Mink lung epithelial cells (Mv.1.Lu cells) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and grown in Minimum Essential Medium (Gibco ®Invitrogen, U.S.A.), supplemented with 10% fetal bovine serum (Invitrogen) at 37°C and 5% CO₂. The canine distemper virus strain CDV-PS (GenBank accession no. JN896331), a low passage isolate (<7 passages) from a morbid dog in 2013 (Yi et al., 2013), was preserved in our laboratory. The virus was propagated in Vero cells. In the study, three additional passages of the virus were performed in Mv.1.Lu cells, resulting in the virus suspension with a titer of 10^3.1 TCID₅₀/mL determined by a 50% tissue culture infectious dose (TCID₅₀) assay (Yamaguchi et al., 1988). Briefly, monolayers of Mv.1.Lu cells in 96-well plates were infected with a 10-fold serial dilution of the supernatant fluids and further incubated for up to 120 h. The wells were assessed for cytopathic effects (CPE) after 3–5 days, and the TCID₅₀ was calculated using the Reed-Muench formula. Because of the low virus titer and the impurity of the virus suspension, virus concentration and purification were performed to improve the virus titer and avoid the effect of non-viral components. The clarified suspension was concentrated by polyethylene glycol 6,000 precipitation and purified by ultracentrifugation in a gradient of sucrose according to standard procedures. Sucrose-purified viruses were then titrated using the TCID₅₀ assay as described above, and the titer of the virus stocks increased to 10^6.9 TCID₅₀/mL. The attenuated CDV vaccine CDV₃ strain was treated the same as PS. The virus stocks were aliquoted and stored at −80°C until further use in the following experiments.

For the establishment of viral kinetics, Mv.1.Lu cells were grown in 6-well plates and subsequently challenged by the virus (PS) at a multiplicity of infection (MOI) of 2, calculated based on the infectious virus particle concentration determined as TCID₅₀. At 6, 12, 24, 36, 48, 60, and 72 hpi, viral propagation was confirmed by observation of the CPE and viral replication and production of PS nucleoprotein for the different time points analyzed was tested by anti-CDV NP antibody. The one-step growth curve, indicating the viral load with the time, was generated according to Chuzo ushimi with slight modifications (Ushimi et al., 1972). Briefly, 200 μL of culture medium was collected at indicated time, followed by the extraction of total RNA from all samples. qRT-PCR was then applied to detect the viral RNA at each indicated time. For iTRAQ labeling, Mv.1.Lu cells were grown in T75 flasks to 70–80% confluence and subsequently infected with the virus (PS) at an MOI of 2. As an uninfected control, a mock-infection was performed. The cells were collected at 24 hpi for the protein extraction. Three biological replicates were prepared for all samples. All experiments were performed under Biosafety Level 2 conditions.

The collected cells were lysed in lysis buffer containing a protease inhibitor cocktail. The lysate was sonicated and centrifuged at 14,000 g for 40 min, and the supernatant was quantified with the BCA Protein Assay Kit (Bio-Rad, U.S.A.). Subsequently, 200 μg of protein for each sample was digested with 4 μg of trypsin (Promega, WI) overnight at 37°C. According to the protocol of the iTRAQ reagents (8 plex, Applied Biosystems), 100 μg of peptide mixture from each sample was labeled follows: the three mock-infected samples were each labeled with iTRAQ 113, 114, or 115, and the three PS-infected samples were labeled with iTRAQ 116, 117, or 118. The labeled samples were then mixed and dried with a rotary vacuum concentrator.

To reduce the complexity of the peptide mixtures, iTRAQ-labeled peptides were fractionated by SCX chromatography using the AKTA Purifier system (GE Healthcare). Briefly, the dried peptide mixture was reconstituted and acidified with buffer A (10 mM KH₂PO₄ in 25% of ACN, pH 3.0) and loaded onto a PolySULFOETHYL 4.6 × 100 mm column (5 μm, 200 Å, PolyLC Inc., U.S.A.). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% of ACN, pH 3.0). The elution was monitored by absorbance at 214 nm, and fractions were collected every 1 min. A total of 15 fractions were collected with screening, and then desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL) and concentrated by vacuum centrifugation.

