Fish & Shellfish Immunology 45 (2015) 184e193

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Identification of regulators of the early stage of viral hemorrhagic septicemia virus infection during curcumin treatment Eun-Hye Jeong a, 1, Bipin Vaidya a, b, 1, Se-Young Cho a, 1, Myoung-Ae Park c, Kusuma Kaewintajuk a, Seok Ryel Kim d, Myung-Joo Oh e, Jong-Soon Choi f, Joseph Kwon f, **, Duwoon Kim a, b, g, * a

Department of Food Science and Technology, Chonnam National University, Gwangju 500-757, South Korea Bioenergy Research Center, Chonnam National University, Gwangju 500-757, South Korea Aquatic Life Disease Control Division, National Fisheries Research and Development Institute, Busan 619-705, South Korea d West Sea Fisheries Research Institute, National Fisheries Research and Development Institute, Incheon 400-420, South Korea e Department of Aqualife Medicine, Chonnam National University, Yeosu 550-749, South Korea f Biological Disaster Analysis Group, Korea Basic Science Institute, Daejeon 305-806, South Korea g Agribio Disaster Research Center, Institute of Environmentally-Friendly Agriculture, 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 29 December 2014 Received in revised form 26 March 2015 Accepted 30 March 2015 Available online 8 April 2015

The effect of curcumin pretreatment (15e240 mM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 mM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Curcumin Viral hemorrhagic septicemia virus Heat shock cognate 71

1. Introduction * 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.: þ82 42 865 3446; fax: þ82 42 865 3419. E-mail addresses: [email protected] (J. Kwon), [email protected] (D. Kim). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.fsi.2015.03.042 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

Virus infection initiates with binding of virus particle to different cell receptors, and delivers its nucleic acid to cytoplasm or nucleus of the cells [1]. Most viruses bind to specific cell receptors, including integrin, fibronectin (FN), vitronectin, laminin and sialic acid, present in the extracellular matrix and enter through plasma

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membranes of cells, which initiates the virus infection. For example, rhabdoviruses bind to FN present in the matrix in fish cells and enter through the plasma membrane of cells by endocytosis into the cytoplasm, where the viral nucleic acid is released [2,3]. However, influenzavirus and herpesvirus bind to specific receptors, including sialic acid and nectin, respectively, to enter into cytoplasm by the fusion of the plasma membrane with virus envelope [1,4]. Moreover, heat shock proteins (HSPs) such as HSP90, HSP70 and heat shock cognate (HSC) 71 present in cells function as part of a receptor complex that mediate viral entry into the cytoplasm [5]. Although the entry of viruses into cells by binding to various receptors has been described well in previous studies, developing a method of interfering virus binding to the cell receptor would be a more applicable study for controlling virus infection. Viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus in the family of Rhabdoviridae, is a negativestrand RNA virus [6] that infects various species of marine and freshwater fishes, including olive flounder, herring, sprat, and cod [7,8]. VHSV infection causes hemorrhaging of skin, muscle, and internal organs with consequent high mortality in fish [9], resulting in severe economic losses. Thus, a preventive or therapeutic agent against VHSV infection is required to preserve aquaculture industries. Although DNA vaccines were developed to control VHSV infection, their application is limited due to the difficulty in mass vaccination and their low availability for commercial purposes [10]. Due to the lack of appropriate anti-viral agents, controlling the early stages of infection could be an applicable approach to reduce outbreaks of VHSV infection in fish. In the early stages of infection, the virus is sensitive to the action of antiviral agents; however, at later stages of infection, the virus becomes partially resistant to such action [11]. The roles of interferons, a group of cytokines, and toll-like receptors have been demonstrated in innate immune responses against VHSV in the early stages of infection [12]. The cytokines are stimulated by polyinosinic: polycytidylic acid (poly I:C), which acts as an immunostimulant against VHSV infection [13]. However, the innate immune system could not respond sufficiently to protect from VHSV infection in different fish species. Therefore, safe and natural therapeutic agents are needed to control VHSV effectively in the early stages of infection. Curcumin (diferuloylmethane), a natural polyphenol derived from turmeric (Curcuma longa), exhibits pleiotropic properties, including anti-inflammatory [14,15], antioxidant [16e18], antiproliferative [19,20], anti-carcinogenic [18], anti-bacterial [21] and anti-viral [22] activities. Curcumin has shown activity against a broad spectrum of viruses, such as human immune deficiency virus (HIV), coxsackievirus, hepatitis B virus (HBV), hepatitis C virus, dengue virus type 2, Rift Valley fever virus, Japanese encephalitis and respiratory syncytial virus [23e30]. To our knowledge, no other study to date has reported the effect of curcumin in fish cells during VHSV infection. Several studies have reported that curcumin possesses different mechanisms, such as downregulating metabolic coactivator PGC1a, viral integrase and Akt-SREBP-1 and suppressing ubiquitinproteasome system for the inhibition of the viruses [22,25e27,29]. Although curcumin has been widely studied in the context of antiviral mechanism, the current literature does not clearly explain the effect of curcumin in the early stages of viral infection. A previous study mentioned that curcumin increased the epithelial barrier for viral entry by enhancing tight junctions [30]. The mechanisms involved in interactions among viruses, cells and antiviral compounds are extremely varied. Therefore, proteomic analysis as an unbiased approach could be an appropriate tool to obtain a comprehensive overview of diverse proteineprotein interactions in host cells during the viral entry stage.

