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Quantitative Proteomics reveals changes in Vero cells in Response to Porcine Epidemic Diarrhea Virus Yu Ye, Jun Zhu, Qiangyun Ai, Chengcheng Wang, Ming Liao, and Huiying Fan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00897 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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Journal of Proteome Research

Quantitative Proteomics reveals changes in Vero cells in Response to Porcine Epidemic Diarrhea Virus Yu Ye1,2, Jun Zhu1,3, Qiangyun Ai1,4,5, Chengcheng Wang1,4,5, Ming Liao 1,3,4,5,*, Huiying Fan1,3,4,5,* 1College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China; 2Department of Preventive Veterinary Medicine, College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China; 3National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, Guangzhou 510642, China; 4Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, Guangzhou 510642, China; 5Key Laboratory of Zoonoses Control and Prevention of Guangdong, Guangzhou 510642, China

*Corresponding Author

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Ming Liao Email:[email protected] Tel/Fax: 0086-20-85280240 Huiying Fan Email: [email protected] Tel/Fax: 0086-20-85280242 Mail address: College of Veterinary Medicine, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China

Abstract: Outbreaks of porcine epidemic diarrhea virus (PEDV) have caused significant lethality rates in neonatal piglets, which pose a serious threat to the swine industry worldwide. Commercial vaccines available fail to protect against the emergence of high virulence of PEDV variants. Therefore, the endemic state of PEDV infection in suckling piglets highlights the urgent need for uncovering the molecular determinants of the disease pathogenesis. In this study, Stable Isotope Labeling by Amino acids in Cell culture (SILAC), combined with high performance liquid chromatography (HPLC)/tandem mass spectrometry, was performed to determine proteomic differences between PEDV-infected and mock-infected Vero cells at 18 h postinfection. The SILAC-based approach identified 4508 host cell proteins, of which 120 were significantly up-regulated and 103 were significantly down-regulated at ≥ 95% confidence. Alterations in the expression of selected proteins were verified by western blot. Several signaling


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metabolic pathways including mevalonate pathway I and superpathway of cholesterol biosynthesis were triggered by the infection of the highly virulent strain, which are linked to host innate immunity. 25-HC, an inhibitor of the mevalonate pathway, exhibited potent antiviral activity against PEDV infection. Meanwhile, cell cycle related function were significantly regulated, which may be likely responsible for viral replication and pathogenicity of PEDV.

Keywords: PEDV, Proteomics, SILAC, Bioinformatics analysis, sterol biosynthesis

Introduction Porcine epidemic diarrhea (PED) is a devastating enteric disease that is characterized with vomiting, anorexia, acute severe watery diarrhea, and dehydration, which results in significantly high morbidity and mortality in less than 7-day-age piglets1. The etiologic agent, porcine epidemic diarrhea virus (PEDV), was first discovered in feeder pigs and fattening herds in England in 19712. The ongoing outbreaks of PED have been reported in European, Asian, and American3-5. In China, since December 2010, highly virulent PEDV variants have emerged, which are major contributors to massive outbreaks of diarrhea with almost 100% mortality in suckling piglets. The CV777-based attenuated vaccine has little effect on protection against this disease6. Subsequently, the first case of PEDV infection was observed in US in 2013. PEDV had continued to spread rapidly to 36 US states and other North American countries, including Canada and Mexico5, 7. The highly virulent PEDV infections were also occurred in South Korea8, Japan9, Vietnam10, Ukraine11, etc. PEDV has caused tremendous economic losses to the global swine production industry. Generally, although diagnostic laboratories could detect PEDV


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rapidly and accurately12, 13, it is still poor knowledge with regard to pathogenesis and development of an effective vaccine against the currently epidemic virus strains. PEDV, a member of the genus Alphacoronavirus family Coronaviridae order Nidovirales, is an enveloped virus with positive-sense RNA genome which is approximately 28 knt in length. The genome comprises a 5’ untranslated region (UTR), a 3’ UTR, and seven known open reading frames (ORFs) that encode four structural proteins (Spike, Envelope, Membrane, and Nucleocapsid) and three non-structure proteins (Replicase 1a and 1b, and accessory protein)7. PEDV S protein makes up the surface projections of the virion, functions as the virus attachment protein interacting with the cell receptor to mediate viral entry and induction of neutralizing antibodies in the host 14. Gene variants in S portion appear to be able to reflect the genetic diversity, which is used for the molecular epidemiology study to investigate the genetic evolution of PEDV15, 16. M protein, the most abundant envelope component, not only plays a vital role in the virus assembly process together with E protein, but also evokes antibodies that neutralize the viruses in the presence of complement17. N protein binds to virion RNA, and provides a structural basis for the helical nucleocapsid. It has been suggested that N protein epitopes may be important for motivation of cell-mediated immunity18. Although the only accessory protein ORF3, identified as an ion channel19, is not essential for viral replication20, other researches have demonstrated that it is related to the virulence of PEDV and adaptation in cell culture21, 22. Moreover, the ORF3 gene can also be applied to molecular epidemiology of PEDV and differentiate between field and vaccine-derived isolates23. Many viruses attempt to subvert host cell processes to increase the efficiency of virus infection, and likewise the cell utilizes a number of responses to generate an antiviral state24. Viral entry and replicate in the cytoplasm of infected cells involve numerous host-pathogen


