc or anti-Flag antibodies. \u003cstrong\u003eB\u003c/strong\u003e The domain of SADS-CoV responsible for interaction with RIG-I was identified. \u003cstrong\u003eC\u003c/strong\u003e The domain of RIG-I responsible for interaction with SADS-CoV N was identified. \u003cstrong\u003eD\u003c/strong\u003e RIG-I 2CARD interacts with SADS-CoV N protein. HEK293T cells were transfected with GST or GST-RIG-I 2CARD together with Myc-SADS-CoV N. The cell lysates were collected and then immunoprecipitated with anti-GST antibodies. The immunoprecipitates were detected by western blotting with the indicated antibodies. \u003cstrong\u003eE\u003c/strong\u003e Co-localization of RIG-I with TRIM25 and SADS-CoV N. HEK293T cells were cotransfected with Flag-RIG-I and HA-TRIM25 or GFP-SADS-CoV N for 24 h. The cells were incubated with rabbit anti-Flag antibodies and mouse anti-HA antibodies before being stained with DAPI. \u003cstrong\u003eF\u003c/strong\u003e SADS-CoV N protein dose-dependently inhibited RIG-I’s interaction with TRIM25. HEK293T cells were cotransfected with Flag-RIG-I and HA-TRIM25 or Myc-SADS-CoV for 24 h. The cell lysates were collected and then immunoprecipitated with anti-Flag antibodies. The immunoprecipitates were detected by western blotting with the indicated antibodies.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/2bddd43a7ab8284365b014ad.png"},{"id":54713010,"identity":"46df5800-89ff-4718-b0ad-dc1c1b722cde","added_by":"auto","created_at":"2024-04-15 15:14:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2070057,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3814773/v1/93fcf210-ea17-4389-9e51-0ce5cd65126d.pdf"}],"financialInterests":"","formattedTitle":"Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibitng TRIM25 oligomerization and functional activation of RIG-I/TRIM25","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoronaviruses (CoVs) infect various animal species and pose a significant threat to both public health and economies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Over the last two decades, severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2, have caused severe respiratory disease in humans [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As an important livestock species, pigs have been particularly susceptible to severe CoV diseases, thus resulting in substantial economic effects [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (PHEV), porcine deltacoronavirus (PDCoV), transmissible gastroenteritis virus (TGEV), and porcine respiratory virus (PRCV) are five distinct swine CoVs.\u003c/p\u003e \u003cp\u003eThe sixth identified porcine coronavirus, swine acute diarrhea syndrome coronavirus (SADS-CoV), was responsible for two significant outbreaks in China [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. SADS-CoV, classified as a swine enteric alpha-CoV, induces swine acute diarrhea syndrome in piglets and leads to vomiting, severe diarrhea and weight loss. The mortality rates of 5-day-old piglets affected by SADS-CoV can reach 90% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. SADS-CoV (family \u003cem\u003eCoronaviridae\u003c/em\u003e, genus \u003cem\u003ealphacoronavirus\u003c/em\u003e) is an enveloped, single-stranded positive-sense RNA virus. The genome of SADS-CoV has a typical CoV structure, seven open reading frames encoding four structural proteins, 16 non-structural proteins and an accessory protein [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Nucleocapsid (N) proteins have important functions in the transcription, replication, and assembly phases of viruses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. N proteins can also act as IFN antagonists. For example, the N protein of PDCoV, PEDV, SARS-CoV-1, SARS-CoV-2 and Mouse hepatitis virus (MHV) suppress IFN production through different mechanisms [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe innate immune response, as the initial barrier against pathogens, encompasses the type I interferon (IFN-I) signaling pathway, which plays an essential role in defending against viral infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. After the identification of viral RNA within the cytoplasm, retinoic acid-inducible gene I (RIG-I)-like receptors initiate the antiviral response through the activation of signaling cascades [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. CoVs replication produce 5\u0026acute;-ppp RNA intermediates and dsRNA, and RIG-I specifically recognized these non-self-RNA signature in the cytoplasm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. RIG-I detects viral 5\u0026acute;-ppp RNA species and subsequently initiates signaling pathways via mitochondrial antiviral signaling (MAVS) protein, thereby inducing the interferon regulatory factors (IRF3 and IRF7) translocation, as well as the IFN and inflammatory cytokines expression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTripartite motif (TRIM) proteins have three domains: an N-terminal Really Interesting New Gene (RING) domain, one or two B-boxes, and a coiled coil domain (CCD) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The RING domain of TRIM proteins acts as an ubiquitin E3 ligase to catalyze the target proteins ubiquitination [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Gack and colleagues first demonstrated that RIG-I caspase activation and recruitment domains (CARDs) ubiquitination involves tripartite motif-containing protein 25 (TRIM25) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. RIG-I