key: cord-0999989-slybrok6
authors: Du, Junzheng; Xing, Shanshan; Tian, Zhancheng; Gao, Shandian; Xie, Junren; Chang, Huiyun; Liu, Guangyuan; Luo, Jianxun; Yin, Hong
title: Proteomic analysis of sheep primary testicular cells infected with bluetongue virus
date: 2016-04-13
journal: Proteomics
DOI: 10.1002/pmic.201500275
sha: 298b1fb8728be8cf99f6397b2bc4a472bb69915c
doc_id: 999989
cord_uid: slybrok6

Bluetongue virus (BTV) causes a non‐contagious, arthropod‐transmitted disease in wild and domestic ruminants, such as sheep. In this study, we used iTRAQ labeling coupled with LC‐MS/MS for quantitative identification of differentially expressed proteins in BTV‐infected sheep testicular (ST) cells. Relative quantitative data were obtained for 4455 proteins in BTV‐ and mock‐infected ST cells, among which 101 and 479 proteins were differentially expressed at 24 and 48 h post‐infection, respectively, indicating further proteomic changes during the later stages of infection. Ten corresponding genes of differentially expressed proteins were validated via real‐time RT‐PCR. Expression levels of three representative proteins, eIF4a1, STAT1 and HSP27, were further confirmed via western blot analysis. Bioinformatics analysis disclosed that the differentially expressed proteins are primarily involved in biological processes related to innate immune response, signal transduction, nucleocytoplasmic transport, transcription and apoptosis. Several upregulated proteins were associated with the RIG‐I‐like receptor signaling pathway and endocytosis. To our knowledge, this study represents the first attempt to investigate proteome‐wide dysregulation in BTV‐infected cells with the aid of quantitative proteomics. Our collective results not only enhance understanding of the host response to BTV infection but also highlight multiple potential targets for the development of antiviral agents.

Proteomic approaches are increasingly recognized as effective tools facilitating comprehensive characterization of virus-host cell interactions. Although BTV, a prototype of the genus Orbivirus within the family Reoviridae, has been extensively studied, the interactions between virus and host cells are not fully understood at present. In this study, we investigated proteomic alterations in BTV-infected ST cells using iTRAQ labeling combined with LC−MS/MS for the first time. The identification of significantly altered proteins in BTV-infected ST cells provides a global overview of the BTV-host cell interaction network. Many of the immune response-related proteins differentially expressed upon BTV infection are novel and have not been reported to date. Elucidation of the functions of these proteins in virus-host cell interactions may be useful for the development of new therapeutic strategies targeting BTV.

protective immunity and have an impact on vaccination strategies [3, 8] . BTV usually causes severe haemorrhage and ulceration of the mucous membranes in ruminants, along with fever, lameness, coronitis, pulmonary oedema, swelling of the head (particularly lips and tongue) and death. In this regard, the pathogenesis of bluetongue is similar to that of hemorrhagic viral fevers of humans, such as Ebola virus [2, 9, 10] .

At least eight different serotypes of BTV (BTV-1, 2, 4, 6, 8, 9, 11 and 16) have emerged in Europe since 1998, collectively representing the largest outbreak of the disease ever recorded that led to the deaths of over 1.8 million animals [11] [12] [13] . BTV caused significant mortality, estimated at ß50% and 15% in clinically affected sheep and cattle, respectively, during the northern European outbreak in 2006-2008 [9, 14] . As a result of its economic importance, BTV has been studied extensively as a model system for related viruses, and represents one of the most well characterized viruses [5, 15, 16] .