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interactions. The complex interplay between host and infected-cells has been demonstrated to result in subversion of specific host cellular processes such as apoptosis, cell cycle, immune response and protein unfolding. In vivo, thin translucent intestinal walls and villus blunting and fusion were visibly observed in regions of small intestines of nursing piglets infected with PEDV7, 25. Meanwhile, structural of tight and adherens junctions in villous and crypt epithelium were impaired26. In vitro, PEDV M protein alters swine intestinal epithelial cell line growth and blocks cell cycle progression at S-phase via the cyclin A pathway27. Extracellular signalregulated kinase (ERK) signaling pathway plays an important role in the PEDV life cycle and is required for viral infection28. TLR2, TLR3 and TLR9 contribute to NF-κB activation in response to PEDV infection29, while N protein and papain-like protease 2 (PLP2) antagonize interferon (IFN) production by sequestering the interaction between IRF3 and TBK1 and interfering with the RIG-I- and STING-mediated signaling pathway, respectively30, 31. Proteomics based studies have been significant in providing insights into how the protein expression profile is altered during viral infection and hence fostered a new branch of study known as viral proteomics32. The methods to study virus-host cell interactions based on quantitative proteomic techniques such as 2D-DIGE, isotope-encoded affinity tag (ICAT), isobaric tags for relative and absolute quantitation (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC) are used widely. This makes it possible to investigate host cellular response to infection and to predict and elucidate the cellular mechanisms involved in viral pathogenesis. SILAC combined with mass spectrometry strategies has demonstrated a great potential method in quantitative proteomics research because it extremely accurate and relatively easy to apply for the quantification of proteins extracted from cultured cells. High throughput


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quantitative proteomics analysis using SILAC has been used in several virus-host interactions, including influenza virus33, infectious bronchitis virus (IBV)34, and dengue virus type 235, etc. In this study, the SILAC-based quantitative proteomic approach was utilized to investigate whether the Vero cell proteome change after infection with PEDV. We succeeded in identifying and measuring nearly 4,500 host proteins, of which 223 were significantly up- or downregulated at 18 h post-infection (h p.i.). Bioinformatics analyses of protein networks and pathways predicted that signaling cascades could be induced in PEDV-infected cells. To our knowledge, this research is the first application of SILAC to study whole host cell proteome changes in response to PEDV infection. Experimental Section Cells and viruses Vero cells were provided by the Collection of Cell Lines in the College of Veterinary Medicine, South China Agriculture University, China. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Life technology) supplemented with 10% fetal bovine serum (FBS, Biological Industry) and 1% streptomycin and penicillin (Solarbio) under the condition of 5% CO2 at 37°C. PEDV strain GD-A36, isolated from a pig farm in the Guangdong Province of China, was grown on a Vero cell monolayer as previously described with minor modification37. In brief, 80% confluent Vero cells in 25 cm2 cell culture flasks were washed twice with the inoculation media (DMEM added 8 µl 0.025% trypsin (Sigma) per ml) and inoculated with 900 µl of inoculation media and 100 µl of the virus supernatants. After 1.5 h incubation at 37°C with 5% CO2, 5 ml maintenance media (DMEM added 4 µl 0.025% trypsin per ml) was added to the flasks. When about 80% cytopathic effects (CPE) were observed, the


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flasks were subject to freeze-thaw for three times. The mixtures were centrifuged at 2,500g for 15 min at 4°C and the supernatants were harvested for next propagation or saved at -80°C until further experiment. Virus titers were performed by 50% tissue culture infective dose per milliliter (TCID50/ml) and calculated by the Reed and Muench method38. Growth curve analysis of PEDV Vero cells were seeded into 6-well plates at a density of 3 × 104 cells/well and incubated at 37°C in a 5% CO2 atmosphere. When reaching 80-90% confluence, cells were washed twice with the inoculation media and infected with PEDV at an MOI of 1. Supernatants were collected from the medium at every 12 h p.i. Viral titers were determined by the Reed-Muench method on Vero cells in 96-well plates. All experiments were performed in triplicate. SILAC labeling and infection For SILAC experiment, cells were grown in media containing either unlabeled arginine and lysine amino acids (R0K0) or containing 13C, 15N-arginin and 2H-lysine (R10K4) and supplemented with 10% dialyzed fetal calf serum (Sigma) and 1% penicillin and streptomycin. The cells were grown at least five rounds of cell division to ensure all the cellular proteins were labeled. Then the unlabeled cells (Light-labeled cells) in T75 flasks were infected with PEDV at a multiplicity of infection (MOI) of 1 while the labeled cells (Heavy-labeled cells) were used as mock-infected controls. The two parallel Vero cells were harvested at 18 h p.i. Protein Digestion The white flocculent precipitate appeared when the cell pellets were re-suspended in cold lysis buffer, so sonication was applied with eight repeats of 0.8s on, 0.8s off on ice. The samples then


