Fish & Shellfish Immunology 39 (2014) 464e474

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Modulation of proteome expression by F-type lectin during viral hemorrhagic septicemia virus infection in fathead minnow cells Se-Young Cho a, 1, Joseph Kwon b, 1, Bipin Vaidya a, 1, Jong-Oh Kim c, Sunghoon Lee d, Eun-Hye Jeong a, Keun Sik Baik b, Jong-Soon Choi b, Hyeun-Jong Bae e, f, Myung-Joo Oh c, **, Duwoon Kim a, f, * a

Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, South Korea Korea Basic Science Institute, Daejeon 305-806, South Korea Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, Jeonnam, South Korea d Personal Genomics Institute, Genome Research Foundation, Suwon 443-270, South Korea e Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, South Korea f Bioenergy Research Center, Chonnam National University, Gwangju 500-757, South Korea b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 March 2014 Received in revised form 13 May 2014 Accepted 30 May 2014 Available online 13 June 2014

Lectins found in fish tissues play an important role in the innate immune response against viral infection. A fucose-binding type lectin, RbFTL-3, from rock bream (Oplegnathus fasciatus) was identified using expressed sequence tag (EST) analysis. The expression of RbFTL-3 mRNA was higher in intestine than other tissues of rock bream. To determine the function of RbFTL-3, VHSV-susceptible fathead minnow (FHM) cells were transfected with pcDNA3.1(þ) or pcDNA3.1(þ)-RbFTL-3 and further infected with VHSV. The results show that the viability of FHM cells transfected with pcDNA3.1(þ)-RbFTL-3 is higher than that of cells transfected with pcDNA3.1(þ) (relative cell viability: 28.9% vs 56.2%). A comparative proteomic analysis, performed to explore the proteins related to the protective effect of RbFTL-3 in the cells during VHSV infection, identified 90 proteins differentially expressed in VHSV-infected FHM cells transfected with pcDNA3.1(þ) or pcDNA3.1(þ)-RbFTL-3. The expression of RbFTL-3 inhibits a vascularsorting protein (SNF8) and diminishes the loss of prothrombin, which are closely associated with controlling viral budding and hemorrhage in fish cells, respectively. Subsequent Ingenuity Pathways Analysis enabled prediction of their biofunctional groupings and interaction networks. The results suggest RbFTL3 modulates the expression of proteins related to viral budding (SNF8, CCT5 and TUBB) and thrombin signaling (F2) to increase the viability of VHSV infected cells. © 2014 Elsevier Ltd. All rights reserved.

Keywords: F-type lectin Viral hemorrhagic septicemia Rock bream Proteomic analysis Expressed sequence tag

1. Introduction Viral infection to farming fishes leads to massive economic losses to the global aquaculture industry. Infection of rhabdovirus such as viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) associates with high mortalities in fish. IHNV can infect only salmonids, whereas VHSV is able

* Corresponding author. Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, South Korea. Tel.: þ82 62 530 2144; fax: þ82 62 530 2149. ** Corresponding author. Tel./fax: þ82 61 659 3173. E-mail addresses: [email protected] (M.-J. Oh), [email protected], duwoonkim@ gmail.com (D. Kim). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.fsi.2014.05.042 1050-4648/© 2014 Elsevier Ltd. All rights reserved.

to infect >80 species in cultured freshwater and marine fishes including salmonids [1,2]. VHSV has a negative and approximately 11 kb single-stranded RNA genome [3]. VHSV-infected fishes often swim erratically and have bulging eyes, expanded abdomen, and extensive internal/external hemorrhaging [4,5]. Olive flounder (Paralichthys olivaceus) and rock bream (Oplegnathus fasciatus) are the most economically important fisheries resources in Korea. The mass mortality of olive flounder is due to VHSV infection [6]. Rock bream is not reported to be infected by VHSV but vulnerable to red sea bream iridovirus (RSIV) infection [7e9]. These differences in the immune systems observed in fish species motivates towards the determination of the mechanism involved in resistance to viral infection. The innate immune system, a defense against viral infection in fish, provides immediate defense mechanism in fish, which

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through homology screening using the Basic Local Alignment Search Tool (BLAST) program available on the NCBI website. The rock bream F-type lectin-3 (RbFTL-3) full-length cDNA was identified via EST analysis. Multiple alignments of the RbFTL-3 amino acid sequences were performed using the Genetyx version 7.0 multiple sequence alignment program (Genetyx Corp., Tokyo, Japan).

