Stromal Antigen 2 Deficiency Induces Interferon Responses and Restricts Porcine Deltacoronavirus Infection

Porcine deltacoronavirus (PDCoV) is a recently discovered enteropathogenic coronavirus and has caused significant economic impacts on the pork industry. Although studies have partly uncovered the molecular mechanism of PDCoV–host interaction, it requires further research. In this study, we explored the roles of Stromal Antigen 2 (STAG2) in PDCoV infection. We found that STAG2-deficient cells inhibited infection with vesicular stomatitis virus (VSV) and PDCoV, whereas restoration of STAG2 expression in STAG2-depleted (STAG2−/−) IPEC-J2 cells line restored PDCoV infection, suggesting that STAG2 is involved in the PDCoV replication. Furthermore, we found that STAG2 deficiency results in robust interferon (IFN) expression. Subsequently, we found that STAG2 deficiency results in the activation of JAK-STAT signaling and the expression of IFN stimulated gene (ISG), which establish an antiviral state. Taken together, the depletion of STAG2 activates the JAK-STAT signaling and induces the expression of ISG, thereby inhibiting PDCoV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of PDCoV replication.


Introduction
Porcine deltacoronavirus (PDCoV) is a recently discovered enteropathogenic coronavirus and has caused significant economic impacts on the pork industry [1][2][3]. PDCoV, similar to other swine enteric coronaviruses, including transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV), have caused frequent occurrences of diarrhea, vomiting, and dehydration in piglets [2,[4][5][6][7][8]. Clinically, PDCoV infection commonly occurs in the form of co-infection with PEDV or TGEV, which has caused significant economic losses to the global swine industry. PDCoV have the potential for cross-species transmission and are causing huge economic losses in the pig industry in China and the world, which therefore needs to be urgently addressed [9].
Innate immunity plays a crucial role in host defense against invading pathogens [10,11]. During viral infection, the innate immune response is often activated, leading to the induction of the type I interferon (IFN-I or IFN α/β). IFN-I is the potent cytokine of critical importance in controlling viral infections and priming adaptive immune responses [12,13]. Following production, IFN-I initiates a positive feed-back loop by binding to their cognate receptors on the cell surface in an autocrine and paracrine manner [14,15] and activates JAK protein tyrosine kinases (JAK1 and Tyk2) which phosphorylate signal transducers and activators of transcription (STAT) 1 (STAT1) and (STAT) 2 (STAT2). STAT1 and STAT2 together with interferon regulatory factor 9 (IRF9) form a transcription factor complex termed IFNstimulated gene factor 3 (ISGF3). Then, ISGF3 is translocated into the nucleus and binds to the IFN-stimulated response elements (ISRE) to induce the expression of IFN-stimulated genes (ISGs), which establish an antiviral state [15][16][17][18].

Plasmids and Antibodies
The full-length sequence of STAG2 was constructed into the pCAGGS-HA vector to obtain the recombinant plasmid of pCAGGS-HA-STAG2. The PCR primers were designed by Primer 5. SgRNAs were designed according to the website http://crispr.mit.edu (accessed on 7 January 2020). The primer sequences and sgRNAs are listed in Table 1. All plasmid construct was confirmed by sequencing.

Virus Infection
Monolayers of IPEC-J2 cells were infected with PDCoV at an MOI of 1 for 1 h at 37 • C. Unbound virus was removed, and cells were maintained in complete medium for various time points until samples were harvested.

Transfection
Cells were transfected with indicated plasmids using X-tremeGENE transfection reagent according to manufacturer's instruction (Roche, Indianapolis, IN, USA). At the indicated times, cell samples were collected and lysed in RIPA buffer (Beyotime, Nantong, China) for Western blot analysis of target proteins.

CRISPR-Cas9 Knockout Cells
STAG2-Cas9 knockout cells were generated by using the CRISPR/Cas9 system. Briefly, the designed single guide RNA (sgRNA) targeting the porcine STAG2 gene was cloned into the Lentiviral vector2 vector. pMD2.G and psPAX2, producing the VSV-G glycoprotein and envelope proteins of the lentivirus, respectively, combined with lenti-guide-puro-sgRNA-STAG2 were co-transfected into HEK293T cells to produce the recombined lentivirus. IPEC-J2 cells were infected with lentivirus, and puromycin (2.5 µg/mL) was added to select the positive clones. The monoclonal cells were obtained with the limited dilution method. Finally, the knockout of STAG2 was confirmed by Western blot at the protein level.