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were centrifuged at 12,000 r/m, 4°C for 20 min. Protein concentration in the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit (Sangon, Shanghai). 250µg protein of PEDV-infected cells and mock-infected cells was reduced, alkylated and trypsin digested as previously described39. The tryptic peptides were extracted and vacuum dried in low temperature and conserved for further study of LC-ESI-MS/MS. LC-ESI-MS/MS The first dimension of high pH-reversed phase (RP) peptide fractionation was employed as briefly described in the following. First, the trypsin-digested peptides were separated using Dionex Ultimate U3000 nanoflow LC system (Thermo Scientific). Then the peptides were further separated by a PepMap C18 reverse phase column (Thermo Scientific) with a linger gradient of 5-35% solvent B over 120 min with a constant flow of 300 nl/min. The Finnigan Sruveyor HPLC system was couple to an LTQ Orbitrap XL (Thermo Scientific) and the spray voltage and the temperature of heated capillary was set to 1.85 kV and 200°C, respectively. Full scan MS survey spectra based on data-dependent mode were acquired in the Orbitrap with a resolution of 60,000 after accumulation of 1,000,000 ions. The five most intense peptide ions from the preview scan in the Orbitrap were fragmented by collision induced dissociation (CID) in the LTQ after the accumulation of 5,000 ions and the parameters were set as follows. Normalized collision energy was 35%, activation Q 0.23 and activation time 30 ms. Maximal filling times were 500 ms for the full scans and 100 ms for the MS/MS scans. The dynamic exclusion was applied with a maximum retention period of 90 s and a relative mass window of 10 ppm. These data were acquired by Xcalibur software (Thermo Scientific, version 2.0.7). Protein Identification and Quantitation


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Protein identification and quantitation were performed with MaxQuant (Max-Planck-Institute of Biochemistry, version 1.1.1.2) and Mascot (Matrix Science, version 2.2) by searching against a concatenated forward-reverse database from UniProt database (http://www.uniprot.org/taxonomy/9528, November 12, 2013, containing 148,822 sequences) and PEDV prototype strain CV777 (http://www.uniprot.org/taxonomy/229032, January 8, 2014, containing 7 entities). In brief, raw MS data were collected by Xcalibru software and the peak lists extracted from raw data were further processed by the MaxQuant and Mascot. The search parameters were determined as follows: MS error tolerance in the Mascot search was set to 10 ppm and MS/MS error tolerance in the MaxQuant and Mascot searches was set to 0.5 Da, enzyme specificity was set to trypsin, the minimum required peptide length was set to six amino acids, a maximum of two missed cleavages was allowed. The variable modifications included oxidation of methionine, N-terminal protein acetylation, carbamidomethyl of cysteine. The false discovery rates (FDR) for sites, peptides and protein identifications were all set to 1%. The maximum posterior error probability (PEP) for peptides was also set to 0.01. A protein group was identified when at least one unique peptide was matched. The minimal total peptide (razor peptide and unique peptide) in a protein group was one. Razor peptides are shared by different proteins within a group and are assigned to the proteins group with most other peptides while unique peptides are not. The peptides, sequence coverage, normalized ratio H/L and significance were automatically produced by MaxQuant software. The proteins whose expression was up- or down- regulated were determined according to the values of “significant B”, a significant score for log protein ratios was calculated on the protein subsets obtained by peptide intensity binning. All significantly PEDV-regulated proteins were uploaded into the bioinformatics software for further analysis.


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Bioinformatics Analysis Due to poor annotation of the monkey genome database, the regulated proteins were converted to human gi number and Uniprot Knowledgebase identifier through BLAST of NCBI and Uniprot, respectively. To determine whether any type of proteins were significantly overrepresented, enrichment analysis of Gene Ontology (GO) terms was performed using the online program DAVID 6.740. Homo sapiens were used as background list, the significance of enrichments was statistically evaluated with a modified Fisher Exact and p-value calculated by applying a Benjamini-Hochberg False Discovery Rate correction. Gi numbers and the SILAC ratios of altered proteins were submitted to Ingenuity Pathway Analysis (IPA®, www.ingenuity.com) (QIAGEN). Photomicrography and Immunofluorescence Assay (IFA) Infected and mock-infected cells in the T25 flask were examined microscopically for cytopathic effect (CPE) at 0, 6, 12, 18, 24, 30, and 36 h p.i. and cells were photographed with inverted microscope & software system (Leica). For immunofluorescence assay, Vero cells were grown in 6-well cell culture dishes. Infected cells were washed with PBS and fixed with 4% paraformaldehyde (Solarbio) at room temperature for 20 min and washed again with PBS. Then the cells were incubated with mouse monoclonal antibody against M protein of PEDV (Median Diagnostic) at 4°C overnight, washed three times with PBS and incubated with secondary antibody of rabbit anti-mouse IgG conjugated to FITC (TransGen Biotech) at 37°C for one hour in the dark, washed with PBS. Images were collected by fluorescence microscopy (Leica). In addition, DAPI (beyotime) was used to visualize the cell nucleus. Western Blot Analysis of Candidate Proteins


