2  Materials and Methods

2.1  Virus, Cells, and Antibodies
The PDCoV CHN-HN-1601 strain (GenBank access no. MG832584) used in this study was isolated from the intestinal contents of a piglet with diarrhea using porcine kidney LLC-PK1 cells in 2017. The eighth passage of PDCoV with a titer of 108.45 50% tissue culture infectious dose (TCID50)/mL was used for virus inoculation. IPEC-J2 cells were generously provided by Prof. Yongchang Cao (School of Life Sciences, Sun Yat-Sen University, Guangzhou, China). The cells were cultivated in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F12) (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Solarbio Life Sciences, Beijing, China). Cells were cultured at 37 °C in a humidified CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA). The monoclonal antibody (mAb), 1A3, raised against the PDCoV nucleocapsid protein was prepared in our laboratory. Mouse anti-β-actin mAb, mouse anti-IFIT1 and rabbit anti-ANAPC7 polyclonal antibodies were bought from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat antimouse/rabbit immunoglobulin G (IgG) were obtained from Sigma-Aldrich. Alexa Fluor 488-conjugated goat antimouse IgG was the product of Thermo Fisher Scientific.

2.2  Virus Inoculation
IPEC-J2 cells approaching ∼80% confluence were washed twice with sterile phosphate buffered saline (PBS; 0.01 M, pH 7.4), and then mock infected or infected with PDCoV CHN-HN-1601 strain at a multiplicity of infection (MOI) of 0.1 TCID50 per cell. After adsorption for 1.5 h at 37 °C, the cells were rinsed once with sterile PBS and serum-free DMEM/F12 medium containing 5 μg/mL of trypsin (Sigma-Aldrich) was added. The cells were further cultured at 37 °C for the specified time points until different assays had been performed. Viral propagation in IPEC-J2 cells was evaluated by observing cytopathic effect (CPE), determination of one-step growth curve and immunofluorescence assay (IFA) using the mAb 1A3 against PDCoV.

2.3  TCID50 Assay
The kinetics of PDCoV multiplication in IPEC-J2 cells was determined by measuring the TCID50 using a microtitration infectivity assay. Briefly, 100 μL/well of 10-fold serial dilutions of PDCoV were inoculated onto IPEC-J2 cells grown in 96-well microplates (Corning, NY, USA). After adsorption at 37 °C for 1.5 h, the inocula were removed, and serum-free DMEM/F12 medium containing 5 μg/mL of trypsin was added to the wells. After an additional 48-h cultivation, virus titers were measured by observing the presence of visible CPEs in the corresponding wells, and calculated using the Reed–Muench method.27

2.4  IFA
When IPEC-J2 cells grown in 96-well microplates reached ∼80% confluence, they were mock infected or infected with PDCoV at an MOI of 0.1 TCID50 per cell. After a 48-h cultivation at 37 °C with 5% CO2, the cells were fixed with prechilled 100% ethanol for 15 min at room temperature. After washing with PBS, the cells were probed with the mAb 1A3 (1:5000 dilution) at 37 °C for 1 h. Following another washing step, 1000-fold-diluted Alexa Fluor 488-conjugated goat antimouse IgG (Thermo Fisher Scientific) was added to the cells and incubated at 37 °C for 1 h. After washing thrice with PBS, cells were observed with an Eclipse Ci-S microscope (Nikon Corp., Tokyo, Japan).