Although the mechanisms of BTV action have been elucidated, the interactions between virus and host cells are not fully understood at present [2, 5, 15] . Proteomic approaches provide effective tools for the comprehensive characterization of virus-host interactions and identification of the cellular proteins involved directly or indirectly in viral infection via detection of the relative changes in protein expression [17] [18] [19] . For example, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption-ionization time-of-flight tandem mass spectrometry (MALDI-TOF/MS) proteomic approaches have been employed to investigate proteomic changes in host cells in response to virus infections, including classical swine fever virus [20] , infectious bursal disease virus [21] , severe acute respiratory syndrome associated coronavirus [22] and porcine circovirus type 2 (PCV2) [23] . Stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry (MS) have additionally been employed for characterization of pathogenic viruses, including PCV2 [24] , influenza virus [25] , adenovirus [26] , Japanese encephalitis virus [27] and human immunodeficiency virus [28] . These studies have provided extensive insights into the infected host cell proteomes and enhanced our understanding of the alterations induced in signaling pathways in response to viral infection.

iTRAQ is a more sensitive and accurate technique than traditional proteomic approaches (2DE, MALDI-TOF/MS and SILAC) for quantifying low-abundance proteins. The method has been employed to investigate virus-host interactions for several viral pathogens, such as transmissible gastroenteritis virus [29] , porcine reproductive and respiratory syndrome virus [30] , PCV2 [31] , porcine epidemic diarrhea virus [32] and equine infectious anemia virus [33] . The mechanisms of BTV pathogenesis and immunomodulation have not been established to date. In the present study, we investigated the host cell response by profiling changes in cellular protein expression patterns in BTV-infected cells using iTRAQ labeling combined with liquid chromatography-mass spectrometry (LC−MS/MS). Alterations in protein expression levels were verified using quantitative real-time RT-PCR and western blot analyses. The possible biological significance of the differentially expressed proteins identified in the host response to BTV infection was further evaluated using bioinformatics tools.

Sheep testes (ST) cells were obtained from three healthy 2 week-old male lambs. Briefly, testes were collected and washed with phosphate-buffered saline (PBS, pH 7.4) supplemented with 100 U/mL penicillin and 100 g/mL streptomycin. Testicular tissues were cut into small pieces and digested with trypsin at 37ЊC for 30 min. Isolated ST cells were washed with PBS three times and resuspended in RPMI-1640 (Hyclone, USA) supplemented with 15% fetal bovine serum (Gibco, USA). Cells were seeded into tissue culture flasks (Corning, USA) at a density of 1×10 5 cells/mL and incubated at 37ЊC under 5% CO 2 for ß1-2 days. Non-adherent and loosely adherent cells were removed by mildly shaking flasks before changing the medium, and the remaining adherent cells further incubated for 3-4 days. The BTV-1 strain (GS/11) was isolated in western China and propagated in BHK-21 cells (ATCC-CCL-10) obtained from the American Type Culture Collection. The viral titer, determined using a plaque formation assay on a monolayer of BHK-21 cells, was estimated as plaque formation units per milliliter (PFU/mL) [16] . All animal experiments were performed according to the protocols approved by the Animal Care and Use Committee of the Lanzhou Veterinary Research Institute (permit no. 2009-26) . All experiments with infectious viruses were conducted in Biosafety Level 3 facilities.

The monolayer of confluent ST cells was dispersed with 0.25% trypsin and 0.02% EDTA and seeded in 6 cm cell culture flasks. After a 24 h incubation period, the culture medium was removed and ST cells washed with PBS. Cells were inoculated with BTV-1 at a multiplicity of infection (MOI) of 0.1. After 1 h of adsorption, infected cells were maintained in RPMI-1640 supplemented with 2% FBS. Uninfected ST cells were used as the mock-infected group. Viral propagation was confirmed with cytopathic effect (CPE) and one-step growth curve of BTV-1 in BHK-21 cells. Three replicates of virus-and mock-infected cultures were prepared at each timepoint. The CPE was observed under a light microscope at 0, 12, 24, 48, and 72 hours post infection (hpi). To further confirm BTV infection in ST cells, RT-PCR was performed using the S7F (5 -GTTAAAAATCTATAGAGATGG-3 ) and S7R (5 -GTAAGTGTAATCTAAGAG-3 ) primers to detect the viral S7 gene. Total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's instructions, and RT-PCR performed with the one-step PrimeScript RT reagent Kit (Takara, Japan). PCR products were verified using 1% agarose gel electrophoresis and sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, USA).