exhibits a specific interaction with TRIM25 via its CARD, which facilitates the transfer of the K63-linked ubiquitin moiety to another CARD and leads to the interaction with MAVS. Furthermore, the RIG-I splice variant (amino acids 36\u0026ndash;80 deletion in the first CARD) is unable to bind TRIM25 and activate downstream effectors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eViruses evade host immune attack by antagonizing antiviral defenses. Specifically, virus-encoded protein impede the innate antiviral responses of the host by selectively targeting the expression of IFN genes or the effector molecules induced by IFN [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The N protein, which is encoded by CoVs, is the main IFN antagonist. For example, MERS-CoV N interacts with TRIM25 to suppress both IFN-I and IFN-Ⅲ production [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, SARS-CoV-1 N protein binds to protein activator of protein kinase R (PACT) and TRIM25 to restrain RIG-I activation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. SARS-CoV-2 N protein plays a role in TRIM25 and RIG-I complex by suppressing TRIM25 E3 ligase activity toward RIG-I [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PRRSV N protein competitively interferes with TRIM25-RIG-I interaction to inhibit RIG-I ubiquitination. However, the detailed roles of SADS-CoV N protein in suppressing type I interferon production and antiviral gene expression have not been directly investigated.\u003c/p\u003e \u003cp\u003eHere, our findings suggested that TRIM25 is upregulated after SADS-CoV infection and significantly inhibits SADS-CoV infection. We identified that SADS-CoV N protein interacts with TRIM25 CCD domain and RIG-I CARDs, which inhibits TRIM25 multimerization and TRIM25-RIG-I interaction, thereby suppresses RIG-I signaling and IFN production. The study offered valuable mechanism that may facilitate therapeutic drugs development against \u003cem\u003eCoronaviridae\u003c/em\u003e family members infection.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and virus\u003c/h2\u003e \u003cp\u003ePorcine intestinal epithelial cells (IPI-2I) and human embryonic kidney cells (HEK293T) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Sigma-Aldrich, St Louis, USA) containing 10% fetal bovine serum (Invitrogen, USA) and 1% antibiotic-antimycotic (Invitrogen, USA). SADS-CoV, SeV and vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP) were stored at our lab.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and antibodies\u003c/h2\u003e \u003cp\u003ePig full-length TRIM25 (GenBank accession number XM_005656971.3) was cloned from IPI-2I cell cDNA and reconstructed with pCMV-Flag and pCAGGS-HA vectors. The deletion mutants of TRIM25 (RING_1\u0026ndash;90 aa, B-boxes_91\u0026ndash;200 aa, CCD_180\u0026ndash;450 aa, and SPRY_451\u0026ndash;630 aa) were constructed on the basis of the full-length TRIM25 plasmid and inserted into the pAcGFP-C1 vector to generate green fluorescent protein (GFP) fusion proteins. TRIM25 DelCCD (lacking the CCD domain) was cloned into the pCAGGS-HA vector, and we named the recombinant plasmid pHA-TRIM25 DelCCD. GFP-SADS-CoV N and N truncated plasmids were stored in our laboratory as previously described [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The plasmids for expression of RIG-I, RIG-IN and RIG-IC with Flag-tag, RIG-I 2CARD with GST-tag, and IFN-β-Luc were preserved at our laboratory. The primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Monoclonal antibodies specific for SADS-CoV N protein were stocked in our laboratory [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We purchased antibody against DDDDK (ab205606), HA (ab9110), TBK1 (ab40676), p-TBK1 (ab109272), IRF3 (ab68481), p-IRF3 (ab76493), GFP (ab290), GST (ab138491) or TRIM25 (ab167154) from Abcam (Cambridge, MA). We purchased antibody against Myc (M4439) or GAPDH (G9545) from Sigma-Aldrich (St. Louis, MO, USA). RNase A, Alexa Fluor 488 goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) and Alexa Fluor 594 goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibodies were purchased from Thermo Fisher Scientific (Carlsbad, CA). Poly(I:C) was purchased from InvivoGen (Hong Kong, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePCR primer sequences used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNames\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSequences(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA-TRIM25 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGATTACGCTGAATTAATGGGCGGAACTGTGCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHA-TRIM25 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCTGCTAGCTCGACTACCTGGTGGAGCAGATGGAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-TRIM25 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGATTACGCTGAATTAATGGCGGAACTGTGCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-TRIM25 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCTGCTAGCTCGACTACCTGGTGGAGCAGATGGAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-RING F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGAATGGCAGAGCTGTGCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-RING R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGTTACCAGACGTCGGCGGGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-B-boxes F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGAACGCCGCCCGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-B-boxes R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGTTAGGCCTCCAGGTCGGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-CCD