IFA
IFA was performed as described previously with slight modification [17]. Briefly, STAG2depleted IPEC-J2 cells lines or WT cells were infected with PDCoV for 24 h, and the cells were fixed and stained with anti-PDCoV-N mouse monoclonal antibody [29] for one hour. After the removal of unbound antibodies, the cells were stained with FITC-conjugated goat anti-mouse IgG for another hour, followed by nuclei staining with DAPI (4,6-diamidino-2-phenylindole; Sigma). After washing the cells, the fluorescence was visualized with an Olympus inverted fluorescence microscope equipped with a camera.

Western Blot
Western blot analysis was performed as previously described [30]. Treated samples were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitors (Roche) and separated by SDS-PAGE under reducing conditions and transferred onto a PVDF membrane (Merck Millipore, Temecula, CA, USA). After blocking, the membranes were incubated with a primary antibody and then probed with an appropriate IRDyeconjugated secondary antibody (LiCor Bio-Sciences, Lincoln, NE, USA). The membranes were scanned using an Odyssey instrument (Li-Cor Biosciences) according to the manufacturer's instructions.

Quantitative RT-PCR
Quantitative RT-PCR analysis was carried out as described previously [31]. Total RNA was extracted from cells and subjected to quantitative RT-PCR using specific primers as listed in Table 1. Relative gene quantification was performed by the method of 2(-Delta Delta C(T)) [32].

TCID 50 Assay
Collected virus samples were frozen and thawed three times and clarified by centrifugation at 8000× g for 10 min prior to titration. TCID 50 assays were performed according to the method of Reed & Muench as previously described [32]. Briefly, cell monolayers were inoculated with 10-fold serial dilutions of each virus stock and incubated for 4 days prior to observation of the presence of cytopathic effect.

Cell Viability Assay
Cell viabilities were assessed using a cell counting kit-8 (CCK-8) (Cat NO. GK10001, GLPBIO, Montclair, CA, USA). Assays were performed according to the manufacturer's instructions. Briefly, the WT cells and the STAG2 −/− cells were incubated in 96-well plates and the cell viabilities were measured at 12 h, 24 h, and 36 h. A total of 10 µL of CCK-8 reagents were added to each well of the plates, and the cells were incubated at 37 • C for 1 h, then the absorbance at 450 nm was measured by a microplate reader.

Statistical Analysis
Variables are expressed as mean ± S.D. Statistical analyzes were performed using student's t-test. Significance is denoted in the figures as follows: *, p < 0.05 and **, p < 0.01.

Establishment of STAG2-Knockout IPEC-J2 Cell Line
To study the role of STAG2 in PDCoV infection, we then generated a single clonal STAG2 knockout in IPEC-J2 cells, porcine intestinal epithelial cell (IEC) line commonly used for PDCoV studies. The knockout effect of STAG2 was determined by Western blot, and the results revealed that the STAG2 protein was knocked out ( Figure 1A). Sanger sequencing confirmed the presence of 1 bp insert in STAG2-depleted (STAG2 −/− ) IPEC-J2 cells line ( Figure 1B). Meanwhile, knockout of STAG2 had no significant effect on the cell viability when compared to the wild type (WT) cells ( Figure 1C). To study the function of STAG2 in PDCoV infection, we generated a stable STAG2 −/− IPEC-J2 cells line, STAG2 −/− IPEC-J2 cells were propagated and the deletion was confirmed by Western blot up to the last passage ( Figure 1D).

Confirmation of STAG2 as a Critical Host Factor for VSV Infection
We next used GFP-expressing VSV to infect WT or STAG2 −/− IPEC-J2 cells. Fluorescence microscopy images showed that VSV infection was evident in WT cells, and conversely VSV infection was significantly inhibited in STAG2 −/− cells compared to WT cells ( Figure 2A). Additionally, the loss of STAG2 resulted in decreased VSV replication, as detected by comparing the level of GFP protein in VSV-infected STAG2 −/− cells to that in the WT cells ( Figure 2B), and the results suggested that VSV was reduced in the absence of STAG2. sequencing confirmed the presence of 1 bp insert in STAG2-depleted (STAG2 −/− ) IP cells line ( Figure 1B). Meanwhile, knockout of STAG2 had no significant effect on th viability when compared to the wild type (WT) cells ( Figure 1C). To study the funct STAG2 in PDCoV infection, we generated a stable STAG2 −/− IPEC-J2 cells line, STA IPEC-J2 cells were propagated and the deletion was confirmed by Western blot up last passage ( Figure 1D).