Each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm^*2 cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (ThermoFisher, U.S.A.) coupled to the Easy nLC chromatography system (ThermoFisher, U.S.A.). The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top 10 method, dynamically selecting the most abundant precursor ions from the survey scan (300–1,800 m/z) for HCD fragmentation. The automatic gain control (AGC) target was set to 3e6, and the maximum inject time was set to 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200, and the isolation width was 2 m/z. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with the peptide recognition mode enabled.

All MS raw data files were analyzed by Proteome Discoverer software 1.4 (ThermoFisher, U.S.A.) using the Mascot 2.2 search engine against a database of mustela putorius furo protein sequences (NCBInr, released March 23, 2017, containing 38, 992 sequences). For protein identification, a mass tolerance of 0.1 Da was allowed for fragmented ions, with permission of two missed cleavages in the trypsin digests: iTRAQ8-plex (Y), oxidation (M) as the potential variable modifications, and carbamidomethyl (C), iTRAQ8-plex (N-term), and iTRAQ8-plex (K) as fixed modifications. The strict maximum parsimony principle was performed, and only peptide spectra with high or medium confidence were considered for protein grouping. A decoy database search strategy was also used to estimate the false discovery rate (FDR) to ensure the reliability of the proteins identified.

For relative quantitation, proteins that involved at least one unique peptide were considered a highly confident identification and used for quantification. Additionally, to guarantee the accuracy of quantification, the proteins with coefficient of variation values <20% for three biological repeats were considered DEPs. The quantitative protein ratios were calculated and normalized by the median ratio in Mascot. For comparison, three identical mock samples, labeled with iTRAQ 113, 114, and 115, were used as references. Between samples, the proteins with fold-change ratios ≥1.20 or ≤0.83 and a p < 0.05 were considered DEPs according to the t-test.

To further explore the impact of the DEP on cell physiological processes and discover internal relations between DEPs, an enrichment analysis was performed. GO enrichment on three ontologies [biological process (BP), molecular function (MF), and cellular component (CC)] was applied based on the Fisher's exact test, considering the whole quantified protein annotations as the background dataset. Benjamini–Hochberg correction for multiple testing was further applied to adjust derived p-values. Only functional categories with p-values under a threshold of 0.05 were considered significant. KEGG pathway annotation was extracted from the online KEGG PATHWAY Database (http://www.kegg.jp/kegg/pathway.html).

The protein–protein interaction information involved in the immune response process of the studied proteins was subsequently retrieved from STRING software (http://string-db.org/). Then, the results were imported into Cytoscape5 software (http://www.cytoscape.org/, version 3.2.1) to visualize and further analyze functional protein-protein interaction networks.

Total RNA was isolated using TRIzol Reagent (Invitrogen, U.S.A.) from Mv.1.Lu cells infected with 2 MOI PS or mock-infected cells at 12 and 24 hpi. After treatment with gDNA Removal (TransGen Biotech, China), 4 μg of each total RNA was used for cDNA synthesis. Real-Time RT-PCR (qRT-PCR) assays were performed on an Applied Biosystems® QuantStudio® 3 System (Thermo Fisher Scientific, U.S.A.) employing the TransStart Top Green qPCR SuperMix kit (TransGen Biotech, China) according to the manufacturer's protocol. The primers for amplifying TRAF6, TRAF2, IRAK4, IRAK2, NFκB2, CCL2, TNF-α, IL-6, and GAPDH are presented in Table 1. Each experiment was performed in triplicate. The relative gene expression was calculated using the 2^−ΔΔCT model, which is representative of n-fold changes compared with mock-infected samples. The data was analyzed by two-way ANOVA followed by Duncan's test.

For testing the production of PS nucleoprotein for the different time points analyzed, cell lysates were harvested at 6, 12, 24, 36, 48, and 60 hpi from PS- and mock-infected samples. For confirmation of the iTRAQ-MS data by western blotting, cell lysates were harvested at 12 and 24 hpi from PS-, CDV₃-, and mock- infected cultures. After measuring the protein concentrations, equivalent amounts of cellular proteins from the triplicates were separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose PVDF membranes (Millipore, U.S.A.). The membranes were blocked with 2% BSA dissolved in TBS, containing 0.05% Tween-20, for 2 h at room temperature, followed by incubation with the corresponding primary antibodies (see below) at 4°C overnight and incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG secondary antibodies (Sangong Biotech, China) at room temperature for 2 h. The protein bands were detected using the ECL Detection Kit (Beyotime, China). The GAPDH protein was used as an internal control.