includes lectins, cytokines, chemokines and antibacterial peptides [10]. Among them, lectins, the carbohydrate-binding proteins, which are frequently reported in fish, have the potential to bind to surfaces of various infectious agents [11]. The lectins found in fish are classified into various groups such as galectins, fucose-binding (F-type), rhamnose-binding, ricin-, lily-, and b-propeller/tectonintypes [11,12]. The F-type lectins (FTLs) are involved in the defense against a wide variety of pathogenic bacteria and in the innate immunity [13]. Many studies reported the presence of the lectins in different types of fishes such as European eel (AAA), striped bass (MsaFBP32), rock bream (RbFTL-1 and RbFTL-2), and gilt head bream (SauFBP32), and sea bass (DlFBL) [14e18]. The RbFTL-1 and RbFTL-2 are sensitive to pathogenic infections and the expressions of their mRNA increase during RSIV infection [17]. Although, many lectins related to immune system have been identified in different fishes, many aspects remain unclear, particularly defense mechanism and proteins involved in immune response by lectin against viral infection. Several studies reported the different approaches to explain immune response mechanism in VHSV infection. Genomic along with transcriptional profiles with DNA microarrays were applied to identify the genes involved in the VHSV infection [19e21]. Though these approaches reveal a large number of differentially regulated genes and mRNA level and their relative expression (RE), they have limitations to identify several important proteins, which are usually modified during viral infection and immune response. Proteomic analysis, an important tool to obtain detailed protein involvement in virus and receptor cells interactions, was used to analyze protein expression in zebrafish [22]. Encinas et al. reported that VHSV infection induces changes in the expression of regulating proteins in the glycolytic pathway, cytoskeletal components and a few immune-related proteins in zebrafish [23]. Similar studies also reported on the changes in protein expression elicited by Streptococcus sp. in olive flounder [24,25]. Although, previous studies have been reported the protein expressions during VHSV infection in fishes, the role of lectin in the protein expression in infected cells and the mechanism in immune response in VHSV infection has not been reported yet. In the present study, an F-type lectin, RbFTL-3 was isolated from rock bream tissues and the function of RbFTL-3 was examined in VHSV-infected fathead minnow (FHM) cells through scrutinizing the proteomic profiling. This study shows the modulation of proteome expression in the VHSV infected cells by inducing RbFTL-3 transfection. To the best of our knowledge, this is the first report to describe the protein interaction network involved in the defensive role of RbFTL-3 against VHSV infection.

To quantify the RE of RbFTL-3 mRNA in rock bream tissues (blood, gill, intestine, kidney, liver, muscle, skin and spleen) total RNA was extracted using TRIzol LS reagent according to the manufacturer's instructions (Invitrogen). Aliquots of the RNA were then reverse-transcribed into cDNA using a First-Strand cDNA Synthesis Kit (Beams Biotech, Seongnam, Korea). Thereafter, realtime PCR was carried out in a Thermal Cycler Dice Real Time System (Takara Bio, Shiga, Japan) using SYBR Premix Ex Tag reagent (Takara). The PCR protocol entailed denaturation at 95  C for 10 s followed by 40 cycles of denaturation at 95  C for 5 s, annealing at 56  C for 10 s and extension at 72  C for 20 s. The primers: RbFTL-610F (5'-TCC TGC AGT CAC ACA AAC AACG-3') and RbFTL-744R (5'-AAT GCG GAT CTC AGC TCC ATTG-3') used for amplification of RbFTL-3. All samples were analyzed in triplicate. The RE of each target transcript was normalized to 18S rRNA according to the manufacturer's instructions (Takara). The expression of the target gene mRNAs were expressed as relative to that of the muscle. For relative quantification of VHSV NV-gene mRNA expression in FHM cell, a fragment of the NV gene was amplified using the qVHSV NV-F (5'-TTG TCC TTC GCG AGA TGA TCG-3') and qVHSV NVR (5'-TTT CTG ACC GAT CGA GGT CAC TG-3') primer pair, The primers: 18S rRNA-F (5'-GAC TCA ACA CGG GAA ACC TC-3') and 18S rRNA-R (5'-AGA CAA ATC GCT CCA CCA AC-3') were used as internal control. The relative expression was determined using real-time PCR.

2. Materials and methods

2.4. Construction of expression vector and transfection of RbFTL-3

2.1. Construction of EST libraries from rock bream and sequencing of RbFTL-3 cDNA

The RbFTL-3 open reading frame (ORF) was amplified using RbFTL-3-for (5'-CCG GAT CCA TGA AAC TCA GTG TTT TC-3') and RbFTL-3-rev (5'-CCC TCG AGC TAA TCC AGT CTGG-3') primers, which contain BamHI and XhoI restriction sites at their N- and Ctermini, respectively. The polymerase chain reaction (PCR) protocol entailed an initial incubation at 95  C for 2 min; 35 cycles of denaturation at 95  C for 30 s, annealing at 58  C for 1 min and extension at 72  C for 1 min; and a final extension at 72  C for 5 min. The amplified product was ligated into the pGEM T-Easy Vector (Promega, Madison, WI, USA) and transformed into Escherichia coli strain JM109, after which the harvested RbFTL-3 gene was subcloned into the BamHI and XhoI sites of pcDNA3.1(þ) vector, yielding pcDNA3.1(þ)-RbFTL-3. The constructed expression vector was then transfected into FHM cells. For transfection of RbFTL-3 plasmid, FHM cells were cultured at 20  C in Leibovitz's L-15 medium supplemented with 10% fetal

A rock bream cDNA sequence database was constructed using a genome sequencer (GS-FLX™, Roche Applied Science, Piscataway, NJ, USA). Initially, Tri Reagent™ (SigmaeAldrich, St. Louis, MO, USA) was used to isolate total RNA from samples of blood, gill, intestine, kidney, liver, muscle and spleen from three healthy rock bream. The mRNA was then purified using an mRNA isolation kit (FastTrack®2.0, Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis and normalization were performed using a Creator™ SMART™ cDNA library construction kit (Clontech, Palo Alto, CA, USA) and a Trimmer-Direct cDNA normalization kit (Invitrogen), respectively. Genome sequencing of rock bream cDNA was carried out according to the manufacturer's manual. A single putative EST gene was identified in the rock bream cDNA sequence library

2.2. Ethical statement All experiments using rock bream were carried out in strict accordance with the recommendations in the Guide for the Institutional Animal Care and Use, the Committee of Chonnam National University, and the protocol was approved by the Committee of Chonnam National University (Permit no. CNU IACUCYS20091). 2.3. Relative expression of RbFTL-3 mRNA and VHSV NV-gene mRNA