F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGACTGGTGGAGCATAAGACCTGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-CCD R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGTTAAGGTCTGGACTTGGCCAGGAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-SPRY F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGAGAGCTCCTGGAGTATTACATTAAAGTCATCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-SPRY R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGTTACTTGGGGGAGCAGATGGAGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyc-SADS-CoV N F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATGGAGGCCCGAATTATGGCCACTGTTAATTGGGGTGACGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMyc-SADS-CoV N R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCCGCGGTACCTCGACTAATTAATAATCTCATCCACCATCTCAACCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2a F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCTCGAGCTCAAGCTAGAAGTGCTTCACGTTCACAGTCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2a R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTAGATCCGGTGGATCAATGTCAACAGACTGTGACGGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2b F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGAGTTGCTGCAGTTAAACAAGCTTTGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2b R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGGACAGCTCTGCTTCTTGGTTTGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2c F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGACTCAGATCTCGATCACCTGCACCTGCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGFP-N2c R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCCCGGGCCCGCGGCGAGGACCAAAGCATTTACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGACGATGACAAGCTTATGACCACCGAGCAGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCTAGAGTCGACTGTCATTTGGACATTTCTGCTGGATCAAATGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGST-RIG-I 2CARD F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCCAAAAATCGATGGTATGACCACCGAGCAGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGST-RIG-I 2CARD R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATCTGCTAGCTCGATTAAGATCTTCTGTTTCAACATCTTTTATACCTTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I-IN F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGACGATGACAAGCTTATGACCACCGAGCAGCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I-IN R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCTAGAGTCGACTGTTATTTAAGATGATGTTCACATATAAGCAGTGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I-IC F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGACGATGACAAGCTTCCAGAATGCCAGAATCTTAGTGAGAATTCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlag-RIG-I-IC R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTCTAGAGTCGACTGTCATTTGGACATTTCTGCTGGATCAAATGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA interference\u003c/h2\u003e \u003cp\u003eThree siRNAs targeting TRIM25 were designed by Shanghai GenePharma (Shanghai, China); the target sequences were as follows: siTRIM25-1 (sense, 5'-GGCTCACATTGATGCTTAT-3'), siTRIM25-2 (sense, 5'-GCTGAGGCATAAACTGACT-3'), and siTRIM25-3 (sense,5'-GCGATCACGGCTTTGTCAT-3'). SiTRIM25 and siNC negative control were transfected into IPI-2I cells with Lipofectamine RNAiMAX reagent (13778150, Thermo Fisher Scientific, USA). After 48 h transfection, we infected the cells with SADS-CoV at a multiplicity of infection (MOI) of 0.1. The levels of SADS-CoV N mRNA and protein in the infected cells were detected at 24 h postinfection (hpi).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDual-luciferase reporter assays\u003c/h2\u003e \u003cp\u003eWe seeded HEK293T cells in 24-well plates, and transfected with luciferase reporter plasmids (IFN-β-Luc) and the indicated plasmid alone or together with SADS-CoV N plasmid for 24 h (pRL-TK \u003cem\u003eRenilla\u003c/em\u003e luciferase reporter plasmid as an internal control). After 24 h transfection, we test the luciferase activity using a dual luciferase reporter assay kit (E1901, Promega, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eConfocal fluorescence microscopy\u003c/h2\u003e \u003cp\u003eThe plasmid-transfected HEK293T cells were fixed with 4% paraformaldehyde (16005, Sigma-Aldrich, USA) at 4\u0026deg;C for 30 min, and permeabilized with 1% Triton X-100 (T8787, Sigma-Aldrich) at room temperature for 15 min. After 5% skim milk blocking at 37\u0026deg;C for 2 h, cells were incubated with different primary antibodies at 4\u0026deg;C overnight. After PBS-Tween-20 (PBS containing 0.05% Tween-20; P1379, Sigma-Aldrich) washing, cells were incubated with Alexa Fluor 594/488 conjugated secondary antibody at room temperature for 1 h. After PBST washing, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 15 min, fluorescence images were directly captured under a LSM880-ZEISS confocal laser scanning microscope equipped with Fast Airyscan (Carl Zeiss AG, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with an RNeasy Mini Kit (52906, Qiagen, Germany), and reverse transcription was performed with PrimeScript\u0026trade; IV 1st strand cDNA Synthesis Mix (6215A, Takara, Japan). Real-time PCR was conducted with TB Green Premix Ex Taq\u0026trade; II (RR820A, Takara, Japan) on a QuantStudio 5 real-time PCR system (Applied Biosystems, Carlsbad, USA). The fold change in gene expression levels was calculated with the comparative CT (ΔΔC\u003csub\u003eT\u003c/sub\u003e) method as described previously [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. All experiments