The following primary polyclonal antibodies were used: anti-CDV NP mouse monoclonal antibody (prepared in our laboratory), NF-κB p65 (RelA) rabbit polyclonal antibody (AN365, Beyotime, China), NFκB1 p105 rabbit polyclonal antibody (4717, CST, U.S.A), NFκBIB (IκB-β) rabbit polyclonal antibody (PA5-40909, ThermoFisher, U.S.A), MHC-I mouse monoclonal antibody (ab23755, Abcam, UK), RPS29 rabbit polyclonal antibody (PA5-41744, ThermoFisher, U.S.A), IκB-α rabbit polyclonal antibody (4812, CST, U.S.A), Phospho-NF-κB p65 rabbit polyclonal antibody (MA5-15181, ThermoFisher, U.S.A), and GAPDH rabbit polyclonal antibody (CW0101M, CWBIO, China).

Mv.1.Lu cells were cultivated on cover glasses in 24-well plates, followed by infection with PS or CDV₃ at an MOI of 2 when the cells reached ~70% confluence. The mock-infected cells were treated with PBS as a negative control. Next, at 24 hpi, the cells were fixed with 4% paraformaldehyde and subsequently permeabilized with 0.1% Triton X-100. Further, the cells were incubated with an NF-κB P65 rabbit polyclonal antibody (Beyotime, China) and a mouse monoclonal antibody specific to CDV N protein and incubated with Cy3-labeled goat anti mouse IgG (Beyotime, China) and FITC-conjugated goat anti-rabbit IgG secondary antibody (ThermoFisher, U.S.A) prior to staining with DAPI. The fluorescent images were analyzed under confocal microscopy (Leica, Germany).

A previous study demonstrated the capacity of CDV growth in Mv.1.Lu cells (Lednicky et al., 2004), thus, we initially confirmed the ability of PS replication in Mv.1.Lu cells and established the growth kinetics of PS replication. An optimal time point under PS infection for proteomic analysis was then identified.

As shown in Figure 1A, CPEs in the infection groups became visible at 24 hpi and progressed thereafter. Up to 36 hpi, an obvious CPE was observed and nearly 50 percent of the cells were detached at 48 hpi. The one-step growth curve revealed that the virus load reached a plateau of ~4.8 log₁₀ copy numbers/μL between 24 and 60 hpi, followed by a gradual decline (Figure 1B). Collectively, 24 hpi was considered the optimal time-point for further proteomic analysis, at which a high viral load was maintained and most cells showed little CPE. Virus replication at 6 and 48 hpi was additionally ensured through RT-PCR. The abundance of the CDV-N gene increased as the infection progressed (Figure 1C). Further validation was performed by sequencing analysis of the PCR products (data not shown). Moreover, the production of nucleoprotein for the different time points analyzed was tested by anti-CDV NP antibody, the result showed quite similar tendency of the viral one-step growth curve (Figure 1D).

The host response to PS infection at 24 hpi was analyzed by examining differences in protein expression. Based on a combination of three biological replicates from mock-infected and PS-infected samples, the iTRAQ-coupled LC–MS/MS analysis identified and measured a total of 37,145 peptides and 6184 proteins. The proteins were designated DEPs based on the following criteria: a p < 0.05 and fold-change ratios ≥1.2 or ≤0.833. Among all the DEPs, 151 and 369 proteins were markedly up-regulated or down-regulated, respectively. Partial DEPs are shown in Table 2 and more detailed information for all DEPs is collated in Table S1.

To characterize the biological functions of the 520 DEPs, canonical Gene Ontology (GO) enrichment were performed using DAVID (Dennis et al., 2003) and UniProt databases to obtain relevant annotations about the cellular components (CC), molecular functions (MF), and biological processes (BP). First, the putative subcellular localizations of the DEPs were analyzed. As depicted in Figure 2, a majority of the DEPs were mainly distributed in the nucleus (45.64%) and cytoplasm (20.09%), followed by extracellular space (10.50%), mitochondria (9.75%), and plasma membrane (9.72%), and a smaller portion were localized in the chloroplast (2.66%), lysosome (0.59%), Golgi (0.30%), cytoskeleton (0.30%), peroxidase (0.30%), and ER (0.15%) (more detailed information is collated in Table S2).