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bovine serum (FBS) and 1% penicillin/streptomycin. After detaching the cell monolayers using trypsin, the cells were washed and resuspended in culture medium supplemented with 10% FBS and dispensed into wells at 4.8  104 cells/well in a final volume of 100 ml. The following day, the cells were transiently transfected with pcDNA3.1(þ) or pcDNA3.1(þ)-RbFTL-3 using FuGene 6 transfection reagent (Roche Applied Science, Indianapolis, IN, USA) in Leibovitz's L-15 medium according to the manufacturer's protocol. The plates were then incubated at 20  C for an additional 48 h prior to cell viability measurements. 2.5. Cell viability assay To assess the effect of RbFTL-3 on cell viability, FHM cells were plated in 96-well plates and transfected for 48 h with pcDNA3.1(þ) or pcDNA3.1(þ)-RbFTL-3 in 100 ml/well culture medium supplemented with 10% FBS. The cells were then infected with VHSV strain JF00Ehi (Genogroup Iva, MOI ¼ 0.01, virus titer ¼ 108.8 TCID50/ml at 20  C for 48 h), and viability was tested using a tetrazolium-based colorimetric method [26]. The control consisted of non-infected FHM cells. After incubating the infected FHM cells at 20  C for 40 h, 10 ml of cell counting kit-8 (Dojindo Molecular Technologies, Gaithersburg, MD, USA) solution was added to each well and incubated at 20  C for an additional 8 h. The reduction of water-soluble tetrazolium salt-8 by mitochondrial dehydrogenases to water-soluble formazan was measured at 450 nm using a microplate spectrophotometer. 2.6. Cytosolic protein extraction and tube-gel protein digestion Cultured cells were rinsed twice with ice-cold phosphate buffered saline (PBS) and centrifuged, after which the cell pellets were lysed in RIPA buffer (25 mM TriseHCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA). The resultant lysates were incubated on ice for 5 min, centrifuged at 12,000  g for 15 min at 4  C, and the supernatants collected. The proteins in the supernatant were then quantified using a Detergent Compatible (DC) Protein Assay Kit (Bio-Rad, Hercules, CA, USA), dried using a Speed Vac and digested using a Tube-Gel digestion protocol [27]. 2.7. Nano UPLC-MSE analysis A nano-ACQUITY Ultra Performance Liquid Chromatograph™ (UPLC) equipped with a Synapt™ HDMS System (Waters Corp., Milford, MA, USA) was used as described previously [28]. Tryptic peptides (5 ml) were loaded onto the enrichment column with mobile phase A (3% ACN in water with 0.1% formic acid) and mobile phase B (97% ACN in water with 0.1% formic acid). A step gradient was then applied at a flow rate of 300 ml/min; the gradient consisted of 40% mobile phase B for 95 min, 40e70% mobile phase B for 20 min, followed by a sharp increase to 80% mobile phase B within 10 min. Sodium formate (1 mmol/min) was used to calibrate the time of flight analyzer in the range of m/z 50e2000, and [Glu1]fibrinopeptide (m/z 785.8426) at 600 nl/min was used for lock mass correction. During data acquisition, the collision energies of the low-energy MS mode and high-energy mode (MSE) were set to 4 eV and 15e40 eV energy ramping, respectively. One cycle of MS and MSE mode acquisition was performed every 3.2 s. MS spectra in each cycle were acquired for 1.5 s with a 0.1 s inter-scan delay (m/z 300e1990), and the MSE fragmentation (m/z 50e2000) data were collected in triplicate.

2.8. Expressed sequence tag analysis and construction of an integrated protein database Analysis of expressed sequence tags (ESTs) is an efficient approach to gene discovery, expression profiling, and development of resources useful for functional genomics. Five cDNA libraries from embryo, brain, spleen plus kidney, liver and whole body were constructed for EST analysis. EST sequences were assembled using the bioinformatics tool, Pipeline for EST Analysis Service (PESTAS). A total of 31,104 ESTs were assembled into 3,515 contigs and 6,632 singletons. All contigs and singletons were annotated using BLAST and incorporated into the database formatted as a Microsoft Excel spread sheet or FASTA format db file. Since ESTs consist of nucleotide sequences, it was necessary to convert them into amino acid sequences for use as a proteomic database. Therefore, all EST sequences were translated into 6 frames using the TransSeq program installed on a Linux system. 2.9. Identification and relative quantitation of protein The nano-UPLC-MSE data were processed and searched using the IDENTITYE algorithm [29] on the ProteinLynx Global Server (PLGS) version 2.3.3 (Waters Corp., Milford, MA, USA). Using PLGS software, data acquired from the nano-UPLC-MSE at alternating low and high energy modes were automatically smoothed, background subtracted, centered, deisotoped, and charge state reduced. In addition, the precursors were aligned and the fragmented data were combined with a retention time tolerance of ±0.05 min. Peptide tolerance was set to 50 ppm and the MS/MS tolerance of the parent ion was set to 0.2 Da. Fixed and variable modifications were set to carbamidomethylation at cysteine and oxidation at methionine, respectively. The peptides were identified using the trypsin digestion rule with one missed cleavage. As a result, protein identification was completed with an arrangement of at least two peptides. For all identified proteins, peptide mass tolerance was based on the IDENTITYE algorithm and set at a minimum of 95% probability [30]. The MSE-based label-free quantitation of proteins, which was based on the peptide ion peak intensity measurements observed in the MS mode in a triplicate set, was performed using Expression Software v2 with the “auto-normalization” function of PLGS. The average fold-change value of the proteins was calculated using multiple tryptic peptides from each protein, and each average fold-change was calculated from the standard deviation of the peptide ion peak intensity measurement. The total number of observed tryptic peptides was determined at the 95% confidence level. Finally, an exact mass retention time (EMRT) table showing quantitative protein and peptide information was generated using Expression Software. All statistical analyses of the proteomic expression data were performed using PLGS 2.3.3 software. 2.10. Bioinformatics and network analysis The function annotation of each EST was conducted using information from the most homologous protein identified from the six-frame translated peptide sequences using the Blastp algorithm (ver. 2.2.26) with the nr database (downloaded on 3 April 2013). A minimum e-value threshold of 10e3 was used in the BLAST search. Protein function annotation was carried out using the homologous gene information using Ingenuity Pathway Analysis (IPA version 9.0; Ingenuity Systems Inc., Redwood City, CA, USA) and manually annotated using the SwissProt databases with the Protein Identifier Cross-Reference (PICR) service accessed at http://www. ebi.ac.uk/uniprot. IPA was used to perform a knowledge-based network analysis of the comparative proteomic data, and the upand down-regulated proteins were categorized into different