2.5  Protein Extraction, Digestion, and iTRAQ Labeling
At 24 h postinfection (hpi), both PDCoV- and mock-infected IPEC-J2 cells grown in T25 flasks (∼5 × 106 cells/flask) were rinsed twice with prechilled PBS, and then harvested with disposable cell scrapers. Three flasks of each group (PDCoV or mock) were harvested and used as three independent biological replicates. After centrifugation at 300g for 10 min, the cell pellets from each flask were lysed with 800 μL of RIPA lysis buffer (Beyotime, Shanghai, China) containing 1% SDS, 8 M urea and protease inhibitors (Beyotime). The cell lysates were further sonicated on ice for 5 min with 10 s bursts and 10 s pauses between cycles. After centrifugation for 20 min at 12 000g and 4 °C, the supernatant was collected and used as the total cellular proteins. The concentration of protein samples was determined using a Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL, USA).
The total cellular proteins from each biological replicate were equally divided into three aliquots, which were used as three independent technical replicates for the LC-MS/MS runs. For tryptic digestion and iTRAQ labeling, an aliquot of each protein sample containing ∼100 μg of total cellular proteins was adjusted to a 100-μL final volume using the RIPA lysis buffer. A final concentration of 10 mM of tris (2-carboxyethyl) phosphine (TCEP) was added to each protein sample, which was then incubated at 37 °C for 1 h. Afterward, iodoacetamide was added at a final concentration of 40 mM, and the protein solution was incubated for 40 min at room temperature shielded from light. Subsequently, prechilled acetone was added to the protein solution in a ratio of 6:1 and precipitated at −20 °C for 4 h. After centrifugation (10 000g, 4 °C) for 20 min, the precipitate was dissolved with 100 μL of 100 mM triethylammonium bicarbonate (TEAB). The processed protein samples were digested with 2 μg/μL of trypsin overnight at 37 °C. Following tryptic digestion, the generated peptides were dried by vacuum centrifugation, and redissolved with 20 μL of 0.5 M TEAB. The prepared peptides were then labeled with an iTRAQ reagents-8 plex kit (AB Sciex, Foster City, CA, USA) as per the manufacturer’s protocol. Briefly, the three independent biological replicates of mock-infected cellular samples were each labeled with iTRAQ-113, iTRAQ-114, and iTRAQ-115; the three independent biological replicates of PDCoV-infected samples were each labeled with iTRAQ-116, iTRAQ-119, and iTRAQ-121. All the labeled samples of each group were mixed with an equal amount, and then fractionated using an ACQUITY ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA, USA) combined with an ACQUITY UPLC BEH C18 column (300 Å, 1.7 μm, 2.1 mm × 150 mm). Finally, a total of 10 fractions of each group were collected. After merging two fractions of each group into one, the pooled 10 fractions were dried by using a rotary vacuum concentrator.

2.6  LC-MS/MS Analysis
LC-MS/MS analyses of the labeled peptides were carried out using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) combined with an EASY-nLC 1200 system (Thermo Fisher Scientific). After loading 2 μg of the labeled peptides onto a C18 reversed phase HPLC column (Thermo Fisher Scientific), peptides were chromatographed for 120 min at a flow rate of 300 nL/min over gradients from 2–80% (mobile phase A comprising 0.1% (v/v) formic acid and 2% (v/v) acetonitrile; mobile phase B comprising 0.1% (v/v) formic acid and 80% (v/v) acetonitrile). The following ion source parameters, including spray voltage 1.8 kV, capillary temperature 275 °C and declustering potential 100 V, were set. The mass spectrometer was run using a data-dependent Top-20 acquisition mode, switching automatically between MS and MS/MS. A full MS scan ranging from 350 to 1300 m/z was conducted at 70 000 resolution with an automatic gain control (AGC) target value of 3 × 106 ions and a maximum ion transfer (IT) of 20 ms. The precursor ions were fragmented by means of high-energy collisional dissociation (HCD), and all MS/MS spectra were scanned using the following parameters: resolution 17 500; AGC 1 × 105 ions; maximum IT 50 ms; dynamic exclusion duration 18 s; normalized collision energy 30%; and intensity threshold 1.6 × 105.

2.7  MS Data Analysis
The original MS/MS raw data were analyzed using the Proteome Discoverer Software 2.1 (Thermo Fisher Scientific, San Jose, CA, USA). The data were searched against the database of UniProt Sus scrofa (February 26, 2017, containing 26 103 sequences, http://www.uniprot.org/proteomes/UP000008227, version: Uniprot-proteome-UP000008227-Sus scrofa (Pig)-26103s-20170226.fasta) and porcine deltacoronavirus Uniprot database (February 26, 2017, containing 442 sequences, http://www.uniprot.org/porcinedeltacoronavirus, version: Uniprot-Porcine deltacoronavirus [1586324]-442s-20170226.fasta). The parameters for database searching were set as follows: instrument, TripleTOF 5600; cysteine alkylation, iodoacetamide; digestion, trypsin; dynamic modification, oxidation (M), acetylation (protein N-terminus), and iTRAQ8plex (Y); static modification, iTRAQ8plex (K), iTRAQ8plex (N-terminus), and carbamidomethyl (C); maximum missed cleavages, 2; precursor mass tolerance, 10 ppm; fragment mass tolerance, 0.05 Da; validation based on, q-value. To guarantee the accuracy of the MS data analysis, the cutoff value for the peptide and protein confidences was set to >95% and >1.20, respectively, coupled with a false discovery rate (FDR) of ≤1% for peptide and protein identifications. The t test function in the R language software was applied to calculate the p-value of the expression difference of cellular proteins between mock- and PDCoV-infected IPEC-J2 cells, and only proteins with a fold change >1.20 or <0.83 and a p-value < 0.05, which have being widely used as the criteria for judging DEPs,28,29 were considered differentially expressed.