were performed in at least triplicate. The primers used in the qRT-PCR assays are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eqRT-PCR primer sequences used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNames\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSequences(5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIFN-β F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGGGAGGCTTGAATACTGCCTCAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIFN-β R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCCTTGGCCTTCAGGTAATGCAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL10 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGGCATTCAAGGAGTACCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCXCL10 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGATGGCCTTCGATTCTGGATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISG56 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCATACATTTCCACTATGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eISG56 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTACTCCAGGGCTTCATTCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehGAPDH F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCATGACCACAGTCCATGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehGAPDH R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGATGACCTTGCCCACAGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esGAPDH F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACCTCCACTACATGGTCTACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esGAPDH R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATGACAAGCTTCCCGTTCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehTRIM25 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCAGGATGTGCGGATGACTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ehTRIM25 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCGTCCAAGAGAGCCTTCAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esTRIM25 F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGCACCGACCTGGAGAACAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esTRIM25 R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCTGCTGTTTAGCTCTCACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSADS-CoV N F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCCTAAACCGGCTCGTAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSADS-CoV N R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAGAATTAGGAACACGCTTCCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c3\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analyses\u003c/h2\u003e \u003cp\u003eWhole cell lysates of IPI-2I cells and HEK293T cells were prepared after SADS-CoV infection or transfection with the indicated plasmids in six-well plates. The contents of each well were lysed with RIPA lysis buffer (R0278, Sigma-Aldrich, USA) with 1 mM PMSF (ST506-2, Beyotime) on ice for 30 min. After centrifuging at 12,000\u0026times;\u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 20 min, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer was added to the supernatant and boiled for 10 min. Proteins were separated with 12.5% SDS-PAGE, then transferred to nitrocellulose membranes at 300 mA for 120 min. After 5% skim milk blocking for 2 h, the membranes were incubated with different primary antibodies at 4\u0026deg;C for 6\u0026ndash;8 h, and then incubated with IRDye 800CW goat anti-mouse lgG (H\u0026thinsp;+\u0026thinsp;L) (1:10,000) (926-32210, LiCor BioSciences) or IRDye 680RD goat anti-rabbit lgG (H\u0026thinsp;+\u0026thinsp;L) (1:10,000) (926-68071, LiCor BioSciences) for 1 h. The membranes were then visualized with Odyssey infrared imaging system (LiCor BioSciences).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eImmunoprecipitation\u003c/h2\u003e \u003cp\u003eWe performed the immunoprecipitation assays as described previously [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Plasmid-transfected HEK293T cells were lysed with IP lysis buffer (87788, Thermo Fisher Scientific), which contained 1 mM PMSF and 1 mg/mL protease inhibitor cocktail (04693132001, Roche) on ice for 30 min. Five percent of the cell lysate was collected as input, and the remainder was incubated with the indicated primary antibody at 4\u0026deg;C overnight, then precipitated with Protein A/G agarose beads (78609, Thermo Fisher Scientific) for 6 h. After five times washing, the beads were collected and analyzed with western blotting.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eAfter 24 h transfection with SADS-CoV N plasmids, HEK293T cells were infected with VSV-GFP for 12 h. Subsequently, we harvested and resuspended the cells in PBS. Basing on the background signal emitted by uninfected cells, cells were subjected to gating for GFP signals. Fluorescence intensity was tested through BD FACSCalibur instrument. The data analysis was performed in FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eViral titration\u003c/h2\u003e \u003cp\u003eIPI-2I cells were infected with SADS-CoV after HA-TRIM25 or siTRIM25 transfection. The culture supernatants were collected at 24 hpi. Vero E6 cells were infected with 10-fold serial dilutions of each supernatant. At 4\u0026ndash;6 days postinfection, cytopathic effects in cells were observed through microscopy. We used the Spearman-K\u0026auml;rber method to calculate the median tissue culture infective dose (TCID\u003csub\u003e50\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperimental infection of piglets\u003c/h2\u003e \u003cp\u003eWe performed the piglets infection experiment as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. We