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To verify the SILAC results, western blot analysis was performed. Several proteins identified were selected on the basis of their representativity and availability of the corresponding antibodies. In brief, Equivalent amounts of cell lysates (50 μg) of virus-infected and mockinfected were denatured in 5× SDS-PAGE loading buffer by boiling for five min and then separated by 12% SDS-PAGE. Proteins were electro-transferred to 0.45 µl nitrocellulose membrane (Bio-Rad) and blocked with 5% nonfat milk one hour at room temperature. After that, the membranes were incubated with rabbit primary antibodies against cathepsin D (Santa Cruz Biotechnology, Inc.), GAPDH (Bioworlde Technology, Inc.), tubulin-β (Bioworlde Technology, Inc.), Bax (Bioworlde Technology, Inc.), caveolin-1 (Bioworlde Technology, Inc.), β-actin (Bioworlde Technology, Inc.), HNRPA1 (Bioworlde Technology, Inc.), and PEX11B (Bioworlde Technology, Inc.) , and anti-PEDV N protein rabbit polyclonal antibody prepared in our lab overnight at 4°C. Then the membranes were washed in TBS containing 0.05% Tween-20 (TBST) and incubated with DyLight488-conjugated goat anti-rabbit IgG (Rockland) diluted to 1:10000 for 1 h. Washed again with TBST and the membranes were visualized using the Odyssey Imaging System (LI-COR Biosciences). Drug treatment assay Cells were pretreated with 0.4μM-10μM 25-hydroxycholesterol (25-HC, Sigma) or solvent (0.4% ethanol) for 8 h prior to infection with 0.1 MOI of PEDV strain GD-A. After 48 h incubation, virus titer of supernatant pooled from biological triplicates were measured by TCID50. All experiments were performed in triplicate. Cytotoxicity assay


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Cell viability was determined using the tetrazolium salt WST-8 (2-(2-methoxy-4-nitrophenyl)3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H tetrazolium, monosodium salt) (Beyotime) according to the manufacturer’s instructions. Vero cells were seeded into 96-well plates at a density of 5 × 103 cells/well and grown at 37°C for 24 h. The culture medium was replaced by medium containing a series of desired concentrations of compounds, and then cells were incubated for 24 h. After CCK-8 was added into each well, the absorbance was measured by Multiskan FC microplate Reader (Thermo Fisher) at a test wavelength of 450 nm. Results were calculated as a percentage of absorbance of treated cell cultures versus untreated ones. All experiments were performed in triplicate. Statistical analysis The data were presented as mean ± standard deviation (SD). Statistical analysis of virus titers among different groups was determined by Student’s t test in GraphPad Prism Software version 5.01. * was considered significantly variable with a p-value of 0.01 to 0.05, and *** denoted highly significant difference with a p-value of < 0.001. Results Kinetics of PEDV-induced cytopathology in Vero cells. Efficient viral replication depends on the strains, the cell type, and the appropriate MOI, especially for PEDV, which has proven difficult to grow in cell culture. Here, we isolated a PEDV strain GD-A from a pig farm and successfully propagated in cell culture stability. A distinct cytopathic effect (CPE), characterized by cell fusion, syncytia formation, and cell detachment, was observed. For SILAC quantitative proteomic analysis, an essential prerequisite is to ensure high proportion of PEDV-infected Vero cells, and then further investigation of interactions between virus and cells could be performed.