BTV-infected and mock-infected ST cells in 25 cm 2 flasks (3×10 6 cells/flask) were collected at 24 and 48 hpi with a cell scraper, centrifuged at 1000 × g for 5 min, and washed three times with ice-cold PBS. Three batches of ST cells were prepared from three lambs, and each batch infected with BTV-1 or mock infected for 24 and 48 h. Equal amounts of cells from each of the three batches of virus-and mock-infected cells were mixed at each time-point. Mixed cell pellets were suspended in lysis buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5, 1 mM PMSF, 2 mM EDTA) and sonicated at 200 watts for 5 min on ice. Proteins were reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide for 1 h, and subsequently precipitated using chilled acetone. After centrifugation at 4ЊC and 30 000 × g, the pellet was dissolved in 0.5 M TEAB (Applied Biosystems, Italy) and sonicated on ice. Following another centrifugation step at 30 000 × g and 4ЊC, an aliquot of supernatant was obtained for determination of protein concentration with the Bradford protein assay using the 2-D Quant kit (Amersham, USA). Proteins in the supernatant were stored at -80ЊC for further analysis.

An aliquot of total protein (100 g) was obtained from each sample solution and digested with Trypsin Gold (Promega, Madison, WI, USA) at a trypsin/protein ratio of 1:30 at 37ЊC overnight. Peptides were dried via vacuum centrifugation, reconstituted in 0.5 M TEAB, and processed according to the manufacturer's protocol for 8-plex iTRAQ reagent labeling. Peptides prepared from BTV-and mock-infected ST cells at 24 hpi were labeled with iTRAQ tag 114 and iTRAQ tag 116, while those prepared from BTV-and mock-infected ST cells at 48 hpi were labeled with iTRAQ tag 118 and iTRAQ tag 119, respectively. Labeled peptide mixtures were pooled and dried via vacuum centrifugation. SCX chromatography was performed with a LC-20AB HPLC Pump system (Shimadzu, Japan). iTRAQ-labeled peptide mixtures were resuspended with 4 mL buffer A (25 mM NaH 2 PO 4 in 25% ACN, pH 2.7) and loaded onto a 4.6×250 mm Ultremex SCX column containing 5 m particles. Peptides were eluted with a gradient of buffer A for 10 min, 5-60% buffer B (25 mM NaH 2 PO 4 , 1 M KCl in 25% ACN, pH 2.7) for 27 min, and 60-100% buffer B for 1 min at a flow rate of 1 mL/min. Elution was monitored by measuring absorbance at 214 nm, and fractions collected every 1 min. A total of 20 fractions were desalted with a Strata X C18 column and vacuum-dried.

Each fraction was resuspended in buffer A (5% ACN, 0.1% formic acid) at a final concentration of 0.5 g/L. Aliquots (10 L) of supernatant were loaded on a LC-20AD nanoH-PLC (Shimadzu, Japan) using the autosampler onto a 2 cm C18 trap column. Peptides were eluted onto a 10 cm analytical C18 column (inner diameter, 75 m) packed in-house. Samples were loaded at 8 L/min for 4 min and a 35 min gradient run at 300 nL/min, starting from 2 to 35% B (95% ACN, 0.1% FA), followed by a 5 min linear gradient to 60%, 2 min linear gradient to 80% and maintenance at 80% B for 4 min, returning to 5% in 1 min.