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groups based on their biological functions and conducted protein interaction network analysis. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base [31]. These genes, called focus genes, were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base. Networks of these focus proteins were then algorithmically generated based on their connectivity. In the graphical representations of the molecular relationships between proteins, products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature or from canonical information stored in the Ingenuity Pathways Knowledge

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Base. Human, mouse, and rat orthologs of peptides are stored as separate objects in the Ingenuity Pathways Knowledge Base but are represented as a single node in the network. Nodes are displayed using various shapes that represent the functional class of the product. Canonical pathway analysis utilizes well characterized metabolic and cell signaling pathways that are generated prior to data input and based on which identified proteins are overlaid. 2.11. Statistical analysis All samples were analyzed in triplicate. Results are expressed as means ± standard deviations, and all statistical analyses were carried out using IBM SPSS ver. 21 (IBM Corp., Armonk, NY, USA).

Fig. 1. Multiple sequence alignment of rock bream F-type lectin 3 (RbFTL-3) with other fish F-type lectins using the Genetyx program. The amino acids are numbered along the right margin. Identical residues are indicated by (*) under the column and conserved amino acids are indicated by (:); semi-conserved substitutions are indicated by (.), and deletions are indicated by dash (). The conserved cysteines are shaded in gray.

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European sea bass (B5AXH0), and yellow perch (C1K9I5) of 90.1%, 80.4% 74.6%, 74.3%, and 71.5%, respectively (Fig. 1). The expressions of RbFTL-3 mRNA were examined in several tissues from healthy rock bream using real-time PCR. The fold expression of RbFTL-3 mRNA was calculated with normalization of the expression of 18S rRNA (Fig. 2). RbFTL-3 transcripts are ubiquitously expressed in rock bream tissues. The highest RbFTL-3 expression was detected in intestine (89.5-fold), followed by liver (31.3-fold), blood (13.0-fold), spleen (4.8-fold), skin (4.1-fold), kidney (2.4-fold), and gill (1.6-fold). The highest levels of RbFTL-3 expression were observed in intestine (Fig. 2), whereas, RbFTL-1 and RbFTL-2 were reported to be most predominately expressed in the kidney and liver, respectively [17], indicating that the expressions of different lectins were not consistent with fish tissues.

Fig. 2. Relative expression of RbFTL-3 mRNA in different tissues. Analysis of RbFTL-3 mRNA was carried out using quantitative real-time polymerase chain reaction. The relative expression was calculated using the 2DDCT method with 18S rRNA as the reference gene. The relative expression in each tissue was compared to the expression in muscle to determine the tissue-specific expression. Bars depict means ± standard deviation for three replicate real-time reactions run using pooled tissue from three individual rock bream. Bars with different superscripts are significantly different (p < 0.05; Tukey's test).

Statistical differences between groups were assessed using Tukey's test; values of p < 0.05 were considered significant.

3. Results and discussion 3.1. Sequencing, multiple sequence alignment and tissue-specific expression of RbFTL-3 The full-length of RbFTL-3 cDNA (GenBank accession no. KC765140) is 1435 bp with an open reading frame of 936 bp that encoded a polypeptide of 312 amino acids with a predicted molecular mass of 34.3 kDa and a theoretical isoelectric point of 5.8. The sequence of RbFTL-3 contains a putative signal peptide of 18 residues that includes 7 completely conserved cysteines (Figure S1). The calculated molecular mass and theoretical isoelectric point of RbFTL-3 are 34.3 kDa and 5.8, respectively. RbFTL-3 showed an identity with FTLs homologues found in rock bream (RbFTL-1), Japanese sea bass (B3RH42), white bass (FBP32),