Confirmation of STAG2 as a Critical Host Factor for VSV Infection
We next used GFP-expressing VSV to infect WT or STAG2 −/− IPEC-J2 cells. Flu cence microscopy images showed that VSV infection was evident in WT cells, and versely VSV infection was significantly inhibited in STAG2 −/− cells compared to WT ( Figure 2A). Additionally, the loss of STAG2 resulted in decreased VSV replicatio detected by comparing the level of GFP protein in VSV-infected STAG2 −/− cells to th the WT cells ( Figure 2B), and the results suggested that VSV was reduced in the ab of STAG2.

Confirmation of STAG2 as a Critical Host Factor for PDCoV Infection
To assess how STAG2 responds to PDCoV infection, We next used PDCoV to STAG2 −/− or WT cells. PDCoV infection was significantly inhibited in the STAG2 −/

Confirmation of STAG2 as a Critical Host Factor for VSV Infection
We next used GFP-expressing VSV to infect WT or STAG2 −/− IPEC-J2 cells. Fluorescence microscopy images showed that VSV infection was evident in WT cells, and conversely VSV infection was significantly inhibited in STAG2 −/− cells compared to WT cells ( Figure 2A). Additionally, the loss of STAG2 resulted in decreased VSV replication, as detected by comparing the level of GFP protein in VSV-infected STAG2 −/− cells to that in the WT cells ( Figure 2B), and the results suggested that VSV was reduced in the absence of STAG2.

Confirmation of STAG2 as a Critical Host Factor for PDCoV Infection
To assess how STAG2 responds to PDCoV infection, We next used PDCoV to infect STAG2 −/− or WT cells. PDCoV infection was significantly inhibited in the STAG2 −/− cells

Confirmation of STAG2 as a Critical Host Factor for PDCoV Infection
To assess how STAG2 responds to PDCoV infection, We next used PDCoV to infect STAG2 −/− or WT cells. PDCoV infection was significantly inhibited in the STAG2 −/− cells compared with the infection in the WT cells, according to the IFA, demonstrating that PDCoV was reduced in the absence of STAG2 ( Figure 3A). As shown in Figure 3B, the viral RNA levels were significantly reduced in STAG2 −/− cells compared to WT cells. Additionally, the inhibitory effect of STAG2 knockout on the PDCoV replication was confirmed by the reduced level of PDCoV N protein expression as determined by Western blot analysis ( Figure 3C). Importantly, PDCoV infectivity was significantly decreased (~1 log) in STAG2 −/− cells compared to WT cells ( Figure 3D), indicating that PDCoV replication was significantly reduced in STAG2 −/− cells.
viral RNA levels were significantly reduced in STAG2 −/− cells compared to WT cells. ditionally, the inhibitory effect of STAG2 knockout on the PDCoV replication was firmed by the reduced level of PDCoV N protein expression as determined by Wes blot analysis ( Figure 3C). Importantly, PDCoV infectivity was significantly decreased log) in STAG2 −/− cells compared to WT cells ( Figure 3D), indicating that PDCoV rep tion was significantly reduced in STAG2 −/− cells.

STAG2 Is Required for PDCoV Replication
To further confirm the effect of STAG2 on PDCoV infection, the infectivit PDCoV was evaluated, followed by exogenous expression of WT STAG2 in STA IPEC-J2 cells. The STAG2 −/− cells were transfected with HA-tagged STAG2 plasm (HA-STAG2) or an empty vector as a control (vector con) for 24 h, and then, the cells w inoculated with PDCoV and cultured for an additional 24 h. Susceptibility to PDCoV fection was restored upon exogenous expression of WT STAG2 in STAG2 −/− IPEC-J2 c suggesting that the effect was specifically due to the loss of STAG2. (Figure 4A). A tionally, an obvious increase in PDCoV N mRNA amount relative to the amount in vector control was proven by quantitative RT-PCR analysis in STAG2 −/− cells transfe with STAG2 plasmids ( Figure 4B). Exogenous expression of WT STAG2 resulted in crease PDCoV replication, as detected by comparing the level of viral nucleocapsid protein in exogenous expression of WT STAG2 in STAG2 −/− IPEC-J2 cells to that in STAG2 −/− IPEC-J2 cells ( Figure 4C). Furthermore, an apparent increase in progeny v was determined by TCID50 assay in the PDCoV-infected STAG2 −/− IPEC-J2 cells tr The results are representative of three independent experiments (the means ± SD). **, p < 0.01. The p value was calculated using Student's t-tests.