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According to previous studies, Vero cells containing unlabeled lysine and arginine were infected with PEDV at an MOI of 1 and microscopically monitored for CPE at 0, 6, 12, 18, 24, 30, and 36 h p.i., and Vero cells containing R10K4 were mock-infected. The results showed the minimal CPE could be visible at 18 h p.i., syncytium formation and cell fusion were clearly observed at 24 h p.i., and cell detachment appeared at 30 h p.i. (Figure 1B), while the mock-infected cells showed neither. In addition, IFA indicated that the fluorescence intensity was weak at 12 h p.i., and then became strong at 18 h p.i. On the other hand, the viral growth curve analysis (Figure 2) demonstrated that a steady increase in PEDV production during the early phage of infection, and the viral titer reached the peak at 36 h p.i. In consideration of these two parameters of minimal CPE and high infected rate cells, 18 h p.i. were chosen as the time point for further proteomics analysis. Cellular Proteins Identification and Quantitative by SILAC method. In this study, 4,508 proteins were successfully detected and 3,679 proteins were quantitated. In addition, 4 viral proteins were also identified, including spike, membrane, nucleoprotein, replicase polyprotein 1ab, and its subunit 1a (Table S1 in Supplementary material). The fact that the replicase polyprotein was detected demonstrated that the viral reproduction was highly active and would have significant impact on Vero cells at 18 h p.i. when we conducted this SILAC experiment. For quantitative analysis, previous investigation using SILAC have applied ratio cutoffs ranging from near 1.0 to 2-fold or biological statistics such as Z-scores and significant value24. In this work, the value of significance B ≤ 0.0541 calculated by MaxQuant as cutoff was used to identify proteins whose expression may have regulated in PEDV-infected cells (Figure S1 in Supplementary material). According to the above criteria and SILAC ratio H/L


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(control/infection), 120 proteins (2.66%) were significantly up-regulated and 103 proteins (2.28%) were significantly down-regulated (Table S1 in Supplementary material). Validation of Protein Identification and Quantification by Western blot. To confirm the SILAC quantitative results, eight proteins (cathepsin D, GAPDH, Bax, β-actin, caveolin-1, HNRPA1, PEX11B, and tubulin-β) were selected to validate by immunoblotting, which represented for up-regulated, unchanged, and down-regulated proteins, respectively. Meanwhile, N protein was chosen to verify PEDV infection. As shown in Figure 3, the Western blot analysis of these proteins between PEDV- and mock-infected cells were consistent with that confirmed by SILAC. Functional Classification of Up- and Down-regulated Proteins in the GO database. Altered proteins were submitted to web-available software DAVID to gain insights into the functional roles based on biological process and cellular component in the GO database (P ≤ 0.05, Table S2 in Supplementary material). The biological process analysis of up-regulated proteins indicated that several functional groups were strongly enriched, including isoprenoid biosynthetic process, cholesterol biosynthetic process, sterol biosynthetic process, and negative regulation of translation based on fold enrichment in Table S2 of Supplementary material. Moreover, several processes such as lipid biosynthetic process, cell death, and regulation of cellular protein metabolic process contained the majority of proteins. In the cellular compartment, the upregulated proteins were localized in ribonucleoprotein complex, nucleolus, organelle lumen, intracellular organelle lumen, cytosol, membrane-enclosed lumen, and nuclear lumen. For the down-regulated proteins, the most overrepresented biological processes were involved in cellular response to oxidative stress, cytoskeleton-dependent intracellular transport, and protein folding, according to fold enrichment. Meanwhile, the majority of these proteins participated in


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macromolecular complex assembly and macromolecular complex subunit organization. In the cellular compartment, most of proteins were located in non-membrane-bounded organelle, cytosol, mitochondrion, ribnucleoprotein complex, and so on. Pathway and Protein Interaction Network Analysis of Altered Proteins. Gene identifications of the significantly regulated proteins were converted to human GI numbers as the monkey genome database is rather poorly annotated compared with human genome database (Table S3 in Supplementary material). These converted GI numbers and the ratios (control/infection) were uploaded into the IPA software to perform a comprehensive analysis of proteomics for a deep understanding PEDV infection. According to functional classification, the significantly altered proteins were divided into three distinctive functional categories: (1) diseases and disorders, (2) molecular and cellular function, (3) physiological system development at statistically significant levels (p ≤ 0.05, see Table S4 in Supplementary material for definitions). The 120 up-regulated proteins (for a more detailed, see “Whole proteome” of Table S1 in Supplementary material) are involved in 22 diseases and disorders, such as hepatic system disease, gastrointestinal disease, and inflammatory response (Figure 4A, left). These up-regulated proteins are also categorized into 26 molecular and cellular function groups, including cell morphology, cellular development, cellular growth and proliferation, cell death and survival, and cell-to-cell signaling and interaction (Figure 4B, left); 22 physiological system development groups, including embryonic development, organismal development, skeletal and muscular system development and function, and tissue development (Figure 4C, left). The 103 down-regulated proteins, which correspond to 24 diseases and disorders, included hematological disease, immunological disease, organismal injury and abnormalities, and gastrointestinal disease (Figure 4A, right). The down-regulated proteins also classified into 25 molecular and cellular function groups, including cell cycle,