3.2. RbFTL-3 and cell viability The transfection of pcDNA3.1(þ)-RbFTL-3 at the concentration of 0.6 mg/ml into the FHM cells showed significant increase in RbFTL-3 mRNA expression, however, the expression did not significantly increase with further increase in the concentration of pcDNA3.1(þ)-RbFTL-3 (0.9 mg/ml) (Fig. 3A). The optimum concentration of pcDNA3.1(þ)-RbFTL-3 was taken as 0.6 mg/ml for cell viability assay. The effect of RbFTL-3 on cell viability was assessed using a tetrazolium-based colorimetric method, in which, the FHM cells were transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL3, and infected with VHSV at a multiplicity of infection (MOI) of 0.01. At the concentration, the relative cell viability was reduced to approximately 80% as compared to non-infected control. The relative cell viability of the cells transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 was 28.9% and 56.2% of control (Fig. 3B). Comparatively higher relative cell viability of the cell transfected with pcDNA3.1(þ)-RbFTL-3 implies the involvement of RbFTL-3 in cell viability. Other study also reported that the lectin found in pearl oyster (F-type lectin) mediates defense activity against pathogens by involving in cell trafficking, immune system regulation, and prevention of autoimmunity [32]. Hence, RbFTL-3 could act as a protection against VHSV infection with increasing cell viability. VHSV-NV gene mRNA transcription in the cell transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 for 1, 2 and 3 h post infection (hpi) is shown in Fig. 3C. The transcription of NV gene was comparatively lower in the cells transfected with pcDNA3.1(þ)RbFTL-3 than that in the cells transfected with pcDNA3.1(þ). The

Fig. 3. Effect of RbFTL-3 on the cell viability of fish cells and the expression of VHSV NV-gene. (A) Optimization of the concentration of pcDNA 3.1 (þ)-RbFTL-3 for transfection of RbFTL-3 in the FHM cells. Different concentration of pcDNA3.1(þ)-RbFTL-3 from 0.1 to 0.9 mg/ml transfected into the cells. (B) Effect of RbFTL-3 on cell viability using tetrazolium colorimetric assay. To determine the effect of RbFTL-3 on the cell viability, FHM cells transfected with RbFTL-3 at the indicated dosages were infected with VHSV (MOI of 0.01). Then using a CCK-8 kit, cell viability was assayed 48 hpi and expressed as a percentage of the viability of non-infected cells. (C) Effect of RbFTL-3 on the expression of VHSV NV-gene. To assess the VHSV-NV gene mRNA expression, the FHM cells were transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 for 1, 2, and 3 hpi. Values are shown as means ± standard deviations (n ¼ 3). Bars with different superscripts are significantly different (p < 0.05; Tukey's test).

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NV gene in VHSV played a significant role in viral replication efficiency and pathogenesis [33]. Thus, the suppression of VHSV-NV gene transcription appears to be related to the protective role of RbFTL-3 in the cells. On the other hand, the virus copy number was increased as increasing infection time from 0 to 46 hpi. However, the virus copy number was not significantly different between control (pcDNA3.1(þ)) and test (pcDNA3.1(þ)- RbFTL-3) at the same hpi (data not shown).

3.3. Identification of proteins differentially regulated by RbFTL-3 in VHSV-infected cells Comparative proteomic analysis was performed to estimate the alteration in RE of the identified proteins in the VHSV infected cells (MOI of 0.01) transfected with pcDNA3.1(þ) or pcDNA3.1(þ)RbFTL-3 at the early (18 h), intermediate (31 h), and late (46 h) stages (Fig. 4) of viral infection (Table 1 and Table S1). The RE

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determined by comparing the protein expressions between the cells transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 at different hpi. Ninety different proteins were identified in the VHSV infected FHM cells transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3. The RE of prothombin (F2) in the VHSV infected cells transfected with pcDNA3.1(þ)-RbFTL-3 was upregulated compared to that in the VHSV infected cells transfected with pcDNA3.1(þ) (Table 1), suggesting that the RbFTL-3 could reduce internal hemorrhage by increasing expression of F2. Furthermore, the vascular-sorting protein that is SNF8, a member of endosomal sorting complexes required for transport (ESCRT) machinery was observed only in the VHSV infected cells transfected with pcDNA3.1(þ) at 46 hpi, indicating enhanced viral budding process at late stage of VHSV infection. The up-regulation of SNF8 was also reported in budding of HIV-1 and Ebola viruses [34]. On the other hand, SNF8 was not observed in the VHSV infected cells transfected with pcDNA3.1(þ)-RbFTL-3 (Table 1), indicating that RbFTL-3 reduces the viral budding process. In addition, in VHSVinfected cells transfected with pcDNA3.1(þ)-RbFTL-3, tubulin beta chain (TUBB) gradually decreased in time-dependent manner (Table 1). Our finding, decrease of TUBB in RbFTL-3 transfected cells, is consistent with the earlier report that TUBB inhibition led to a reduction in influenza virus replication [35]. On the other hand, Zheng et al. found that TUBB expression was up-regulated in vero cells during bursal disease virus infection [36]. Hence, the result indicated that the RbFTL-3 could reduce the up-regulated TUBB in the cells during VHSV infection. Moreover, cytosolic T-complex protein 1 subunit epsilon (CCT5) was only observed in the VHSV infected cells transfected with pcDNA3.1(þ) in 46 hpi, indicating that it supports to form the proper functional structure of tubulins during late stage of VHSV infection (Table 1) [37,38]. However, CCT5 was not observed in the VHSV infected cells transfected with pcDNA3.1(þ)-RbFTL-3. This reveals that the inhibition of CCT5 by RbFTL-3 leads to a reduction of tubulin in VHSV-infected cells. To identify the potential biological functions and mechanism of different proteins, the identified protein in VHSV infected cells transfected with pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 were divided in different groupings associated with different biological functions and disease using IPA (Table 1). A number of proteins were associated with specific biological functions and diseases including hematological disease, inflammatory disease, infection disease, organismal injury and abnormalities, and protein folding. 3.4. Protein network composed of identified proteins regulated by RbFTL-3

Fig. 4. Workflow and application of nano-UPLC-MSE. Shown is a flow chart of the steps involved in the comparative proteomics based on nano-UPLC-MSE analysis of proteins from FHM cells transfected with pcDNA3.1(þ) or pcDNA3.1(þ)-RbFTL-3. Proteins were collected during early (18 hpi), intermediate (31 hpi) and late (46 hpi) stages of virus infection. Comparative proteomics was carried out using Expression™ (PLGS version 2.3.3), and pathway analysis was performed using Ingenuity Pathways Analysis (IPA version 9.0; Ingenuity Systems Inc.).

