The interactome of porcine epidemic diarrhea virus nucleocapsid protein CURRENT STATUS: POSTED

Background Many viral proteins specifically interact with cellular proteins to facilitate virus replication. Understanding these interactions can decipher the viral infection mechanism and provide potential targets for antiviral therapy. Porcine epidemic diarrhea virus (PEDV), the agent of PED, causes numerous economic losses for the swine industry each year. Till now, no effective vaccine or drugs are available to contain this disease. As a result, it is critical urgent to elucidate the PEDV interactome. The nucleocapsid (N) of PEDV plays an important role in viral replication. Results In this study, the N gene was cloned into pEGFP-C1 and transfected into 293T cells. The interactome of N was elucidated by label-free mass spectrometry. A total of 125 cellular proteins interacting with PEDV N protein were discovered, of which 4 cellular proteins, DHX9, NCL, KAP1, TCEA1, were confirmed by pull down, immunoprecipitation, and co-localization.


Background
Many viral proteins specifically interact with cellular proteins to facilitate virus replication.
Understanding these interactions can decipher the viral infection mechanism and provide potential targets for antiviral therapy. Porcine epidemic diarrhea virus (PEDV), the agent of PED, causes numerous economic losses for the swine industry each year. Till now, no effective vaccine or drugs are available to contain this disease. As a result, it is critical urgent to elucidate the PEDV interactome. The nucleocapsid (N) of PEDV plays an important role in viral replication.

Results
In this study, the N gene was cloned into pEGFP-C1 and transfected into 293T cells. The interactome of N was elucidated by label-free mass spectrometry. A total of 125 cellular proteins interacting with PEDV N protein were discovered, of which 4 cellular proteins, DHX9, NCL, KAP1, TCEA1, were confirmed by pull down, immunoprecipitation, and co-localization.

Conclusions
The interactome of N protein supplied a powerful tool to explore the role of N in PEDV infection and therapeutic targets.

Background
Porcine epidemic diarrhea (PED) is an acute and highly contagious enteric disease characterized by watery diarrhea, vomiting, dehydration, severe enteritis, and weight losses [1,2]. Inactivated and live-attenuated CV777-based vaccines have been used as a major strategy to control PED for many years until the outbreak of PED in China in October 2010 [3][4][5]. This outbreak occurred on vaccinated and non-vaccinated pig farms and caused nearly 100% morbidity and mortality rates among suckling piglets leading to great economic losses to the swine industry. In April 2013, a PED outbreak emerged in the United States causing high mortality in piglets and also huge economic loss [6,7]. These reemerging outbreaks indicated PED is a serious threat to the swine industry worldwide.
PED is caused by PED virus (PEDV), a large-enveloped RNA virus, which is belonging to the order Nidovirales, family Coronaviridae, subfamily Coronavirinae, and genus Alphacoronavirus [8][9][10]. Its genome is about 28 kb in length, with a 5' cap and a 3' polyadenylated tail and comprises a 5' untranslated region (UTR) and a 3' UTR, encoding two replicase polyproteins (pp1a and pp1ab), spike (S), envelope (E), membrane (M), and nucleocapsid (N) four structural proteins, and one hypothetical accessory protein [11]. N protein is a multifunctional viral protein and plays a key role in PEDV infection, such as the RNA-binding protein, viral RNA synthesis and modulating host cell processes [12][13][14]. It can subvert innate immunity by antagonizing beta and lamda interferon production [15,16], prolonging the host cell S phase, inducing endoplasmic reticulum stress and up-regulating interleukin-8 expression [17].
As obligate intracellular parasites, the successful replication of viral pathogen in a host is a complex process involving many interactions to achieve viral invasion, replication, and packaging processes.
Proteome study is a powerful tool to uncovering the cellular proteins taking part in the viral life cycle by interacting specific viral protein, and also by using which to find new therapeutics against virus infection [18][19][20][21]. In this study, to explore the biological function of PEDV N protein and the role of N protein in viral replication, the interactome of N protein was uncovered, which supplied useful information on the further study the function of N proteins and also gave hints for antiviral drug targets.

Cells and viruses
Human Embryonic Kidney 293T (HEK293T) cells and Vero E6 cells were obtained from the cell bank of Shanghai Academy of Sciences and grown in Dulbecco's modifed Eagle's medium (DMEM; sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; GemCell. the USA) at 37 ᵒC with 5% CO 2 .
The PEDV LJX01/2014 strain was isolated and preserved by our laboratory.