randomly separated six 3 days old specific pathogen free (SPF) piglets into the challenge and control group. The challenge group was orally infected with 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of SADS-CoV, whereas the control group was orally infected with the same volume DMEM. We recorded the clinical symptoms (vomiting and diarrhea), and euthanized all piglets at 36 hpi. Intestinal tissues were collected for qRT-PCR analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe figures display the mean and standard deviation (SD) of results obtained from three independent experiments. Data were analyzed in Graph Pad Prism 8.0. Error bars represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ns, not significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eUpregulation of TRIM25 by SADS-CoV infection\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious studies have indicated that multiple virus infection affect the expression of TRIM25 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To determine the expression level and potential role of TRIM25 in SADS-CoV infection, we infected IPI-2I cells with 0.1 MOI SADS-CoV. After qRT-PCR and western blotting analysis, we observed marked upregulation of both TRIM25 mRNA and protein at 24 and 48 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In addition, SADS-CoV significantly upregulated the expression of TRIM25 with increasing SADS-CoV MOI values (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Subsequently, we assessed the expression of TRIM25 in the ileum in piglets infected with SADS-CoV. Compared with the control group, TRIM25 mRNA levels were higher in infected group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Together, these results suggested that TRIM25 was upregulated by SADS-CoV infection \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTRIM25 affects SADS-CoV replication\u003c/h2\u003e \u003cp\u003eTo determine the role of TRIM25 in SADS-CoV infection, IPI-2I cells were transfected with TRIM25 expression plasmid and infecte with SADS-CoV. Western blotting showed that the N protein levels of SADS-CoV significantly decreased in a dose-dependent manner by TRIM25 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In agreement with the observed changes in protein levels, increased TRIM25 expression was associated with decreases in mRNA levels of N protein ranging from 34.2\u0026ndash;76.43% in IPI-2I cells, according to qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Furthermore, TCID\u003csub\u003e50\u003c/sub\u003e assays indicated that the titer of released virus decreased after TRIM25 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Together, these results indicated that TRIM25 upregulation inhibited SADS-CoV replication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further ascertain the effect of TRIM25 on SADS-CoV replication, three siRNAs targeting TRIM25 were synthesized. The qRT-PCR results showed that TRIM25 mRNA levels decreased significantly after transfection of IPI-2I cells with siTRIM25-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). In contrast, the mRNA level of the viral N gene exhibited an increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). IPI-2I cells was transfected with siTRIM25-1 at different concentrations, and subsequently infected with 0.1 MOI SADS-CoV for 36 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, siTRIM25-1 promoted SADS-CoV propagation. The qRT-PCR results indicated lower TRIM25 mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and higher SADS-CoV N mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI) than observed in the siNC group. Furthermore, TCID\u003csub\u003e50\u003c/sub\u003e assays indicated that the viral titer was greater after TRIM25 knockdown than control siNC transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). These results indicated that TRIM25 silencing promoted SADS-CoV replication. Together, our results suggested that TRIM25 acts as an antiviral factor inhibiting SADS-CoV infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTRIM25 interacts with SADS-CoV N\u003c/h2\u003e \u003cp\u003eCoVs N protein is highly abundant in infected cells and has a crucial function in viral transcription and assembly [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. SARS-CoV-1 and PRRSV N protein inhibit IFN-I production by competitively interfering with TRIM25-RIG-I interaction and suppressing RIG-I ubiquitination [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. SADS-CoV, SARS-CoV-1, and PRRSV belong to the \u003cem\u003eNidovirales\u003c/em\u003e order, and TRIM25 also has important roles in the cellular anti-SADS-CoV response, we next investigated whether SADS-CoV N protein might interact with TRIM25 and antagonize its antiviral effects. To test this possibility, we assessed the interaction between TRIM25 and SADS-CoV N protein. We found that Myc-tagged SADS-CoV N interacted with HA-tagged TRIM25 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, we immunoprecipitated virus-infected IPI-2I cells with mAb 3E9, then performed western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, endogenous TRIM25 co-immunoprecipitated with SADS-CoV N protein. To eliminate the effects of RNA interference on the interaction between TRIM25 and the N protein of SADS-CoV, we used RNase A to remove any RNA molecules present in the experimental system. The SADS-CoV N-TRIM25 interaction was not impeded in the presence of RNase A, suggesting that the interaction