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In the present study, the effect of curcumin against VHSV infection was evaluated in fathead minnow (FHM) cells in vitro. Proteomic analysis was conducted in VHSV-infected cells pretreated with curcumin to shed light on the mode of action of curcumin in an early stage of VHSV infection. This study also describes the protein network involved in the inhibitory role of curcumin against VHSV infection. 2. Materials and methods 2.1. Compound Curcumin (SigmaeAldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, SigmaeAldrich) to prepare stock solutions at a concentration of 240 mM, and was stored at 20  C. The stock solution was diluted in Leibovitz's L-15 medium (Welgene Inc., Daegu, South Korea) and filtered through a sterilized syringe filter (Sartorious Biotech, GmbH, Germany) with a 0.2-mm pore size immediately before each experiment. Rabbit polyclonal anti-HSC71 antibody and b-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Cell culture and virus Fathead minnow (FHM) cells were grown in Leibovitz's L-15 supplemented with 10% fetal bovine serum (FBS, Gibco®, Grand Island, NY, USA), 100 U/ml penicillin G and 100 mg/ml streptomycin (denoted as L-1510). The cell culture was maintained at 20  C. VHSV (Genogroup IV) stock was stored at 80  C until use. The titer of the virus stock was 108.8 TCID50/ml obtained by the limiting-dilution method. 2.3. Cytotoxicity and cell viability Cell viability was evaluated by the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) following the manufacturer's protocol. First, FHM cells were plated in 96-well plates (SPL Lifescience, Seoul, Korea) at a density of 1.0  105 cells/well in L-1510 medium, and incubated for 24 h at 20  C. After incubation, the culture medium was removed and replenished with 100 ml of L1510 containing 15, 30, 60, 120, and 240 mM curcumin, or 1 and 10 mM heat shock protein inhibitor, benzylidene lactam compound (KNK437, Calbiochem, San Diego, CA, USA). Cells treated with curcumin or KNK437 were incubated for 12 or 1 h, respectively. Subsequently, the medium was removed, replaced with the same medium containing 100 ml of VHSV suspension (MOI ¼ 0.2), and incubated for 41 h at 20  C. After incubation, 10 ml of CCK-8 solution per well was added to the plates and incubated for 8 h at 20  C. The solution was reduced by mitochondrial dehydrogenases of viable cells to a soluble colored formazan, and the intensity of the color was measured at 450 nm using an ELISA reader. The cytotoxicity of curcumin was measured, and calculated using the following equation: (AB)/(CeB)  100, where A, B and C are the absorbance of the test sample (curcumin-treated cells), background (medium without cells), and negative control (untreated medium with cells), respectively. Relative cell viability was calculated using the following equation: (DE)/(FE)  100, where D, E, and F are the absorbance of the test sample (curcumin-treated VHSV-infected cells), positive control (VHSV-infected cells for 12 h) and background (medium only) [31]. 2.4. Cytopathic effect The FHM cells were incubated in 96-well plates at a density of 1.0  105 cells/well for 24 h. After incubation, cultured cells were