Plasmids and transfection
The full length of the N gene was amplified from PEDV LJX01/2014 strain with the primers: 5'-AGGATCCATGGCTTCTGTCAGCTTTC-3' and 5'-GGCTCGAG TTAATTTCCTGTATCGAAG-3'. The purified N gene was cloned into the pEGFP-C1 vector with the BamH I and Xho I restriction enzymes to generate the pEGFP-N recombinant plasmid. The plasmids pEGFP-N and pEGFP-C1 were transfected into 293T cells with 50-70% confluency using the X-tremeGENE HP DNA Transfection Reagent (Roche) at 1:3 ratio according to the procedure. Monolayer Vero E6 cells were transfected with Lipofectamine 2000 (Life Technologies) according to the instruction.

GFP pull down
pEGFP-N and pEGFP-C1 were transfected respectively into 293T cells and harvested 24 h posttransfection. The cells were resuspended by lysis buffer (0.5% NP40; 10 mM Tris/Cl pH 7.4; 0.5 mM EDTA; 150 mM NaCl) supplemented with cOmplete™, EDTA-free protease inhibitor cocktail (Roche) and lysed on ice for 30 min. After centrifugation at 14,000 g for 10 min, the supernatant was collected and mixed with GFP-Trap (Chromotek) and incubated for 6 h in a shaker at 4 ᵒC. The mixtures were centrifuged at 2,500g for 2 min and washed 2 times with wash buffer (10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA) supplemented with cOmplete™, EDTA-free protease inhibitor cocktail (Roche).
For samples for mass spectrometry, after the removal of wash buffer, the cellular proteins were eluted with 50 μL of elution buffer (200 mM Glycine pH 2.5). The supernatants were separated by centrifugation and transferred to a new 1.5 mL centrifuge tube. This step was repeated 2 times to ensure the maximum elution to get 100 μL of eluted proteins, and then 10 μL of Tris-base buffer (pH 10.4) was added to neutralize the eluate. The eluted proteins were analyzed by label-free mass spectrometry. For samples for Western blot analysis, after the removal of wash buffer, the beads were resuspended with 2×SDS-Sample buffer.

Liquid Chromatography (LC) -Electrospray Ionization (ESI) Tandem MS (MS/MS) Analysis by Q Exactive.
The GFP pulldowns (250 μg for each sample) were purified with 200 μl UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0) to remove the detergent, DTT and other low-molecular-weight components by repeated ultrafiltration (Microcon units, 10 kD). Then 100 μL UA buffer with 0.05 M iodoacetamide was added to block reduced cysteine residues and the samples were incubated for 20 min in darkness.
Each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100μm*2cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3μm resin) in buffer A (2% acetonitrile and 0.1% Formic acid) and separated with a linear gradient of buffer B (80% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) that was coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) over 120 min. MS data were acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300-1800 m/z) for HCD fragmentation. Determination of the target value is based on predictive Automatic Gain Control (pAGC). Dynamic exclusion duration was 25 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. The 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 peptide recognition mode enabled. MS experiments were performed triply for each sample.
Then the protein G resin (GenScript) were mixed overnight at 4 ᵒC on a rotator. The immunoprecipitated samples were collected by centrifugation and washing, finally, eluted with 100 μL of the 2×SDS-sample buffer.

Western blot analysis
The protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore).
After blocking with 5% skim milk for 2 h at room temperature, the specific primary cellular antibodies or anti-N monoclonal antibody prepared by our laboratory [22] were added for 2 h at room temperature. After washing 3 times, horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse secondary antibodies (Kang Wei Century; 1:5000 dilution) were added and incubated for 1 h at room temperature. After another 3 times washing, the target bands were developed using a developing reagent ClarityTM Western ECL Substrate (Bio-Rad).

Confocal imaging
The plasmids pEGFP-N and pEGFP-C1 were transfected in 293T and Vero E6 cells. After 24 h, cells were fixed with 4% paraformaldehyde for 30 minutes, washed 3 times. The 0.1% (v / v) Triton X-100 was used to permeabilize cells for 15 min. After washing 3 times, specific antibodies were added, incubated for 2 h at room temperature, and washed 3 times with PBS. Then PE-labeled goat antirabbit IgG (Southern Biotech) secondary antibody was added and incubated for 1 h at room temperature. Cell nucleus was stained with DAPI (Vectorlabs; H-1200) for 10 min at room temperature then observed under a laser confocal microscope (Leica; Germany).