between TRIM25 and SADS-CoV N protein did not rely on RNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, the co-localization of SADS-CoV N and TRIM25 was observed through indirect immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These data indicated that TRIM25 interacted with the SADS-CoV N protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAmino acids 215\u0026ndash;249 of SADS-CoV N protein interact with TRIM25 CCD domain and inhibit CCD-dependent TRIM25 oligomerization\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo clarify the specific region of SADS-CoV N protein required for SADS-CoV N-TRIM25 interaction, we cotransfected HEK293T cells with GFP-tagged truncated fragments of SADS-CoV N protein (aa 1\u0026ndash;146, 147\u0026ndash;249, and 250\u0026ndash;376) with HA-TRIM25. Co-IP assays revealed that the N2 domain of SADS-CoV N (aa 147\u0026ndash;249) was the essential region for SADS-CoV N-TRIM25 interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). In contrast, the other truncated regions of SADS-CoV N protein were not responsible for this association. To further determine the necessary amino acid residues in SADS-CoV N2 domain (aa 147\u0026ndash;249) directing the SADS-CoV N-TRIM25 interaction, we constructed a series of overlapping recombinant N2 truncated mutant proteins and determined their interaction with TRIM25 through Co-IP assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, GFP-N2c and GFP-N2b\u0026thinsp;+\u0026thinsp;N2c were responsible for the interaction with TRIM25. These results further suggested that S\u003csup\u003e215\u003c/sup\u003ePAPAPKPARKQMDKPEWKRVPNSEEDVRKCFGPR\u003csup\u003e249\u003c/sup\u003e of N protein is the key region for SADS-CoV N-TRIM25 interaction. TRIM25 consists of a N-terminal RING domain, two B-boxes, a central CCD and a C-terminal SPRY domain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To enhance understanding of the SADS-CoV N-TRIM25 interaction, we comprehensively analyzed the structural domains necessary for TRIM25 binding to SADS-CoV N proteins, by using a series of GFP-tagged deletion mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, the CCD domain of TRIM25 interacted with SADS-CoV N, whereas the other domains did not show any interaction. Furthermore, the CCD deletion TRIM25 mutant (TRIM25 Del CCD) was unable to bind SADS-CoV N protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). The coiled coil region of the TRIM family proteins primarily participated in homo-oligomeric interactions, and promoted the formation of a hypersecondary structure with multiple a helices [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Mutants lacking the CCD region of TRIM25 cannot multimerize [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given that SADS-CoV N interacted with the CCD domain of TRIM25, we next tested whether SADS-CoV N might interfere with TRIM25 multimerization. Indeed, SADS-CoV N expression effectively inhibited CCD-mediated TRIM25 multimerization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSADS-CoV N protein interferes with TRIM25-RIG-I interaction\u003c/h2\u003e \u003cp\u003eGiven that TRIM25 is a RIG-I regulatory partner, our subsequent studies were designed to determine SADS-CoV N-RIG-I interaction. To test this possibility, we investigated the SADS-CoV N-RIG-I interaction through Co-IP. On the basis of precipitation with antibodies against Flag or Myc tag, we determined that RIG-I interacted with SADS-CoV N protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Further Co-IP assays indicated that only full-length SARS-CoV N interacted with RIG-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We also generated Flag-tagged N-terminal (Flag-RIG-IN) and C-terminal (Flag-RIG-IC) constructs and assessed them in Co-IP experiments. Co-IP assays revealed that the SADS-CoV N protein interacted with Flag-RIG-I and Flag-RIG-IN, but not Flag-RIG-IC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). To further establish that the RIG-I 2CARD was sufficient for interaction with SADS-CoV N, GST-RIG-I-2CARD and Myc-SADS-CoV N were co-transfected in HEK293T cells. The results indicated that the N-terminal CARD of RIG-I was crucial for SADS-CoV N-RIG-I interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). To explore the localization of the N/RIG-I, and TRIM25/RIG-I, and N/TIRM25/RIG-I complexes, we performed confocal microscopy. Imaging showed that RIG-I was colocalized with SADS-CoV N and TRIM25 in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). These results further suggested that SADS-CoV N interacts with the RIG-I.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStudy showed that TRIM25 SPRY domain interacted with RIG-I, which induced K63-linked ubiquitination of the RIG-I N-terminal CARDs, thereby markedly activating RIG-I downstream signaling [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Given the association of the RIG-I N-terminal CARDs with SADS-CoV N protein, we speculated that the binding of RIG-I to TRIM25 might be affected by SADS-CoV N protein. To examine this possibility, we coexpressed HA-TRIM25 and Flag-RIG-I in HEK293T cells, both with and without Myc-SADS-CoV N. Co-IP assays revealed that overexpression of SADS-CoV N protein attenuated TRIM25-RIG-I interaction, which was a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Thus, SADS-CoV N protein competitively binds RIG-I N-terminal CARDs and interferes with the TRIM25-RIG-I interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSADS-CoV N protein