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treated with 120 mM curcumin. After 12 h of treatment, the cells were infected with VHSV (MOI ¼ 0.2), and incubated at 20  C for different interval of time. Virus-induced cytopathic effect was observed using an inverted microscope (Optinity KI 400, Korea) attached with a digital microscope camera (DCM310). 2.5. ROS assay using 20 ,70 -dichlorofluorescin-diacetate (DCF-DA) The FHM cells were incubated in 96-well plates at a density of 1.0  105 cells/well for 24 h. After incubation, cultured cells were treated with 120 mM curcumin. After 12 h of treatment, the cells were infected with VHSV (MOI ¼ 0.2) and incubated at 20  C for 36 h. ROS levels were assayed using 20 ,70 -dichlorofluorescin-diacetate (DCF-DA, SigmaeAldrich). DCF-DA stock solution (10 mM in DMSO) was freshly prepared immediately before the start of the experiment. After two washes with PBS, the cells were treated with 10 mM DCF-DA at 20  C, and subsequently washed with PBS after 1 h. The fluorescence was measured using a SPECTRAmax (Molecular Devices, Sunnyvale, CA, USA) microplate reader with excitation and emission wavelengths of 480 and 530 nm, respectively [32]. 2.6. Apoptosis assay using the Muse cell analyzer Cell apoptosis was analyzed by the Muse Cell Analyzer (Merck Millipore, Billerica, MA, USA) according to the manufacturer's protocol. FHM cells were exposed to VHSV with or without curcumin treatment for 48 h. The cells were harvested and stained with the Annexin V and Dead Cell Reagent (7-aminoactinomycin D, 7-AAD). After staining, the cells were incubated in the dark for 20 min and the events for dead, late apoptosis, early apoptosis, and live cells were counted using Muse Cell Analyzer. 2.7. Viral replication using real-time PCR Total RNA was recovered from VHSV-infected FHM cells treated with curcumin (15e120 mM) or KNK437 (1e10 mM) using Trizol LS reagent (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. Aliquots of the RNA was reverse transcribed into cDNA using the Superscript First-Strand cDNA Synthesis Kit (Bioneer, Daejon, South Korea). The specific primer pair, q-VHSV-NF (50 - ATC GAA GCC GGA ATC CTT ATG C-30 ) and q-VHSV-NR (50 - CCT TGA CGA TGT CCA TGA GGT TG-30 ) was designed based on a highly conserved region of the VHSV N gene. PCR reactions were performed using the SYBR Green Master Kit (Takara Bio) in a Thermal Cycler Dice Real Time System (Takara Bio) at the following conditions: initial denaturation at 95  C for 10 s followed by 40 cycles of denaturation at 95  C for 5 s, at 60  C for 30 s, and 1 cycle of dissociation at 95  C for 15 s, 60  C for 30 s and 95  C for 15 s. To create the standard curve, a fragment of the N gene was amplified with the primer pair VHSV-N-ORF-F (50 -ATG GAA GGG GGA ATC CGT GC-30 ) and VHSVN-ORF-R (50 -TTA ATC AGA GTC CCC TGG GTA GTC GT-30 ) and was cloned into the pGEM T-easy vector according to the manufacturer's protocol (Promega, Madison, WI, USA). The copy number of the plasmid DNA template was calculated according to the plasmid molecular weight and then converted into copy numbers based on Avogadro's number. Ten-fold dilutions of the plasmid were used to create the calibration curve for absolute quantification of the viral samples [33]. 2.8. Cytosol protein extract and tube-gel protein digestion Cultured cells were rinsed twice with ice-cold PBS. To create cell lysates, cell pellets were resuspended in RIPA buffer (25 mM TriseHCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) plus a protease inhibitor cocktail (Roche Applied

Science, Indianapolis, IN, USA). The samples were incubated on ice for 5 min and centrifuged at 12,000  g at 4  C for 15 min, and the supernatants were collected. Protein quantification was performed using a Detergent Compatible Protein Assay Kit (Bio-Rad, Hercules, CA, USA) and dried using a SpeedVac. The shape of the polymerized gel matrix is a miniaturized tube, thus named “Tube-Gel”, and the proteins were digested using “Tube-Gel digestion” protocol [34]. The cytosol protein pellet was resuspended in 50 ml of 6 M urea, 5 mM EDTA, and 2% (w/v) SDS in 0.1 M triethylammonium bicarbonate (TEABC) and incubated at 37  C for 30 min for complete dissolution. Proteins were chemically reduced by adding 10 ml of 20 mM tris (2-carboxyethyl) phosphine and alkylated by adding 20 ml of 20 mM iodoacetamide at room temperature for 30 min. To incorporate proteins into the gel directly in the microtube, 18.5 ml of 40% (v/v) of acrylamide/bisacrylamide (29:1), 2.5 ml of 10% (w/v) of ammonium persulfate, and 1 ml of 100% tetramethylethylenediamine were then applied to the protein solution. The gel was cut into small pieces and washed out three times with 1 ml of TEABC containing 50% (v/v) acetonitrile (ACN). The dehydrated gel samples were then digested with 15 ml trypsin (protein: trypsin, 10:1, w/ w) in 25 mM TEABC with incubation at 37  C overnight. The digested peptides were recovered twice with a solution containing 50 mM ammonium bicarbonate in 50% ACN, and 5% trifluoroacetic acid (TFA). The resulting peptide extracts were pooled, lyophilized in a vacuum centrifuge, and dissolved in 0.5% TFA prior to LC-MS/ MS analysis. 2.9. Nano UPLC-HDMS analysis Complex tryptic peptide mixtures were separated using nanoACQUITY Ultra Performance Liquid Chromatograph (UPLC) equipped with a Synapt G2-Si High Definition Mass Spectrometer (HDMS, Waters Corp., Milford, MA, USA), which was operated in a data-independent manner coupled with ion mobility [35]. The MS was operated in positive electrospray ionization (ESI) resolution mode with a resolution of >250,000 full-width at half maximum. During data acquisition, the MS and MS/MS were set at low (4 eV) and elevated (14e40 eV) collision energies, respectively, on the transfer collision cell, using a scan time of 0.5 s per function over 100e2500 m/z. MS and MS/MS spectra data were collected in triplicate. Tryptic peptides (1.5 mg) 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 stepwise gradient was applied at a flow rate of 250 nl/min. The gradients consisted of 97% mobile phase A for 1 min, 85% mobile phase A for 10 min, 65% mobile phase A for 150 min, and finally a sharp increase to 97% mobile phase A over 25 min. Sodium formate (1 mM/min) was used to calibrate the time-of-flight analyzer in the range of m/z 100e2500, and [Glu1]fibrinopeptide (m/z 785.8426) at 2 ml/min was used for lock mass correction. 2.10. Protein identification and relative quantification Protein identification and quantification information were obtained using Progenesis QI for Proteomics (QIP) version 1.0 (Nonlinear Dynamics, Newcastle, UK) [36] and lists of proteins were identified by EST database (22/03/2012), including 58,350 entries [37]. Prominent ion features were used as vectors to match each dataset to a common reference chromatogram. An analysis window of 0e160 min was selected, which encompassed a total of 5108 features (charge states of þ2, þ3, and þ4), and was separated by ion-mobility separation. Protein identifications and quantitative information were extracted using the dedicated algorithms in Progenesis QIP. The initial ion-matching requirements were as