The identified proteins were assigned into different molecular and cellular functional classes by Ingenuity Pathways Analysis (IPA). IPA highlighted several canonical pathways in the both VHSVinfected cells transfected with both pcDNA3.1(þ) and pcDNA3.1(þ)-RbFTL-3 including the pathways related to clathrinmediated endocytosis signaling, remodeling of epithelial adherens junctions, mechanisms of viral exit from host cells, oxidation phosphorylation, thrombin signaling, ERK/MAPK signaling, protein ubiquitination and EIF2 signaling (Table 1). The proteomic data was further integrated to IPA to visualize the multidirectional interaction network composed of the up-regulated and down-regulated proteins with their most significant relations to function and disease. Proteins and their possible interrelationships with each other are presented in Fig. 5. IPA also revealed the involvement of protein ubiquitination pathway, as indicated by the presence of polyubiquitin (UBB) and HSC71, glucose-regulated protein 78 and HSP90. The presence of HSC71 (also called HSC70) during VHSV infection network analysis showed that the ubiquitin-proteasome system in the cells was affected during VHSV infection (Fig. 5).

Accession No.a

Protein name

Related human gene

Scoreb

Ratio c

d

F 18/P 18

F31/P31

F46/P46

Sequence Coverage (%)

Diseases and functionse

Canonical pathway

Clathrin-mediated endocytosis signaling, Remodeling of epithelial adherens junctions, Mechanisms of viral exit from host cells Clathrin-mediated endocytosis signaling, Remodeling of epithelial adherens junctions, Mechanisms of viral exit from host cells Clathrin-mediated endocytosis signaling, Remodeling of epithelial adherens junctions, Mechanisms of viral exit from host cells Clathrin-mediated endocytosis signaling, Remodeling of epithelial adherens junctions, Mechanisms of viral exit from host cells

Actin, alpha 1, skeletal muscle

ACTA1

860.2

P18f

F31

P46

47.5

Inflammatory disease

JF3336_4

Similar to actin, alpha skeletal muscle B

ACTA2

2199.7

11.11

0.12

0.38

67.1

Inflammatory disease

JF0014_5

Similar to actin, cytoplasmic 2

ACTB

2348.67

4.17

2.86

1.10

55.2

Infection disease, Inflammatory disease

JF0019_4

Similar to actin, alpha skeletal muscle B

ACTC1

2711.92

0.65

1.52

1.22

75.7

JF0337_5

Similar to ATP synthase subunit beta, mitochondrial

ATP5B

326.39

1.47

1.18

1.11

60.1

JF2925_2

CCT5

212.19

eg

e

P46

18.4

EEF1A1

994.46

0.74

0.96

1.10

74.9

JF8748_3 JF0164_3

Similar to T-complex protein 1 subunit epsilon Similar to elongation factor 1-alpha Elongation factor 2 Similar to prothrombin

DNA Replication, Recombination and Repair; Infection disease DNA Replication, Recombination and Repair; Infection disease Endocrine system disorder

EEF2 F2

285.66 528.35

e 1.03

P31 1.35

0.89 1.32

30.9 42.5

JF0372_4

H3 histone, family 3A

H3F3A

1503.48

0.15

0.59

0.29

58.2

JF2690_2

Histone H2A

HIST2H2AB

5562.53

0.83

0.75

0.54

79.8

JF0215_1

Similar to heat shock protein HSP 90-beta

HSP90AB1

174.26

e

F31

P46

7.5

JF0620_5

Similar to 78 kDa glucoseregulated protein

HSPA5

152.7

0.89

1.23

0.99

36.1

JF0135_4

Similar to heat shock cognate 71 kDa protein

HSPA8

495.71

1.30

1.03

1.19

56.8

JF0003_3

Similar to intermediate filament protein ON3

KRT8

1157.36

2.08

1.75

0.93

71.9

JF0199_6

Similar to MAP kinase-interacting serine/threonine-protein kinase 2 Similar to 60S ribosomal protein L18a Similar to 40S ribosomal protein S13 40S ribosomal protein S14 Ribosomal protein S27a Ribosomal protein S3

MKNK2

208.86

F18

F31

e

RPL18A

149.02

F18

e

RPS13

148.73

e

RPS14 RPS27A RPS3

212.81 715.49 128.08

P18 5.56 1.79

JF0034_3

JF0063_5 JF0263_3 JF0116_6 JF0665_5 JF0497_4

Oxidation phosphorylation

e

6.8

Infection disease, Inflammatory disease Inflammatory disease Hemorrhage disease, Cellular growth and proliferation, Infection disease, Inflammatory disease Infection disease, Inflammatory disease Infection disease, Inflammatory disease Infection disease, Protein folding, Endocrine system disorder Hemorrhage disease, Infection disease, Inflammatory disease, Protein folding DNA Replication, Recombination and Repair; Inflammatory disease, Protein folding Cellular growth and proliferation, Protein folding, Endocrine system disorder Cellular growth and proliferation