STAG2 Is Required for PDCoV Replication
To further confirm the effect of STAG2 on PDCoV infection, the infectivity of PDCoV was evaluated, followed by exogenous expression of WT STAG2 in STAG2 −/− IPEC-J2 cells. The STAG2 −/− cells were transfected with HA-tagged STAG2 plasmids (HA-STAG2) or an empty vector as a control (vector con) for 24 h, and then, the cells were inoculated with PDCoV and cultured for an additional 24 h. Susceptibility to PDCoV infection was restored upon exogenous expression of WT STAG2 in STAG2 −/− IPEC-J2 cells, suggesting that the effect was specifically due to the loss of STAG2. ( Figure 4A). Additionally, an obvious increase in PDCoV N mRNA amount relative to the amount in the vector control was proven by quantitative RT-PCR analysis in STAG2 −/− cells transfected with STAG2 plasmids ( Figure 4B). Exogenous expression of WT STAG2 resulted in increase PDCoV replication, as detected by comparing the level of viral nucleocapsid (N) protein in exogenous expression of WT STAG2 in STAG2 −/− IPEC-J2 cells to that in the STAG2 −/− IPEC-J2 cells ( Figure 4C). Furthermore, an apparent increase in progeny virus was determined by TCID 50 assay in the PDCoV-infected STAG2 −/− IPEC-J2 cells transfected with HA-STAG2. Taken together, these data suggest that the loss of STAG2 likely leads to an alteration of signaling pathways within host cells that is commonly shared by PDCoV. fected with HA-STAG2. Taken together, these data suggest that the loss of STAG2 lik leads to an alteration of signaling pathways within host cells that is commonly shared PDCoV.

Loss of STAG2 Activates IFN and ISG Expression
To identify the mechanism by which the loss of STAG2 leads to a suppression PDCoV growth, we first performed an unbiased RNA-sequencing analysis, using different platforms, to profile the transcriptome of WT and STAG2 −/− IPEC-J2 cells. G ontology pathway analysis revealed a distinct IFN signature in the STAG2 −/− IPEC-J2 c (data not shown). Several antiviral proteins in STAG2 −/− IPEC-J2 cells, including IFN IFN-λ1, IFN-λ3, OAS1, IL-54, IL-15, IL-56, and OASL, were significantly increased determined by quantitative RT-PCR ( Figure 5A-H).  50 . The results are representative of three independent experiments (the means ± SD). **, p < 0.01. The p value was calculated using Student's t-tests.

Loss of STAG2 Activates IFN and ISG Expression
To identify the mechanism by which the loss of STAG2 leads to a suppression of PDCoV growth, we first performed an unbiased RNA-sequencing analysis, using two different platforms, to profile the transcriptome of WT and STAG2 −/− IPEC-J2 cells. Gene ontology pathway analysis revealed a distinct IFN signature in the STAG2 −/− IPEC-J2 cells (data not shown). Several antiviral proteins in STAG2 −/− IPEC-J2 cells, including IFN-β, IFN-λ1, IFN-λ3, OAS1, IL-54, IL-15, IL-56, and OASL, were significantly increased, as determined by quantitative RT-PCR ( Figure 5A-H).

STAG2 Deletion Triggers IFN Production by Activating the Levels of Phosphorylated STAT1
We next sought to determine mechanistically how the cell-intrinsic IFN activation occurred in the STAG2 −/− cells. We assayed the phosphorylation status of signaling pathways, based on the IFN-stimulated response elements to induce the expression of IFNstimulated genes, which establish an antiviral state. The relative quantities of STAT1 mRNA in STAG2 −/− cells relative to the expression in WT cells were up-regulated by quantitative RT-PCR ( Figure 6A). Strong phosphorylation of STAT1 was observed in STAG2 −/− cells by Western blot analysis ( Figure 6B).

STAG2 Deletion Triggers IFN Production by Activating the Levels of Phosphorylated STAT1
We next sought to determine mechanistically how the cell-intrinsic IFN activation occurred in the STAG2 −/− cells. We assayed the phosphorylation status of signaling pathways, based on the IFN-stimulated response elements to induce the expression of IFN-stimulated genes, which establish an antiviral state. The relative quantities of STAT1 mRNA in STAG2 −/− cells relative to the expression in WT cells were up-regulated by quantitative RT-PCR ( Figure 6A). Strong phosphorylation of STAT1 was observed in STAG2 −/− cells by Western blot analysis ( Figure 6B).