suppresses IFN-I production\u003c/h2\u003e \u003cp\u003eTo explore whether the presence of SADS-CoV N protein in the RIG-I/TRIM25 complex might inhibit RIG-I downstream signaling and IFN-β production, HEK293T were transfected with Myc-N or Myc expression plasmid for 24 h, and then infected with Sendai virus (SeV) or stimulated with poly(I:C) for another 12 h. SADS-CoV N protein inhibited the IFN-β promoter activity inducing by SeV and poly(I:C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). qPCR-RT analysis indicated that the expression of SADS-CoV N protein decreased the transcription of IFN-β, CXCL10, and ISG56 inducing by SeV and poly(I:C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Consistently, ectopic expression of SADS-CoV N inhibited the phosphorylation of IRF3 and TBK1 stimulated by SeV and poly(I:C) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). These data indicated that SADS-CoV N protein suppresses IFN-β production. To examine whether SADS-CoV N protein regulated RIG-I mediated IFN-β activation, HEK293T cells were transfected with firefly luciferase reporter plasmid, empty vector, or SADS-CoV N and RIG-I expression plasmid. The RIG-I induced IFN-β promoter activation was significantly suppressed in a dose-dependent manner by SARS-CoV N protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Similarly, N protein of SADS-CoV inhibited RIG-I mediated IFN-β transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Furthermore, qRT-PCR indicated that TRIM25 further enhanced IFN-β transcription levels by acting on RIG-I; however, SADS-CoV N decreased the TRIM25-mediated RIG-I signaling enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). We next investigated whether SADS-CoV N protein regulated VSV-GFP infection and proliferation. Fluorescence microscopy, flow cytometry, and western blotting showed that Myc-N plasmid-transfected HEK293T cells, compared with vector-treated cells, facilitated the replication of VSV-GFP, as measured by GFP signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ-L), thus suggesting that SADS-CoV N inhibits the secretion of antiviral factors. Collectively, these data indicated that SADS-CoV N inhibits the TRIM25-mediated enhancement of RIG-I signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSADS-CoV infection entails a multifaceted interplay between virus and host. Understanding the SADS-CoV and host protein interaction can not only elucidate the mechanism of viral infection but also identify antiviral factors. In this study, we found TRIM25 was a new host restriction factor in SADS-CoV infection. The experiments provided clear evidence that SADS-CoV N interacted with TRIM25 and RIG-I protein. This interaction inhibits RIG-I signaling via TRIM25, which is essential for full RIG-I activation [20].\u003c/p\u003e\n\u003cp\u003eA multitude of TRIM family proteins have significant functions in the inhibition of viral replication. After viral infection, the mRNA and protein level of several TRIM protein are upregulated; these proteins subsequently act as host factors promoting the production of IFNs and inhibiting viral replication. TRIM14 is upregulated after HSV-1 infection and positively regulates type I IFN signaling [37]. TRIM22, a restriction factor, is upregulated at both the mRNA and protein levels in influenza A virus infected human alveolar epithelial A549 cells [38]. In the current study, SADS-CoV infection upregulated the levels of TRIM25 mRNA and protein \u003cem\u003ein vitro\u003c/em\u003e and\u003cem\u003e in vivo\u003c/em\u003e. The most prominent role of TRIMs is inhibition of viral replication. TRIM56 impairs hepatitis B virus, PEDV, yellow fever virus, and human coronavirus OC43 infection [39-41]. TRIM5\u0026alpha; inhibits human immunodeficiency virus type 1 (HIV-1) infection [42]. We found that upregulation of TRIM25 restricts SADS-CoV infection, whereas downregulation of this protein enhances SADS-CoV infection (Figure 2). Therefore, TRIM25 is a novel host factor that exerts inhibitory effects on SADS-CoV replication. \u003c/p\u003e\n\u003cp\u003eThe N proteins of different coronaviruses are highly conservated and perform multiple functions during viral infection [6]. TRIM25 is targeted by MERS-CoV N [23] and SARS-CoV N protein [32] for immune evasion. Similarly, SADS-CoV N protein also interacts with TRIM25 (Figure 3). The N\u003csup\u003eS215-R249\u003c/sup\u003e domain of SADS-CoV N was critical for binding TRIM25\u003cem\u003e in vitro\u003c/em\u003e. Despite variations in length and primary sequence among nucleoproteins from different coronaviruses, a conserved three-domain organization has been identified. Notably, domain Ⅱ of the N protein is highly conserved, as compared with domains I and Ⅲ [43]. The N\u003csup\u003eS215-R249\u003c/sup\u003e domain of SADS-CoV N protein is in domain Ⅱ. TRIM25 consists of a RING-finger domain, two B-box domains, a central CCD, and a C-terminal SPRY domain [18]. SADS-CoV N protein directly binds TRIM25 and targets the TRIM25 CCD, thereby disrupting TRIM25 multimer formation (Figure 4H). The formation of TRIM25 multimers acts a pivotal role in RIG-I CARDs ubiquitination\u0026mdash;a crucial modification necessary for the optimal IFN production in response to viral infection [20]. SADS-CoV N directly binds TRIM25, and inhibits the signal transduction of RIG-I and IFN-\u0026beta; production. The similarity of the observed mechanism to those used by paramyxovirus V [44] and influenza A virus NS1 [36] demonstrates that TRIM25 may be a common viral target for RIG-I antagonism. Interestingly, despite SADS-CoV N and paramyxovirus V both targeting TRIM25 , our data indicated that SADS-CoV N utilized a different mechanism to inhibit TRIM25 and RIG-I compared with paramyxovirus V. Paramyxovirus V targets TRIM25 SPRY domain, whereas SADS-CoV N protein interacts with TRIM25 CCD domain. Despite these differences, SADS-CoV N, paramyxovirus V, and influenza A virus NS1 proteins ultimately similarly suppress RIG-I signaling ubiquitination/activation by precluding TRIM25 binding to RIG-I.