Expression of N protein
The N gene was amplified directed by the template of PEDV LJX01/2014 strain and cloned into the pEGFP-C1 vector to make recombinant plasmid pEGFP-N. The pEGFP-N and blank vector were transfected into 293T cells. The expression of the N gene was confirmed by observing under fluorescence microscopy (Fig. 1A) and Western blot analysis (Fig. 1B).

Identification of the potential cellular interacting partners of N proteins
In order to obtain the interactome of N protein, the plasmids pEGFP-N and pEGFP-C1 were transfected in 293T and the cellular binding partners were pulled down using the GFP-trap (Fig. 2A). These proteins were identified by label-free MS. The results showed that approximately 1200 cellular proteins were initially identified and quantified, which represented both specific and nonspecific interactions. Under the criteria of more than 2 unique peptides, a false discovery rate (FDR) ≤ 1%and a p value <0.05 for the t-test analysis, 125 cellular potential proteins were identified (Table 1). After analyzed by the biological database STRING (https://string-db.org/), 79 proteins were in the nucleus, 4 proteins were in cytosolic part and 17 proteins exist in both parts. Four KEGG pathways were significantly enriched: ribosome, spliceosome, RNA transport, and non-homologous end-joining.
Biological function GO-terms significantly enriched with RNA processing including mRNA metabolic process, translational initiation, translation, RNA catabolic process, mRNA catabolic process, gene expression and so on. Molecular functions of RNA binding, structural constituent of ribosome, heterocyclic compound binding, and structural molecule activity was enriched.

Validation of cellular proteins interacting with N proteins
Four cellular proteins including transcription elongation factor A protein 1(TCEA1), ATP-dependent RNA helicase A (DHX9), nucleolin (NCL) and transcription intermediary factor 1-beta (KAP1) were selected to validate the cellular proteins potentially interact with N proteins. Independent GFP pull down were carried out on 293T cells, the pulldowns were analyzed by Western blot. The results showed that DHX9, NCL, KAP1, and TCEA1 were all in pull downs which pulled down by N protein (Fig.   2B). The same procedure was applied onto Vero E6 cell, PEDV susceptible cell line, to further confirm their interaction. The results showed that in the N pulldowns, DHX9, NCL, KAP1, and TCEA1 were all detected (Fig. 2C). These results showed that N could pull down all these four cellular proteins.
IP assay was employed to confirm the interaction of the cellular proteins with N protein in both 293T cells and Vero E6 cells. The IPs were immunoprecipitated by anti-DHX9, NCL, KAP1, and TCEA1 antibodies, respectively. The Western blot results showed that N protein was in the immunoprecipitated products of DHX9, NCL, KAP1, and TCEA1 (Fig. 3A, 3B).
Above results showed that all four cellular proteins of DHX9, NCL, KAP1, and TCEA1 were pulled down by N protein and all four proteins could immunoprecipitated N proteins, indicating the interaction of the four cellulars with N protein.

Co-localization of pEGFP-N with cellular proteins
The plasmids pEGFP-N and pEGFP-C1 were transfected in 293T and Vero E6 cells. At 24h posttransfection, the cells were fixed and probed by anti-DHX9, NCL, KAP1, and TCEA1. The co-localization of pEGFP-N with cell proteins was observed by laser confocal technique. Results showed that N protein co-localized with NCL in both 293T and Vero E6 cells. While no colocalization between DHX9, KAP1, TCEA1, and N protein was observed (Fig. 4A, 4B).