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cellular assembly and organization, gene expression, and cell morphology (Figure 4B, right); 23 physiological system development groups, including connective tissue development and function, embryonic development, tissue development, and tissue morphology (Figure 4C, right). According to the ingenuity canonical pathways analysis, many regulated proteins were involved in mevalonate pathway I (p value = 1.12×10-4), superpathway of cholesterol biosynthesis (p value = 1.35×10-4), superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate) (p value = 3.16×10-4), apoptosis signaling (p value = 0.010), regulation of eIF4 and p70S6K signaling pathway (p value = 0.014), and so on. To generate potential protein network connections, proteins that expressed significantly in PEDV-infected cells could be assigned to 10 specific functional networks by the IPA tool. Each network contains 14 or more focus molecules with score ≥ 20 and several common members (“Network” of Table S4 in Supplementary material). The six most strongly connection network of interest correspond to (1) cellular development, cellular growth and proliferation, gene expression, (2) cellular assembly and organization, cell morphology, cancer, (3) hereditary disorder, neurological disease, cell death and survival, (4) neurological disease, organismal injury and abnormalities, cell death and survival, (5) cell cycle, cellular assembly and organization, organismal injury and abnormalities, (6) cell death and survival, organismal development, dermatological diseases and conditions (Figure 5). 25-HC impairs PEDV replication. According to the mevalonate pathway, 25-HC involves in regulation of sterol metabolism. Previous studies suggest that 25-HC inhibits the production of enveloped viruses. To evaluate the effect of 25-HC on PEDV infection, we examined the titers of PEDV in Vero cells pretreated with serial concentrations of 25-HC. Figure 6 showed that PEDV replication was significantly inhibited in the presence of 25-HC in a dose-dependent manner. In


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contrast, no appreciable cytotoxicity was observed for Vero cells (Figure 7). Taken together, it is suggested that 25-HC exhibits a potent antiviral activity against PEDV. Discussion Viruses could have developed multiple strategies to modulate cellular processes such as signaling pathways and morphology to facilitate infection in host cells. On the other hand, cells would activate the immune system in response to infection. The interactions between virus and host cell are complex as it correlated with many proteins and integrated cellular processes. For the reason, high throughput quantitative proteomic combined with SILAC have been widely applied for a better characterization and understanding of virus-host cell interaction24. In this study, we utilized the SILAC method to investigate the interaction between PEDV and Vero cell, which improves our knowledge of PEDV pathogenesis and potential effects on viral infection. Systematic analysis of whole-cell proteome response has revealed insights into many aspects of host defense and biological imperative modulated during infection with PEDV. Through Gene Ontology and IPA analysis, many of the pathways and biological processes changed significantly in our work, which were mainly involved in mevalonate pathway I, superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate), superpathway of cholesterol biosynthesis, isoprenoid biosynthetic process, cholesterol biosynthetic process, and sterol biosynthetic process. The potential roles of functional clusters are discussed below. Sterol related pathways and biological processes. The mevalonate pathway, crucial for cholesterol, sterols, and isoprenoids synthesis, is an essential metabolic pathway in multiple cellular processes, and regulates the inflammation and innate immune response by protein geranylgeranylation42. Deregulation of this pathway is correlated with cholesterol abnormalities


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and mitochondrial dysfunction, which involved lower mitochondrial membrane potential and increased release of pro-apoptotic factors 43. It has become increasingly recognized that the mevalonate pathway has important implications for pathogen invasion and host defense. For example, influenza virus infection induces the rapid activation of human gammadelta T lymphocytes via the mevalonate pathway 44. Sterols are ubiquitous and essential membrane components in all eukaryotes, affecting membrane rigidity, fluidity, and permeability45. The activation of pathogen recognition receptors and downstream effector functions in host defense are critically sensitive to cholesterol. Conversely, microorganisms can modulate the sterol/lipoprotein network in order to facilitate self-replication 46. According to previous reports, cholesterol plays distinct roles in the viral life cycle. Under certain circumstances, cholesterol is vital for virus entry and replication in host cells, including mouse hepatitis virus (MHV)47, severe acute respiratory syndrome coronavirus (SARS-CoV)48, canine coronavirus (CCoV)49, transmissible gastroenteritis virus (TGEV)50, West Nile virus (WNV)51, and hepatitis C Virus (HCV)52. Further, whether cholesterol homeostasis could stimulate or inhibit viral replications depends on the result of a carefully orchestrated modulation of host cells. In our study, six proteins (HMGCS1, MVD, IDI1, FDFT1, SQLE, and LSS) involved in sterol/cholesterol biosynthesis were all up-regulated (Table S4 in Supplementary material). SQLE is suggested to be one of the rate-limiting enzymes in this pathway, which catalyzes the first oxygenation step in sterol biosynthesis53. Meanwhile, the majority of these proteins also participated in mevalonate pathway I and superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate). On the basis of these results, we presume that it may result in the up-regulated sterol/cholesterol biosynthesis and thus contribute to the modification of host immune response to create a more favorable milieu for PEDV infection.