ERK/MAPK signaling

e

2.4

Inflammatory disease

EIF2 signaling

F31

e

3.8

EIF2 signaling

1.14 F31 1.23

P46 1.15 1.04

Infection disease Inflammatory disease Infection disease Infection disease Inflammatory disease

16.8 14.8 42.9

e p70S6K signaling Clathrin-mediated endocytosis signaling, Thrombin signaling, Coagulation System, Extrinsic Prothrombin Activation Pathway, Intrinsic Prothrombin Activation Pathway ERK/MAPK signaling ERK/MAPK signaling Protein ubiquitination pathway

Protein ubiquitination pathway

Clathrin-mediated endocytosis signaling Protein ubiquitination pathway

e

EIF2 signaling EIF2 signaling EIF2 signaling

S.-Y. Cho et al. / Fish & Shellfish Immunology 39 (2014) 464e474

JF0021_4

470

Table 1 Relative expression of proteins associated with different diseases, functions, and canonical pathways in VHSV-infected FHM cells transfected pcDNA3.1(þ)-RbFTL-3 and pcDNA3.1(þ)..

JF0036_6

JF0210_2 JF1416_5

Similar to 40S ribosomal protein SA Similar to vacuolar-sorting protein SNF8 Tropomyosin2 Similar to tubulin alpha chain

377.71

F18

1.67

1.14

27.5

SNF8

143.51

e

e

P46

2.9

TPM3 TUBA1A

166.99 2250.82

P18 0.80

e 0.82

e 3.23

15.3 46.4

JF0234_3

Similar to tubulin alpha chain

TUBA1C

3640.71

8.33

2.50

6.67

89.5

JF6530_2

Similar to tubulin alpha chain

TUBA3C

3887.56

0.07

0.30

0.28

79.2

JF4335_3

Similar to tubulin beta chain

TUBB

1389.1

16.67

0.11

0.06

47.2

JF0316_6 JF0389_2

Similar to tubulin beta-4 chain Similar to tubulin beta-1 chain

TUBB2B TUBB4B

1933.63 2728.61

F18 1.12

0.38 1.05

1.14 0.90

17.7 78.0

JF1664_5

Polyubiquitin

UBB

715.49

e

P31

F46

49.4

JF1494_2 JF7467_1

Similar to vinculin Transitional endoplasmic reticulum ATPase

VCL VCP

260.35 195.32

0.90 e

0.56 F31

1.33 e

43.6 11.5

JF6209_1

a b c d e f g

This initial stands for Jeonnam National University Flounder database. Scores based on PLGS 2.3.3 software. These proteins were expressed at 18, 31 and 46 h post infection (hpi) in FHM cells transfected with pcDNA3.1(þ)-RbFTL-3. These proteins were expressed at 18, 31 and 46 hpi in FHM cells transfected with pcDNA3.1(þ). Derived from human gene database using IPA. Identified only in a particular sample. Not detectable.

Inflammatory disease

EIF2 signaling

Infection disease

Mechanisms of viral exit from host cells

Inflammatory disease Infection disease, Inflammatory disease, Endocrine system disorder Infection disease, Inflammatory disease, Endocrine system disorder Infection disease Inflammatory disease, Endocrine system disorder Hemorrhage disease Infection disease Inflammatory disease Infection disease Inflammatory disease, Endocrine system disorder Inflammatory disease

e Remodeling of epithelial adherens junctions

Inflammatory disease Cellular growth and proliferation DNA Replication, Recombination and Repair; Inflammatory disease

Remodeling of epithelial adherens junctions

Remodeling of epithelial adherens junctions

Remodeling of epithelial adherens junctions Remodeling of epithelial adherens junctions Remodeling of epithelial adherens junctions

Clathrin-mediated endocytosis signaling Protein ubiquitination pathway Remodeling of epithelial adherens junctions e

S.-Y. Cho et al. / Fish & Shellfish Immunology 39 (2014) 464e474

RPSA

471

472

S.-Y. Cho et al. / Fish & Shellfish Immunology 39 (2014) 464e474

Fig. 5. Functional network analysis of the identified proteins in FHM cells. The identified proteins are associated with hemorrhage disease (F2, HSP5A and TUBB), infection disease (ACTB, ACTC1, ATP5B, EEF1A1, F2, H3F3A, HIST2H2AB, HSP90AB1, HSPA5, RPS13, RPS14, RPS27A, SNF8, TUBA1A, TUBA1C, TUBA3C, TUBB and TUBB4B), inflammatory disease (ACTA1, ACTA2, ACTB, EEF1A1, EEF2, F2, H3F3A, HIST2H2AB, HSP5A, HSP8A, RPL18A, RPS13, RPS3, RPSA, TPM3, TUBA1A, TUBA1C, TUBA3C, TUBB2B, TUBB4B, UBB, VCL and VCP), DNA replication, recombination and repair (ACTC1, ATP5B, HSPA8 and VCP), endocrine system disorder (CCT5, HSP90AB1, KRT8, TUBA1A, TUBA1C, TUBA3C and TUBB4B), cellular growth and proliferation (F2, KRT8, MKNK2 and VCP) and protein folding (HSP90AB1, HSPA5, HSPA8 and KRT8). Proteins shaded in gray color are the proteins identified by proteomic analysis, and other proteins are associated with the identified proteins based on the IPA.