Discussion
The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces IFNs and cytokines that perform antiviral functions to eliminate invading viruses [33][34][35][36]. However, during coevolution with their host, viruses have developed new strategies to evade host antiviral defense programs [37][38][39][40].
Coronaviruses has acquired multiple mechanisms to antagonize the host innate immune system by either targeting viral sensors or blocking downstream antiviral sig-

STAG2 Deletion Triggers IFN Production by Activating the Levels of Phosphorylated ST
We next sought to determine mechanistically how the cell-intrinsic IFN activa occurred in the STAG2 −/− cells. We assayed the phosphorylation status of signa pathways, based on the IFN-stimulated response elements to induce the expressio IFN-stimulated genes, which establish an antiviral state. The relative quantities of ST mRNA in STAG2 −/− cells relative to the expression in WT cells were up-regulate quantitative RT-PCR ( Figure 6A). Strong phosphorylation of STAT1 was observe STAG2 −/− cells by Western blot analysis ( Figure 6B).

Discussion
The innate immune system is the first line of the host defense program ag pathogens and harmful substances. Antiviral innate immune responses can be trigg by multiple cellular receptors sensing viral components. The activated innate imm system produces IFNs and cytokines that perform antiviral functions to eliminat vading viruses [33][34][35][36]. However, during coevolution with their host, viruses have veloped new strategies to evade host antiviral defense programs [37][38][39][40].
Coronaviruses has acquired multiple mechanisms to antagonize the host in immune system by either targeting viral sensors or blocking downstream antiviral

Discussion
The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces IFNs and cytokines that perform antiviral functions to eliminate invading viruses [33][34][35][36]. However, during coevolution with their host, viruses have developed new strategies to evade host antiviral defense programs [37][38][39][40].
Coronaviruses has acquired multiple mechanisms to antagonize the host innate immune system by either targeting viral sensors or blocking downstream antiviral signaling molecules. For example, Nsp1 proteins of Severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), murine hepatitis virus (MHV), TGEV and PEDV suppresses host gene expression [41][42][43]. Of the several known viral evasion strategies, the cleavage of crucial innate immune molecules including adaptors, kinases, and transcriptional factors are considered to be a particularly powerful way for viruses to escape the innate immune response. The 3C-like protease of PEDV and PDCoV, disrupts type I IFN signaling by cleaving the NF-κB essential modulator (NEMO) [44,45]. In addition, PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2, an essential factor for IFN responses [46]. IFNs generate an antiviral state through ISG induction as a defense mechanism against viral infection. To combat these antiviral effects of ISGs, many viruses, including CoVs, have evolved elaborate mechanisms, such as altering subcellular localization or inducing ISG degradation, to antagonize their antiviral functions. To our knowledge, some proteins encoded by CoVs, such as PEDV N protein, PEDV nsp1, PDCoV nsp5, PDCoV nsp6, and MHV nsp15, have been demonstrated to hijack IFN signaling to reduce ISG production indirectly [47]. However, viruses are not limited to the aforementioned strategies to antagonize IFN responses.
Cohesin is a multi-subunit nuclear protein complex that coordinates sister chromatid separation during cell division. Highly frequent somatic mutations in genes encoding core cohesin subunits have been reported in multiple cancer types, and its loss of function has been believed to induce aneuploidy [48]. STAG2, a cohesin family gene, is among the most recurrently mutated genes in cancer [49,50]. In contrast to the implication of STAG2 in cancer, less information has been reported on the interplay between STAG2 and microorganism infection. It has been reported that the loss of STAG2, an important component of the cohesin complex, confers resistance to RV replication in cell culture and human intestinal enteroids. In addition, STAG2 deficiency results in spontaneous genomic DNA damage and robust IFN expression via the cGAS-STING cytosolic DNA-sensing pathway. The resultant activation of JAK-STAT signaling and ISG expression broadly protects against virus infections, including RVs [26].
In the present study, we first identified that PDCoV and VSV replication were significantly reduced in the STAG2 −/− cells by establishing of STAG2-knockout IPEC-J2 cell line. To identify the mechanism by which the loss of STAG2 leads to a suppression of PDCoV growth, we first performed an unbiased RNA-sequencing analysis, using two different platforms, to profile the transcriptome of WT and STAG2 −/− IPEC-J2 cells. RNAseq dataset revealed that STAG2 depletion elicits an excessive IFN expression. Moreover, STAG2 deficiency results in robust IFN expression via the JAK-STAT signaling pathway. Our work may facilitate a better understanding of PDCoV infection and pathogenesis.