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25-HC, an inhibitor of the mevalonate pathway, is in charge of the cholesterol negative feedback regulation. Stronger evidence that 25-HC produces important effects on innate and adaptive immunity, which can be induced by type I IFN signaling and has a broad ability to neutralize viral replication including WNV, HCV, vesicular stomatitis virus (VSV), Ebola virus, and HIV51, 54-56. Moreover, sterol regulatory element-binding protein 2 (SREBP2) promotes the expression of related sterol biosynthesis and uptake 57. Interferon antiviral defense responses can mediate perturbation of sterol biosynthesis through reduction of SREBP2 at both the protein and transcription level 58. On the other hand, limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response 59. Taken together, these studies further illustrate a metabolic-inflammatory circuit that links impediments of cholesterol biosynthesis with activation of innate immunity. Significantly, 25-HC also involves antagonism of SREBP2, which might mediate inhibition of sterol biosynthesis and repression of viral replication 54. In our data, SREBF2 (alterative name: SREBP2) and 25-HC is predicted to be activated in the upstream regulators analysis (Table S4 in Supplementary material). What’s more, the latest research indicated that cholesterol 25-hydroxylase (CH25H), which catalyzes oxidation of cholesterol to 25-HC, inhibited porcine reproductive and respiratory syndrome virus (PRRSV) infection via two different mechanisms: the enzyme activity through producing 25-HC to prevent virus entry or the ubiquitin–proteasome pathway by degrading viral protein60, 61. To date, the role of CH25H during PEDV replication is still unclear. It was therefore speculated that pharmacologic manipulation of these regulatory factors and pathways may have a therapeutic effect in PEDV infection. In further studies, the exact impact of sterol related pathways and biological processes remains to be elucidated.


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Cell cycle related function. It has been clear that many viruses subvert the cell cycle control machinery in order to create a more favorable milieu for viral gene expression and replication 62. In coronavirus research, N protein of SARS-CoV and p28 of MHV interact with the cyclincyclin-dependent kinase (cyclin-CDK) complex, resulting in a cell cycle block in the S phage through hypophosphorylation of retinoblastoma (RB) protein 63, 64. According to the results, proteins involved in functional cluster of cell cycle were significantly altered in PEDV-infected cells. Some proteins were down-regulated, including STIL, SKA1, UBE2S, and RB1. For instance, the expression of protein STIL (SCL/TAL1 interrupting locus) was identified at about 11-fold reduction after PEDV infection, which is ubiquitously expressed in proliferating cells. Previous studies have determined that STIL is vital in cell proliferation, mitotic regulation and centrosome integrity65. Reciprocally, depletion of STIL leads to loss of centrioles and abrogates PLK4-induced centriole overduplication66, 67. Spindle and kinetochore-associated protein 1 (SKA1) is integral to chromosome segregation and timely anaphase onset during mitosis68. UBE2S (ubiquitin-conjugation enzyme E2S), a cell cycle-regulated ubiquitin ligase that controls progression through mitosis, acts as an essential factor of the anaphases promoting complex/cyclosome (APC/C)69. RB1 is one of cell cycle regulatory proteins, phosphorylation of which regulates G1 to S phase transition63. Inactivation of RB1 can restore G1 to S phase progression 70, whereas RB1enhances the ability to induce a G1 cell cycle arrest when combined with negative G1 regulators 71. In addition, other proteins were increased, such as TPX2 (Targeting protein for Xenopus kinesin-like protein 2), which is required for spindle formation and microtubule nucleation around the chromosomes72. The latest studies indicate that M and N protein of PEDV elicit cell cycle arrest at S phase with a decrease of cyclin A27, 73, and ORF3 gene also prolongs S phase to facilitate formation of vesicles and virus production 74. Thus, it


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could be inferred that these changed proteins might induce cell cycle disorder. In such circumstance, it may be advantageous for virus to replicate by predominating aberrant cell cycle progression in PEDV infected cells. Further research need to focus on the mechanism of the regulated proteins related to cell cycle in the pathological process of PEDV. Conclusion To sum up, a quantitative proteomic approach with SILAC method combined by HPLCMS/MS was applied to investigate the interaction between PEDV and Vero cells. Comprehensive functional analysis of host proteome profiles in response to PEDV infection revealed that many proteins regulated were involved in multiple host cell pathways and biological processes of sterol biosynthesis and cell cycle related function. The present study could thus yield important insight into the modulation of host metabolism in PEDV infection and have the potential to improve our understanding the pathogenicity of PEDV. Acknowledgements This work was supported by the Natural Science Foundation of Guangdong Province (2014A030313462); the Science and Technology Program of Guangzhou (201508020088); the “Three High” agricultural special fund of Guangdong Province (5500-F15071); the Public Agriculture Specific Research Program (Grant 284 No.201303034). Conflict of Interest Disclosure The authors have declared no conflicts of interest. Abbreviations


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CPE, cytopathic effects; Da, dalton; DMEM. Dulbecco’s Modified Eagle’s Medium; FDR, false discovery rate; HIV, human immunodeficiency virus; IFN, interferon; IgG, immunoglobulin G; LC-ESI-MS/MS, liquid chromatography combined with tandem mass spectrometry using electrospray ionization; MOI, multiplicity of infection; nt, nucleotide; ORF, open reading frame; p.i., post-infection; PEP, posterior error probability; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SD, standard deviation; SILAC, stable isotope labeling by amino acids in cell culture; TLR, toll-like receptor; UTR, untranslated region.