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follows: 1 fragment per peptide, 3 fragments per protein, and 1 peptide per protein. The enzyme specificity was trypsin allowing one missed cleavage, carbamidomethyl of cysteine (fixed) and oxidation of methionine (variable). Parent- and fragment-ion parts per million errors were calculated empirically, and decoy databases were used to estimate the identification error rate. Scoring of the database searches was refined by correlation of physicochemical properties of fragmented peptides from theoretical and experimental data. Peptide identifications were imported to Progenesis QIP and filtered to exclude peptides with scores less than 0.5.

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were blocked for 1 h at room temperature with 5% skim milk in trisbuffered saline (TBS)/0.1% Tween 20 (TBS-T), and then incubated with the anti-HSC71 primary antibody (1:1000). The horseradish peroxidase-linked secondary antibodies was obtained from Dako (Carpintaria, CA, USA). The blots were re-probed for total HSC71 (1:1000) and b-actin (1:10,000). The signals were scanned for quantitative analysis with Tina 2.0 software (Raytest, Straubenhardt, Germany). The ratio of F-actin to G-actin was measured by western blotting techniques previously described by Song et al. [38]. 2.13. Statistical analysis

2.11. Bioinformatics analysis Ingenuity Pathway Analysis (IPA version 9.0; Ingenuity Systems Inc., Redwood City, CA, USA) was used to analyze upregulated and downregulated proteins and perform knowledge-based network analysis of comparative proteomics data.

All samples were analyzed in triplicate, and results are expressed as means ± standard error. Statistical analysis was performed using the IBM SPSS Statistics software (version 21.0, IBM Corp., Armonk, NY, USA). The differences were considered significant at p < 0.05 with Duncan's multiple range test.

2.12. Western blot analysis

3. Results and discussion

FHM cells were treated with the HSP inhibitor KNK437 for 1 h and then infected with VHSV. The virus inoculum was removed after 24 h, and fresh basal medium was added into the culture. The culture was allowed to continue a further incubation for 18 h. Proteins were separated in 10% SDS-polyacrylamide gels, and transferred to a PVDF membrane for 60 min at 350 mA. The blots

3.1. Inhibitory effect of curcumin pretreatment on VHSV infection The cytotoxicity of curcumin to FHM cells was measured based on relative cell viability by a CCK-8 assay (Data not shown). The cell viability was >90% with the curcumin treatment within the concentration range of 15e240 mM, indicating low cytotoxicity of the