\u003c/p\u003e\n\u003cp\u003eViruses evolving to inhibit antiviral activity induced by IFN is well-documented. For instance, viral protein specifically impedes RIG-I- mediated the signal transduction. Influenza A virus NS1 inhibits the IFN-I induction via the interaction with RIG-I [45]. We found SADS-CoV N binds two N-terminal CARDs of RIG-I, thus suppressing the TRIM25-RIG-I interaction (Figure 5). TRIM25-SADS-CoV N-RIG-I interaction supports the hypothesis that N protein may potentially impede the progression of TRIM25-RIG-I downstream signaling pathway. Different coronavirus N proteins have been indicated to inhibit IFN production. PDCoV N protein suppresses Riplet-induced ubiquitination of RIG-I through interaction with porcine RIG-I and TRAF3 protein [46]. SARS-CoV-1 and MHV N proteins interaction with PACT protein attenuates the activation of RIG-I and MDA5 [8]. PEDV N protein interferes with TBK1-mediated IRF3 phosphorylation and thus inhibits IFN-\u0026beta; expression [11]. Our current results indicated that ectopic expression of SADS-CoV N protein significantly inhibited mRNA expression of IFN-\u0026beta;, CXCL10 and ISG56 induced by SeV or poly I:C, and RIG-I activity. Overexpression of SADS-CoV N protein in HEK293T cells enhanced the replication of VSV-GFP (Figure 6). Thus, the SADS-CoV N protein facilitates viral replication by inhibiting the host interferon response.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our results suggested that TRIM25 impeded SADS-CoV replication. Furthermore, SADS-CoV N protein inhibits TRIM25 multimerization and TRIM25-RIG-I interaction to antagonize antiviral activity. In addition, SADS-CoV N suppressed IFN-I production and facilitated VSV-GFP replication. Our findings elucidate a significant molecular mechanism through which SADS-CoV utilizes its N protein to evade innate immune response mediated by TRIM25, thus providing an explanation of the natural viral defense mechanism and potentially facilitating the development of more effective strategies for controlling SADS-CoV infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: JZ, DS, and LF. Methodology: JZ, ZJ, XZ, DL, XY, MZ, DS, and LF. Experiment operation: JZ, LZ, ZJ, and TF. Supervision: DS and LF. Data analysis: JZ, HS, JC, XZ, DS, and LF. Writing\u0026mdash;original draft: JZ. Writing\u0026mdash;review and editing: JZ, DS, and LF. All authors read and approved the final manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Regional Innovation and Development Joint Fund of National Natural Science Foundation of China, grant number U23A20236; the National Key R\u0026amp;D Program of China, grant number 2021YFD1801105; and the Central Public-interest Scientific Institution Basal Research Found, grant number 1610302022015.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the authors on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by Harbin Veterinary Research Institute. The animal ethics committee approval number was\u0026nbsp;231108-05-GR.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579:270\u0026ndash;\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Guo H, Zhou P, Shi ZL (2021) Characteristics of SARS-CoV-2 and COVID-19. Nature reviews. 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Cell 122:669\u0026ndash;682\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Swine acute diarrhea syndrome coronavirus, Nucleocapsid, Interferon, RIG-I, TRIM25","lastPublishedDoi":"10.21203/rs.3.rs-3814773/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3814773/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSwine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging \u003cem\u003eAlpha-coronavirus\u003c/em\u003e, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed IFN production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I tandem caspase activation and recruitment domain and TRIM25 CCD domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-I production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.\u003c/p\u003e","manuscriptTitle":"Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibitng TRIM25 oligomerization and functional activation of RIG-I/TRIM25","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-29 04:52:42","doi":"10.21203/rs.3.rs-3814773/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adff40b2-b8a9-439f-8ed2-e0c0f8b7fbf9","owner":[],"postedDate":"January 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-15T15:11:13+00:00","versionOfRecord":{"articleIdentity":"rs-3814773","link":"https://doi.org/10.1186/s13567-024-01303-z","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2024-04-08 15:02:11","publishedOnDateReadable":"April 8th, 2024"},"versionCreatedAt":"2024-01-29 04:52:42","video":"","vorDoi":"10.1186/s13567-024-01303-z","vorDoiUrl":"https://doi.org/10.1186/s13567-024-01303-z","workflowStages":[]},"version":"v1","identity":"rs-3814773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3814773","identity":"rs-3814773","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}