Figure Legends Figure 1. A, indirect immunofluorescence analysis to monitor PEDV infection at an MOI of 1 in Vero cells at different time points (0, 6, 12, 18, and 24 h). The bar in each graph shows 100 μm. B, typical CPE caused by PEDV were showed by optical microscope hours at 6, 12, 18, 24, 30, and 36 h p.i. Syncytium formation and cell fusion were clearly observed at 24 h and cell detachment appeared at 30-36 h. Red arrow indicates the characterization of syncytia formation. The bar in graphs shows 200 μm. C, Mock-infected Vero cells in the fluorescence microscopy. D, poly-nuclear formation caused by virus infection. Cell nuclear was stained by DAPI and the primary anti-PEDV-M-protein antibody was used to detect virus infection. The button raw were virus-infected and the upper as mock control. Green indicates the distribution of PEDV, and blue indicates the nucleus of Vero cells. The bar in pictures shows 50 μm. Figure 2. One-step growth curve of PEDV measured through the TCID50 endpoint dilution assays at 12, 24, 36, and 48 h p.i. Cells were infected with PEDV strain GD-A at an MOI of 1. The data are expressed as mean ± SD from triplicate measurements.


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Figure 3. Western blot analysis of host proteins in mock-infected and PEDV-infected Vero cells at 18 h p.i. The SILAC ratios (Ratio H/L) obtained by mass spectrometry analysis are displayed at right-side. Figure 4. Functional cluster of up- and down-regulated proteins. (A) Diseases and disorders; (B) molecular and cellular functions; (C) physiological system development and functions. Grey bar denotes the up-regulated proteins, and black bar denotes the down-regulated proteins. Each graph bar indicates the number of the regulated protein enriched. Figure 5. Network analysis of PEDV regulated proteins with IPA. (A) cellular development, cellular growth and proliferation, gene expression; (B) cellular assembly and organization, cell morphology, cancer; (C) hereditary disorder, neurological disease, cell death and survival; (D) neurological disease, organismal injury and abnormalities, cell death and survival; (E) cell cycle, cellular assembly and organization, organismal injury and abnormalities; (F) cell death and survival, organismal development, dermatological diseases and conditions. In our study, according to SILAC ratio H/L (control/infection), the down-regulated proteins are shaped green, the up-regulated are shaped red, and other proteins that were not identified or changed are shaped white. Based on the expected influence of the molecules that are altered, orange nodes indicate predictions of activation, and blue nodes indicate prediction of inhibition. The intensity of color indicates the degree of the change in protein expression level. The shapes define the type of protein families. Lines connecting the molecules indicate molecular relationships. Orange lines represent state of activation, blue lines represent state of inhibition, yellow lines represent finding inconsistent with state of downstream molecules, and gray lines represent the effect not predicted. Dashed lines indicate indirect interactions, and solid lines indicate direct interactions.


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Figure 6. Vero cells were pretreated with medium containing serial concentrations of 25-HC (0.4μM, 2μM, and 10μM) or ethanol for 8h and infected with PEDV strain GD-A at 0.1 MOI. Viral titer was determined by TCID50 at 48 h p.i. Values represent means of biological triplicate samples. Figure 7. Vero cells were treated with indicated concentrations of 25-HC or ethanol. Cell viability was measured by Enhanced Cell Counting Kit-8 dye. Values represent means of biological triplicate samples. Supporting Information The following supporting information is available free of charge at ACS website (http://pubs.acs.org). Figure S1. Proteome-wide accurate quantitation and significance. Signal intensities (log10) of all quantified proteins in the PEDV infected experiment are shown as a function of their fold change. The spread of the cloud is lower at high abundance, indicating that quantification is more precise. The criteria as being identified as a significantly regulated protein can be evaluated by the significance B level indicated in blue, red, green and purple, respectively. Table S1. A comprehensive proteome map of PEDV infected Vero cells and MS/MS spectra of all single-peptide-based proteins. Table S2. GO characterization of Up- and Down-regulated Proteins by DAVID software. Table S3. Gi number and Uniprot Knowledgebase identifier conversion of altered proteins between monkey and human database.


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Figure 1 258x124mm (300 x 300 DPI)



Figure 2 204x80mm (300 x 300 DPI)


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Figure 3 155x177mm (300 x 300 DPI)



Figure 4 249x203mm (300 x 300 DPI)


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Figure 5 262x186mm (300 x 300 DPI)



Figure 6 97x90mm (300 x 300 DPI)


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Figure 7 107x112mm (300 x 300 DPI)



Table of Contents Graphic 209x83mm (300 x 300 DPI)


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