Fig. 1. Inhibition of viral hemorrhagic septicemia virus (VHSV) infectivity in fathead minnow (FHM) cells by curcumin. (A) Effect of curcumin pretreatment in cell viability of VHSVinfected FHM cells. The FHM cells were pretreated with 0, 15, 30, 60, 120, and 240 mM curcumin for 12 h prior to VHSV infection, and cell viability was determined by Cell Counting Kit (CCK)-8 assay. (B) Effect of curcumin treatment on VHSV replication. Curcumin at the indicated concentrations was added 12 h before VHSV infection. After 46 h post-infection, supernatants of infected cultures were harvested and the copy number of the recovered VHSV N gene was estimated by RT-PCR. (C) Effect of curcumin (120 mM) on reactive oxygen species production in VHSV-infected FHM cells. The relative fluorescence intensities of oxidized 20 ,70 -dichlorofluorescin-diacetate (DCF-DA) exposure to different cells were measured by a SPECTRAmax microplate reader. The excitation and emission wavelengths were 480 and 530 nm, respectively. The result was expressed as fold change in intensity compared to mock (untreated and non-infected samples) (D) Effect of curcumin on apoptosis in VHSV-infected FHM cells using the Muse Annexin V and Dead Cell Assay. The different cell samples were stained with 7-aminoactinomycin D and dead, late apoptotic, early apoptotic, and live cells were counted using the Muse Cell Analyzer. Bars represent means ± SE (n ¼ 3), and those labeled with different letters significantly differ from one another (p < 0.05; Duncan's multiple range test).

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compound to FHM cells. A previous study also reported that the compound did not show cytotoxicity in human lung, kidney, prostate, cervix, CNS malignancies and melanoma cell lines, whereas the compound induced apoptosis in malignant tumor cell lines, such as leukemia, breast, colon, hepatocellular, and ovarian cell lines [39]. To determine the effect of curcumin on the viability of VHSV-infected cells, the relative cell viability of the infected cells pretreated with 15, 30, 60, 120, and 240 mM curcumin for 12 h were measured by a CCK-8 assay (Fig. 1A). The results indicate that curcumin pretreatment increased the cell viability of VHSV-infected cells in a concentration-dependent manner, and the viability of the infected cells was completely restored at >120 mM curcumin. Moreover, the changes in morphology of VHSV infected FHM cells at different h post-infection (hpi) with and without curcumin treatment were observed (Supplementary Figure 1). The cell morphology was not markedly different until 30 hpi, however the cytopathic effects in cells, such as increase in size and change into round shape, could be observed in VHSV infected cells from 36 hpi. The cell clumps were detached, and circular void areas (without cells) were formed due to cell lysis from 42 hpi. At 48 hpi, size and number of clear circular areas increased. Whereas, curcumin treatment could reduce the cytopathic effect such as changing in shape and lysis of cells. To identify the effect of curcumin in viral replication, the viral copy number was investigated by RT-PCR in the VHSV-infected cells treated with different concentrations of curcumin (Fig. 1B). The copy number as measured by the nucleocapsid (N) gene expression in the cells was not significantly reduced with increasing concentrations of curcumin; however, the level was significantly reduced at 120 mM curcumin pretreatment. The VHSV N gene plays a significant role in the viral replication cycle [40]. In response to curcumin pretreatment, a 0.2-log reduction in N gene expression was observed at 120 mM. These results suggest that curcumin pretreatment inhibits VHSV infection by reducing viral replication at higher concentrations. Consistent with our study, curcumin significantly suppressed the enterovirus 71, HIV, and HBV replication in infected cells [41e43]. The effect of curcumin on reactive oxygen species (ROS) production in VHSV-infected FHM cells was measured by changes in

fluorescence intensity of 20 , 70 -dichlorofluorescein diacetate. Curcumin pretreatment (120 mM) significantly decreased the fluorescence intensity as compared to VHSV-infected cells, indicating that the curcumin treatment decreased ROS production (Fig. 1C). Increased ROS levels are a major pathogenic factor in cell death [44]. Viral infections have been shown to increase ROS production and decrease antioxidant levels inside cells [45]. Suppression of ROS could be due to upregulation of natural antioxidants, including superoxide dismutase, catalase and glutathione levels in cells by curcumin treatment [46]. Curcumin reduces ROS levels, which results an increase in cell viability by suppressing the oxidative stress induced by VHSV infection [45,47]. Hence, suppressing ROS production by curcumin treatment is a potential approach to decreasing VHSV pathogenesis in fish. Furthermore, apoptosis in VHSV-infected cells affected by curcumin (120 mM) was measured as the percentages of live and apoptotic cells by Annexin V and dead cell staining, as shown in Fig. 1D. The percentages of live, early apoptotic and late apoptotic cells in the VHSV-infected (V) samples were 30.7, 66.9 and 2.4%, respectively, whereas those in curcumintreated VHSV-infected samples (CV) were 72.7, 15.5 and 11.0%, respectively. The percentage of apoptotic cells in CV was significantly lower than that in V. This result suggests that curcumin pretreatment reduced the infectivity of VHSV in cells. Consistent with the present result, a previous study reported that oral administration of curcumin significantly decreased apoptosis in Schwann cells [48]. However, apoptosis was reported to be induced in carcinoma cells by curcumin treatment [39]. Collectively, these results suggest that curcumin pretreatment suppressed the severity of VHSV infection by increasing cell viability, inhibiting viral replication, and suppressing ROS production. 3.2. Identification of proteins differentially regulated by curcumin pretreatment in VHSV-infected cells To understand the modulation of proteins by curcumin pretreatment in VHSV infection, proteomic analysis was performed in four samples (mock, M; curcumin-treated only, C; VHSV-infected, V; and curcumin-treated VHSV-infected, CV) in triplicates in the early stage of infection. Out of 185 proteins detected, twenty