HSC71 plays crucial roles in host cell entry, virus assembly, disassembly, and replication [39e41]. Protein ubiquitination and subsequent degradation are critical mechanisms for regulating numerous essential cellular functions including viral pathogenesis [42]. Viruses can utilize this system to degrade intracellular proteins that prevent viral infection or to release their virus progeny [43e45]. Moreover, the proteomic result shows that HSC71 was overexpressed in pcDNA3.1(þ)-RbFTL-3 compared to pcDNA3.1(þ) at all stages, however, the trend of change in HSC71 expression decreased from early (F18/P18 ratio 1.30 at 18 hpi) to late (F46/P46 ratio 1.19 at 46 hpi) stage (Table 1). Furthermore, our network analysis showed that the relationship between blood coagulation regulating proteins, such as F2, fibrin and coagulation factor XI (F11). The presence of F2 indicates the involvement of thrombin signaling pathway. In our study, RbFTL-3 transfection in cells up-regulated F2 leading to conditions favoring blood coagulation, which could be the cause of protection of hemorrhagic syndrome observed in VHSV infection. The protection

could be due to involvement of RbFTL-3 in regulation of F2 and formation of fibrin. F2 converts to thrombin, which catalyzes the conversion of soluble fibrinogen to insoluble fibrin, and hemostatic pathways are completed by the deposition of fibrin. The internal bleeding seen in VHSV-infected fish could be related to the reduced availability of F2 or fibrin. VHSV was able to create an F2 deficiency leading to conditions favoring anticoagulation, which could be the cause of the hemorrhagic syndrome observed in VHSV infection with blocking the actions of multiple clotting factors involved in the formation of the F2 activator complex [46]. Thus, the formation of thrombin plays a key role in protection from hemorrhage [47]. Lin et al. also reported the binding of F2 or thrombin by dengue virus nonstructural protein, thereby blocking their activities [48]. Moreover, the up-regulation of prothrombin induced by RbFTL-3 suggests the lectin enhances protection in part by reducing internal hemorrhage. For identification of protein, the database was not available for specific fish species. Despite limited proteomic and genomic

S.-Y. Cho et al. / Fish & Shellfish Immunology 39 (2014) 464e474

information on fish species, the combined use of an EST database for olive flounder prepared in-house and proteomic analysis enabled acquisition of an optimal number of peptide sequences required for protein identification. Olive flounder was selected for preparing EST database because the fish species is vulnerable to VHSV infection and the species will be used further in vivo study. Moreover, although the fish protein database was not exactly the same as the set used in our experiments, the proteomic analysis of the fish cell model elucidates the regulation of protein in the cells by RbFTL-3. 4. Conclusion In conclusion, the F-type lectin, RbFTL-3, exerts significant defensive role against VHSV in FHM cells. RbFTL-3 appears to increase in cell viability and decrease NV mRNA expression of VHSV infected fish cells by modulating the expression of proteins related to ubiquitination, viral budding and prothrombin signaling. The expression of RbFTL-3 diminishes the loss of prothrombin and inhibits a vascular-sorting protein (SNF8) in VHSV infected fish cells. We anticipate that in vivo studies in future will identify new biomarkers for diagnosis and therapeutic targets that can serve as the basis for the protection from VHSV infection in olive flounder. Acknowledgments This research was supported in part by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2010e0013133), by a project fund (C34703) to J.S. Choi from the Center for Analytical Research of Disaster Science of Korea Basic Science Institute, and by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Project No. 2010e0020141). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2014.05.042. References [1] Kim WS, Oh MJ, Nishizawa T, Park JW, Kurath G, Yoshimizu M. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene. Arch Virol 2007;152:2119e24. [2] Pierce LR, Willey JC, Palsule VV, Yeo J, Shepherd BS, Crawford EL, et al. Accurate detection and quantification of the fish viral hemorrhagic septicemia virus (VHSV) with a two-color fluorometric real-time PCR assay. PloS one 2013;8:e71851. [3] Biacchesi S. The reverse genetics applied to fish RNA viruses. Vet Res 2011;42: 12. [4] Skall HF, Olesen NJ, Mellergaard S. Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming e a review. J Fish Dis 2005;28: 509e29. [5] Winton J, Einer-Jensen K. Molecular diagnosis of infectious hematopoietic necrosis and viral hemorrhagic septicemia. In: Cunningham C, editor. Molecular diagnosis of salmonid diseases. Dordrecht: Kluwer Academic Publishers; 2002. pp. 49e79. [6] Kim T, Jung T, Lee J. Expression and serological application of a capsid protein of an iridovirus isolated from rock bream, Oplegnathus fasciatus (Temminck & Schlegel). J Fish Dis 2007;30:691e9. [7] Do JW, Cha SJ, Kim JS, An EJ, Park MS, Kim JW, et al. Sequence variation in the gene encoding the major capsid protein of Korean fish iridoviruses. Arch Virol 2005;150:351e9. [8] He JG, Zeng K, Weng SP, Chan SM. Experimental transmission, pathogenicity and physicalechemical properties of infectious spleen and kidney necrosis virus (ISKNV). Aquaculture 2002;204:11e24. [9] Kim YJ, Jung SJ, Choi TJ, Kim HR, Rajendran KV, Oh MJ. PCR amplification and sequence analysis of irido-like virus infecting fish in Korea. J Fish Dis 2002;25: 121e4.

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