Discussion
Viral proteins often interact with cellular proteins to facilitate finishing the viral life cycle or creating a favorable environment for viral replication. Studying these interactions will help for the analysis of viral pathogenesis and the function of viral proteins to reveal the viral infection mechanism and provides more options for antiviral targets [23,24]. In the present study, the interactome of PEDV N protein was discovered, which would supply a great platform for studying the role of N protein in PEDV infection and the selection of anti-PEDV therapies.
We used the combination of EGFP-trap with Label-free LC-MS/MS approach to elucidate the N protein interactomes which have been successfully used on other viral proteins, including human respiratory syncytial virus, infectious bronchitis virus, porcine reproductive and respiratory syndrome virus and Ebola virus VP24 [18,20,21,23,24]. The specific interaction partners of N would be selectively enriched in the pEGFP-N samples. To provide a statistically robust data set, pull downs with both the pEGFP control and pEGFP-N were conducted independently in triplicate. Selected under stringent criteria, 125 cellular proteins were listed ( Table 1). The interaction of these proteins was analyzed using the STRING algorithm and found that most of these proteins were in the nucleus. The function of these proteins was mostly related with RNA including mRNA metabolic process, translation, RNA binding and so on, which was coincident with the characteristic of nucleocytoplasmic trafficking of N protein the reported function of RNA-binding protein, viral RNA synthesis [12][13][14]. This interactome also gives us hints for the novel role of N protein and cellular proteins, for example, analyzed by UniProt database showed that 107 among 125 cellular proteins were related to acetylation. This showed us a new way about the epigenetic changes during the expression of N protein and PEDV infection.
Because nearly 80% cellular proteins of N partners were in the nucleus part, nuclear proteins of NCL, DHX9, KAP1, and TCEA1 with different fold changes were selected to validate the MS results. Firstly, the interaction was confirmed by pull down and IP in 293T and Vero E6 cells. N protein was found to interact with all four cellular proteins by pull down and all four proteins interacted with N protein by IP in both cell lines. These results supply strong proof of their interaction. NCL is a multifunctional DNA/RNA-binding protein widely conserved among eukaryotes. It is involved in RNA metabolism, in particular in rRNA maturation [25]. It also plays multiple and important roles during virus infection by helping the formation of infectious virus particles, virus replication, virus internalization, trafficking, immune evasion and so on, such as Epstein-Barr virus (EBV), dengue virus, feline calicivirus, influenza A virus, herpes simplex virus 1 [26][27][28][29][30]. The most important thing is that the N protein of Avian infectious bronchitis virus within the same family Coronaviridae with PEDV interacted with NCL.
DHX9 is a multifunctional ATP-dependent nucleic acid helicase which unwinds DNA and RNA and that plays important roles in DNA or RNA processes [31]. It also takes part in the virus life cycle to regulate viral RNA synthesis by interacting with N protein of porcine reproductive and respiratory syndrome virus [32]. DHX9 is a component of virus replication complexes of chikungunya virus (KSHV) and also EBV and hepatitis B virus [33][34][35]. KAP1 is a ubiquitously expressed protein involved in many critical functions which are dependent upon post-translational modifications, such as phosphorylation or sumoylation [36]. Its function also can be hijacked by the virus to mediate viral gene expression and play a role during viral latency, such as KSHV, Murine Leukemia Virus [37,38]. TCEA1 is a transcription elongation factor S-II which stimulates mRNA chain elongation catalyzed by RNA polymerase II [39]. Little information is available on its function and virus infection. Based on the role of these proteins on other viruses, we believe that these four cellular proteins had a role in PEDV life cycle. However, these proteins may form a complex with other cellular proteins which are highly structured and dynamic nuclear organelle which may a reason for no colocalization of DHX9, KAP1, and TCEA1 with N proteins. The real role of cellular proteins in N expression or PEDV infection needs more study. Other cellular proteins which were not selected for confirmation also take parts in the virus life cycle, such as LARP1, G3BP1, SERBP1, SRP14, IGF2BP1, YBX1, HMGB2. All these indicated that the interactome of N proteins was reliable.
In the present research, the interactome of PEDV N protein was elucidated and confirmed by pull

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Funding
This work was funded by State Key Laboratory of Veterinary Etiological Biology (SKLVEB2015KFKT0015) and the Funds of Shandong "Double Tops" Program.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Authors' contributions
MT and GD conducted the research and interpreted the results. MT, GD, XC, SC, FC, JL, LL, YZ, SL, GL and YX participated in data collection. MT, GL and YX contributed to data analysis and helped draft the manuscript. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.

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Ethics approval and consent to participate
Not applicable.  Figure 1 Identification of the pEGFP-N expression in 293T cells by fluorescence microscopy (A) and

Tables
Western blot (B). The predicted molecular size was 27, 81kDa for EGFP and EGFP-N.   Validation of the partners of N protein by co-localization A Co-localization of cellular proteins with N in 293Tcells. 293Tcells were transfected with pEGFP-N or pEGFP-C1. The cellular proteins were probed by anti-DHX9, KAP1, NCL, and TCEA1 antibodies and visualized by PElabeled goat anti-rabbit IgG (Red). Nuclei were stained with DAPI (blue). The colocalization was determined by the yellow signal in the merged images. B The same procedure of confocal technique was applied on Vero E6 cells.