Fig. 2. Identification of viral entry-associated proteins expressed during viral hemorrhagic septicemia virus (VHSV) infection in fathead minnow (FHM) cells. Differentially expressed proteins associated with viral entry in VHSV infection are illustrated in a spider graph representing the average ratio of significantly differentially expressed proteins between the V:M and CV:M groups at two hpi in FHM cells (M, mock; V, VHSV; CV, curcumin-treated VHSV-infected). Asterisk (*) indicates proteins that were significantly differentially expressed between two groups.

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Fig. 3. Label-free protein profiling of fathead minnow (FHM) cells using Progenesis QI software. (A) Total ion chromatograms of protein from viral hemorrhagic septicemia virus (VHSV)-infected cells (V, pink) and curcumin-treated VHSV-infected cells (CV, green). (B) Alignment grid according to the liquid chromatography retention time and the m/z of all the detected peptides. (C) Representation of the abundance of one peptide from heat shock cognate 71 protein (HSC71) within VHSV-infected (purple) and curcumin-treated VHSVinfected (blue) samples. JF0186_3, similar to actin cytoplasmic 2 (gene symbol ACTG1); JF0135_4, similar to heat shock 70 protein 8 (gene symbol HSPA8); JF0034_3, similar to elongation factor 1-alpha (gene symbol EEF1A1).

proteins were identified in all samples (Supplementary Table 1). In order to investigate the roles of these proteins during curcumin treatment for the prevention of VHSV infection in the early stage, we compared the protein expression between V and CV samples using Progenesis QI software. Quantitative protein information was obtained for 12 proteins detected in all 4 LC-MS/MS triplicate datasets by Progenesis software using an algorithm based on the

pairwise feature detection at the LC-MS level. The fragmentation spectrum of each quantified peptide was introduced into the analysis, so that the proteins were identified and compared their abundance-level among samples. Then, for each differentially expressed protein, peptides were quantified and normalized to the abundances of all the peptides. The IPA indicated that three proteins, heat shock cognate 71 kDa protein (HSC71 or HSPA8), actin

Fig. 4. Network pathway analysis based on proteome data for entry stage of VHSV infection. The protein networks of VHSV-infected cells and curcumin-treated VHSV-infected cells are illustrated in left and right panels, respectively. Associations among proteins shown by solid or dashed lines represent direct or indirect interactions, respectively. Upregulated and downregulated proteins are shown in red and green, respectively. The orange and blue colors indicate the predicted activation and inhibition status of the connected proteins, respectively, based on Ingenuity Pathway Analysis (IPA) database.

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cytoplasmic 2 (ACTG1), and elongation factor 1-alpha (EEF1A1) showed significant differential expression between V:M and CV:M (Fig. 2). The total ion chromatograms of peptides from V (pink) and CV (green) samples are shown in Fig. 3A. The alignment grid of peptides corresponding to these major 3 proteins (JF0186_3, JF0135_4, and JF0034_3) is shown in Fig. 3B. The peak areas of the peptides corresponding to JF0135_4 (HSPA 8) were greater than that of other proteins. In addition, the ion intensities of peptides of HSC71 were compared between V and CV samples. A decrease in the relative abundances of peptides by curcumin treatment was observed in CV sample (Fig. 3C). Therefore, HSC71 was confirmed as the strongest candidate for the prevention of viral entry in VHSV infection via our comparative proteomics. 3.3. Protein network composed of identified proteins regulated by curcumin The proteomic data were further integrated in IPA to visualize the multidirectional interaction network composed of the upregulated and downregulated proteins. Fig. 4 shows the protein networks in response to VHSV infection in the left panel and curcumin pretreatment in VHSV-infected cells in the right panel. The