Interestingly, the GO analysis showed that most proteins were assigned to functions involved in similar molecular functions and biological processes. As shown in Figure 3A, most DEPs were closely related to binding and catalytic activity when infected by PS infection (more detailed information is provided in Table S3). The BP annotation showed that DEPs associated with various biological processes, including cellular process, metabolic process, biological regulation, immune system process and process of response to stimulus (Figure 3B) (more detailed information is provided in Table S4). Collectively, these categories consisted of the following proteins: CCL2, IRAK4, UBE2L6, NFκB1, NFκB2, TNF-a, IRAK2, IL-6, TRAF6, APOA1, TNFAIP3, TRAF2, RelA, and VCAM1 (up-regulated proteins) and CCR7, CXCR7, SMURF1, NFκBIB, MAPK7, RBM15, IGF2, TSC1, and CD59 (down-regulated proteins). To further investigate the pathways involving the identified DEPs, KEGG pathway analysis was performed. According to the results, DEPs were mainly involved in the NF-κB and NOD-Like receptor (NLR) signaling pathways. In addition, several proteins could be mapped to apoptosis and specific disease associations, consisting of infectious and respiratory diseases (Figure 3C) (more detailed information is shown in Table S5).

In the present study, we detected a total of 27 DEPs involved in the immune response process. To further investigate the interaction network associated with the immune response, these 27 proteins were imported into STRING software and further analyzed by Cytoscape5. As shown in Figure 4, 13 strongly interacting proteins were interestingly grouped into a functional set chiefly associated with the NF-κB signaling pathway. The interaction network provides clues for further illumination of the pathogenic mechanism and immunomodulation between CDV and the mink host.

To confirm the iTRAQ-MS data, we selected significantly changed proteins, including NFκB1, RelA, MHC-I, RPS29, and NFκBIB, which reliably cross-reacted with polyclonal antibodies to the corresponding human proteins for western blotting analysis. As shown in Figure 5A, the five representative proteins showed up-regulated or down-regulated expression in PS-infected Mv.1.Lu cells at 12 and 24 hpi (the original blots are shown in Figure S1), in accordance with the results of the iTRAQ analysis (Figure 5B). However, due to the limitation of the availability of antibodies to Neovison vison proteins, the confirmation of DEPs by immunoblotting was restricted. Thus, eight other proteins involved in the immune response process were selected and tested using real-time RT-PCR. As illustrated in Figure 5C, compared to the mock group, mRNA expression of TRAF6, TRAF2, IRAK4, IRAK2, NFκB2, CCL2, TNF-a, and IL-6 in PS-infected cells was significantly up-regulated in a time-dependent manner, which further confirmed the iTRAQ-MS data.

The activation of the NF-κB signaling pathway requires a series of cascade reactions, followed by the recruitment and phosphorylation of NF-κB protein and subsequent translocation from the cytoplasm to the nucleus, as well as the proteasome degradation of IκB proteins, which ultimately induces the production of inflammatory cytokines and type I IFN. Therefore, the degradation of IκB proteins (typically represented by IκB-α) and phosphorylation and nuclear accumulation of the NF-κB proteins (typically represented by NF-κB P65) are distinct features of NF-κB signaling pathway activation. The network analysis of the DEPs involved in the immune response has preliminarily indicated the induction of the NF-κB pathway by PS infection. To further validate this speculation, Mv.1.Lu cells were infected with PS at 2 MOI, after incubation for 12 or 24 h, total proteins were collected to measure the expression of IκB-a and phosphorylated NF-κB P65 proteins. As shown in Figure 6A, compared to that in mock-infected cells, phosphorylated NF-κB P65 (P-P65) and IκB-a proteins were obviously increased and decreased in PS-infected cells, respectively (the original blots are shown in Figure S2). To assess whether PS infection facilitates NF-κB P65 nuclear translocation, Mv.1.Lu cells were infected with PS at an MOI of 2 or mock infected for 24 h. As shown in Figure 6B, NF-κB P65 showed evident nuclear translocation in PS-infected cells but remained in the cytoplasm of mock-infected cells. Further, to determine whether other CDV strains could activate NF-κB P65, the expression of phosphorylated p65 and IκB-α was also detected in CDV₃-infected cells, which was increased and decreased, respectively (Figure 6A). Additionally, the nuclear translocation of NF-κB P65 was also observed in CDV₃-infected cells (Figure 6B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.