2.8  Bioinformatics Analysis
The data for the identified DEPs of PDCoV-infected IPEC-J2 cells were submitted to Gene Ontology (GO) Terms (http://geneontology.org/) for GO analysis, by which the DEPs were assigned into three branches of ontology—biological process (BP), cellular component (CC), and molecular function (MF). GO enrichment analysis for the DEPs was done using the Goatools software (https://github.com/tanghaibao/GOatools) with Fisher’s exact test, and those with a p-value < 0.05 were thought to be significantly enriched. The protein database of clusters of Orthologous Groups (COG; http://www.ncbi.nlm.nih.gov/COG/) was also used to assign possible functions to the identified DEPs. The Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) pathway analyses were performed to reveal the potential functions of the DEPs, which were annotated using the BlastP program of the Diamond software against the KEGG database with a cutoff E-value ≤ 1 × 10–5 and identity ≥0.98. The pathway enrichment statistics were performed using the KOBAS software (http://kobas.cbi.pku.edu.cn/home.do/) with the Fisher’s exact test, and those with p-values < 0.05 were regarded as statistically significant.30,31 Furthermore, the protein–protein interaction networks were created using the online STRING database (http://string-db.org/) and visualized by Cytoscape (https://cytoscape.org/), which was widely used for analyzing the relationships between DEPs.32

2.9  Quantitative Real-Time PCR (qPCR)
To verify the DEPs identified by iTRAQ at the transcriptional level, the mRNA of two representative DEPs, the downregulated ANAPC7 and the upregulated IFIT1, were detected by qPCR. Total cellular RNA of both PDCoV- and mock-infected IPEC-J2 cells was extracted using the TaKaRa MiniBEST universal RNA extraction kit (Dalian, China). The first strand cDNA was synthesized using 2 μg of cellular RNA and with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) following the manufacturer’s instructions. The qPCR assays were conducted using a TaKaRa SYBR Premix Ex Taq kit on an ABI 7500 real-time PCR machine along with β-actin as a housekeeping gene. The primers used for the qPCR assay were designed based on the NCBI reference sequences for ANAPC7, IFIT1 and β-actin genes under GenBank accession numbers NM_016238, HQ679904 and NM_001101, respectively. The information for the designed primers was as following: ANAPC7 (Forward: 5′-CTTTGCTGAGGAACGCACTG-3′, Reverse: 5′-TCCATGTCGTCCACATCCTC-3′); IFIT1 (Forward: 5′-GAAGATTTAACCCAACAAGAACATA-3′, Reverse: 5′-CTTTCGATACGTAAGGTAATACAGC-3′); β-actin (Forward: 5′-TCCCTGGAGAAGAGCTACGA-3′, Reverse: 5′-AGCACTGTGTTGGCGTACAG-3′). The qPCR assays were carried out in a 20-μL reaction volume comprised 10 μL of TaKaRa SYBR Premix Ex Taq, 0.4 μL of each forward and reverse primer (10 μM), 7.2 μL of nuclease-free water, and 2 μL of template. The qPCR amplifications were carried out using a thermal profile with an initial step at 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 10 s (for ANAPC7 and β-actin) or 55 °C for 10 s (for IFIT1) and 72 °C for 40 s. Fold-change values were calculated in term of the 2–ΔΔCt method and normalized to the β-actin reference gene.33

2.10  Western Blot Analysis
To further validate the identified DEPs, Western blot was performed to detect ANAPC7 and IFIT1 protein expression levels using β-actin as an internal control. Briefly, total cellular proteins were extracted from both mock- and PDCoV-infected IPEC-J2 cells at 24 hpi. The resulting protein samples were separated on 12% SDS-PAGE gels and transferred onto 0.22 μm poly(vinylidene fluoride) (PVDF) membranes (Millipore, Bedford, MA, USA) using a semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% (w/v) nonfat dry milk overnight at 4 °C and then probed with either anti-ANAPC7 (1:1000), anti-IFIT1 (1:1000), or anti-β-actin (1:5000) primary antibodies at 37 °C for 1 h. After thorough washing with PBST, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000) for 1 h at 37 °C. The target protein blots on the membranes were developed with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific), and images were taken using a ProteinSimple FluorChem E image system (Santa Clara, CA, USA).

2.11  Data Availability
The proteomics data obtained by LC-MS/MS in this study were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) by means of the Proteomics Identifications (PRIDE) partner repository with the data set identifier PXD019975.

2.12  Statistical Analysis
The methods used for the statistical analysis of proteomic data and the corresponding threshold criteria were described in detail in the corresponding sections of MS data and bioinformatics analyses. The data from three independent qPCR runs were expressed as means ± standard deviation (SD), and were statistically analyzed by Student’s t test using the GraphPad Prism software (Version 5.0; La Jolla, CA, USA). Differences with a value of p < 0.05 were regarded as statistically different.