proteins, especially fibronectin (FN) 1, HSC71, ACTB, ACTG1, F-actin, and gelsolin (GSN) are involved in VHSV infection. In our study, IPA predicted that FN1 was upregulated in the V sample. Among several virus-binding components present in the extracellular matrix, FN1 represents the main receptor for fish rhabdoviruses such as VHSV and IHNV [2], and promotes VHSV migration and invasion of cells. Many viruses, including hepatitis A virus, HBV, HIV, and Rous sarcoma were previously demonstrated to bind to FN [49e52]. On the other hand, IPA predicted that curcumin downregulated FN1 in the CV sample, which could be due to interference of curcumin with FN1 expression, with resulting blocking of VHSV entry. The network reveals that the plausible approaches to blocking viral entry could be the identification of the cell receptors, such as FN, and the interference with the expression of the receptors in cells. The analysis also predicted the involvement of HSPs, including HSC71, HSP70, and HSPA5 in VHSV infection. The expression of HSC71 was upregulated by viral infection. The network showed that HSC71 plays a pivotal role for the regulation of different functions by interacting with various proteins, including FN1, GSN, and actins. Several studies reported that HSC71 could interact with viral proteins such as the Bornavirus protein X, the papillomavirus protein L229, and the influenza virus protein M1 [53]. HSC71 has

Fig. 5. Effect of curcumin and KNK437 on HSC71 expression. (A) Western blot analysis of heat shock cognate 71 protein (HSC71) expression in fathead minnow (FHM) cells. The cells were treated with curcumin (120 mM) for 12 h in FHM cells before virus infection. The cells were infected with VHSV (MOI ¼ 0.2) and incubated for 1e4 h at 20  C. (B) Effect of KNK437 on viability of VHSV-infected FHM cells. The cells were treated with KNK437 (1e10 mM) for 1 h before VHSV infection. Cell viability was determined by CCK-8 assay. (C) Effect of KNK437 on the replication of the VHSV N gene. KNK437 at the indicated concentrations was added 1 h before infection. After 46 h of infection, supernatants of infected cultures were harvested and the copy number of the recovered VHSV N gene was estimated by RT-PCR. (D) Effect of KNK437 (1e10 mM) on the expression of HSC71. TINA 2.0 software was used for densitometry analysis of HSC71 expression before and after VHSV infection. The expression levels were normalized to b-actin and expressed relative to those of mock (untreated and non-infected sample). Bars represent means ± SE (n ¼ 3), and labeled with different letters significantly differ from one another (p < 0.05; Duncan's multiple range test).

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been reported to perform crucial roles in viral entry to cells, viral assembly, disassembly, and replication [5,54]. HSPs act as actinbinding proteins and control the transition between monomeric (G-actin) and filamentous (F-actin) states. Furthermore, IPA predicted that F-actin decreased in the V sample, indicating that Factin could be depolymerized to G-actin in the cell. Consequently, VHSV infection could disrupt the actin cytoskeleton. Actin is responsible for many other cellular functions, such as motility and maintenance of cell [55]. The network also showed that the binding of HSPs to actin filaments is independent of Ca2þ, whereas GSN binds actin filaments in the presence of Ca2þ. GSN activity is required at sites where rapid modification of the membrane actin cytoskeleton occurs. GSN is upregulated in VHSV infection, which may induce the depolymerization of F-actin [56,57]. However, the role of GSN is inconsistent as it was also reported to modulate the structural dynamics of the actin cytoskeleton by formation and stabilization of F-actin [58]. In our network analysis, G-actin was predicted to be downregulated in the CV sample with upregulated F-actin. The HSP inhibitor KNK437 was predicted to downregulate HSC71 by inhibiting the expression of heat shock factor (HSF) 1. HSF1 is considered a stress-responsive factor that can be activated during stress conditions such as VHSV infection and enhanced HSP responses [59]. KNK347 treatment inhibits HSF1, resulting in reduction of HSC71 synthesis, suggesting that HSF1 is a negative regulator of the HSP response [60].

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3.4. HSC71 expression in curcumin-pretreated FHM cells To validate the proteomic results, we investigated the effects of curcumin and HSP inhibitor on HSC71 expression by western blotting. The expression of HSC71 in FHM cells during VHSV infection at 1, 2, 3, and 4 hpi is shown in Fig. 5. In FHM cells, VHSV infection led to an increase in HSC71 expression, as expected, and curcumin pretreatment suppressed HSC71 expression in CV sample (Fig. 5A). Similarly, a previous study also reported that quercetin treatment reduced hepatitis C virus RNA replication by downregulating HSP72 expression [61]. The HSC71 expression level in CV sample was lower than V sample by 1.5-fold in both 2 and 3 hpi. However, the expression was not significantly different at 4 hpi, which could be due to degradation of curcumin in cells [62,63]. Moreover, to confirm the involvement of HSC71 in cell viability and VHSV replication, the effect of KNK437 on the viability of VHSVinfected cells and VHSV N gene copy number was investigated by RT-PCR. Fig. 5B shows that treatment with 10 mM KNK437 significantly increased (42.5% higher than untreated sample) cell viability without cytotoxic effects, and elicited a 1.4-log reduction in VHSV N gene copy number as compared to untreated sample (Fig. 5C). The effect of KNK437 on cell viability and virus replication was observed due to inhibition of HSP expression during VHSV infection. In addition, the effect of the HSP inhibitor, KNK437, in HSC71 expression with and without VHSV infection is shown in Fig. 5D. The increasing concentration of KNK437 treatment caused a

Fig. 6. Effects of curcumin and KNK437 on the expression ratio of F-actin and G-actin (F/G ratio). Western blot analysis of F/G actin expression was performed with the curcumin pretreatment (120 mM) in fathead minnow (FHM) cells for 12 h prior to VHSV infection. The cells were treated with KNK437 (10 mM) for 1 h before VHSV infection. The cells were infected with VHSV (MOI ¼ 0.2) and incubated at 20  C for different time intervals of 1e4 h. At each time point, the expression of both actins was quantified by densitometry and expressed in terms of ratio of F-actin and G-actin. Bands indicate the expression of F-actin and G-actin in left and right panels, respectively. Bars represent means ± SE (n ¼ 3). Asterisks indicate significant differences from the mock determined using Student's t-test (*p < 0.05).

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gradual decrease in HSC71 expressions in both infected and noninfected cells. The expression was upregulated to 1.4-fold by VHSV infection; however, KNK437 treatment markedly decreased the level of HSC71 expression. Similarly, KNK437 was reported to inhibit various HSPs, specifically HSP70 at the mRNA level [64]. These results reveal that HSC71 acts as a target protein for antiviral activity by downregulating its expression using the HSP inhibitor, KNK437. Similar to KNK437 treatment, curcumin inhibited HSC71 expression (Fig. 5A), with a significant increase in viability of VHSVinfected FHM cells as described before (Fig. 1A). The suppression of HSC71 expression with increasing cell viability by curcumin pretreatment in the VHSV-infected cells was comparable to those patterns observed with KNK437-treated cells (Fig. 5A and B). Curcumin pretreatment also resulted in inhibition of VHSV replication through the suppression of HSC71 expression. These results suggest that HSC71 could be a therapeutic target molecule for controlling VHSV infections in FHM cells. Chen et al. also predicted that HSP72 is directly involved in enhancement of viral RNA replication [65]. 3.5. Effect of curcumin pretreatment on F/G-actin ratio To validate the prediction of alterations in F- and G-actin content by IPA, we further quantified the ratio of F- and G-actin (F/G ratio) by western blotting. Fig. 6 shows that the F/G ratio significantly decreased at 3 hpi in VHSV infected cells as compared to mock, indicating that F-actin was degraded during VHSV infection presumably by depolymerization of F-actin, with a minor change in the amount of G-actin. Similarly, previous researchers demonstrated the depolymerization of F-actin in cells by infection of rabies and herpes simplex viruses at the early stage of infection [38,66]. On the other hand, curcumin pretreatment significantly increased F/G ratio at the same hpi in the cells. Additionally, KNK437 treatment significantly increased the F/G ratio to levels similar to those observed in the curcumin pretreatment at 3 hpi. The role was possibly played by HSC71 to induce actin depolymerization during VHSV infection; however curcumin and KNK437 can prevent the depolymerization by suppressing HSC71 expression during the early stages of VHSV infection. The reason could be that the suppression of HSC71 reduces the chances of interaction of HSC71 and an actin-binding factor, cofilin [67]. 4. Conclusion Curcumin pretreatment significantly increased relative cell viability and inhibited VHSV replication without cytotoxic effects in FHM cells. Curcumin pretreatment resulted in inhibition of VHSV infection through suppression of viral entry and ROS production. IPA revealed that HSC71 could play a central role in different cellular processes during VHSV infection, and FN may act as a main receptor, allowing VHSV to bind at an early stage to enter the cell. Furthermore, the remodeling of the actin cytoskeleton could receive more attention as a mechanism for controlling the entry of VHSV by rearrangement of the F/G ratio. Thus, this study will encourage further research toward application of curcumin as a safe and preventive intervention against VHSV infection in aquaculture. Acknowledgments This research was supported in part by a grant from the National Fisheries Research and Development Institute (NFRDI), Republic Korea (RP-2014-AQ-145), by a project fund (C3373A) 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

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Identification of regulators of the early stage of viral hemorrhagic septicemia virus infection during curcumin treatment.

The effect of curcumin pretreatment (15-240 μM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell vi...
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