Data acquisition was performed with a TripleTOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA, USA). Data were acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150ЊC. The MS was operated with a RP of greater than or equal to 30 000 FWHM for TOF MS scans. For IDA, survey scans were acquired in 250 ms, and as many as 30 product ion scans collected if exceeding a threshold of 120 counts per second (counts/s) and with a 2+ to 5+ charge state. The total cycle time was fixed to 3.3 s. The Q2 transmission window was 100 Da for 100%. Four time bins were summed for each scan at a pulser frequency value of 11 kHz through monitoring of the 40 GHz multichannel TDC detector with four-anode/ channel detection. The sweeping collision energy was set to 35 ± 5 eV, and dynamic exclusion was set for 1/2 of peak width (15 s).

Acquired MS raw data files were converted into MGF files using Proteome Discoverer 1. [34] . For protein identification, mass tolerance of 0.05 Da (ppm) was permitted for intact peptide masses and 0.1 Da for fragmented ions, with allowance for one missed cleavage in the trypsin digests. Gln→pyro-Glu (N-term Q), Oxidation (M), Deamidated (NQ) were the potential variable modifications, and Carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K) were fixed modifications. The charge states of peptides were set to +2 and +3. Specifically, an automatic decoy database search was performed in MASCOT by choosing the decoy checkbox in which a random sequence of database is generated and tested for raw spectra as well as the real database. To reduce the probability of false peptide identification, only peptides at the 95% confidence interval (above the "identity" threshold) defined by MASCOT probability analysis were counted as identified. Each confident protein identification involved at least one unique peptide. For quantitation, a protein was required to contain at least two unique spectra. The quantitative protein ratios were weighted and normalized by the median ratio in MASCOT. We specifically used ratios with p-values < 0.05, and only fold changes >1.5 or <0.667 were considered significant.

To verify the differentially expressed proteins identified using iTRAQ at the transcriptional level, ST cells infected with BTV-1 or mock-infected with culture medium were harvested at 24 and 48 hpi, and cellular total RNA extracted with TRIzol (Invitrogen, USA). After treatment with RNase-free DNase, 4 g of each total RNA was reverse-transcribed with Primer-Script RT Enzyme Mix I, Oligo dT(18) primer and random primer (TaKaRa, Japan). Real-time PCR was performed on Mx3500p (Agilent Technologies, Germany) using a SYBR R Premix Ex Taq TM kit (TaKaRa, Japan) following the manufacturer's instructions. The primers used for amplifying cDNA of CASP8, DDX58, eIF4a1, HSP27, IFIH1, IFIT3, ISG20, OAS1, RSAD2, STAT1 and ␤-actin are presented in Table 1 . Sheep ␤-actin was selected as the internal reference gene. Real-time RT-PCR for each gene was performed in triplicate. The relative expression level of each target gene in infected samples was calculated using 2 -⌬⌬CT , representing n-fold change relative to the mock-infected sample [35] .

Cell lysates of BTV-1-infected and mock-infected cultures were harvested at 24 and 48 hpi, and protein concentrations were determined. Equivalent amounts of cell lysates from the three replicates were denatured in 1×protein loading buffer by heating at 100ЊC for 5 min and separated via 12% SDS-PAGE. Protein bands in each gel were electrophoretically transferred onto PVDF membranes (Millipore, USA). Membranes were blocked with 5% fat-free milk and 0.5% Tween-20 in PBS for 1 h at room temperature, and incubated with rabbit polyclonal antibodies against HSP27, ␤-actin, STAT1, and eIF4a1 (Bioworld, USA) at 4ЊC overnight. After washing three times with PBS containing Tween-20, membranes were incubated with alkaline phosphatase-conjugated secondary antibodies (Sigma, USA) at room temperature for 1 h. Protein bands were detected using BCIP/NBT (Sigma, USA), and the intensity of the bands was measured using ImageJ software.

The differentially expressed proteins identified in this study were converted to human orthologous proteins, and the lists submitted to the database for annotation, visualization, integrated discovery (DAVID) online server (http://david.abcc.ncifcrf.gov) and UniProt databases. Categories belonging to biological processes (GO-BP), molecular functions (GO-MF), and cellular components (GO-CC) identified at a confidence level of 95% were included in the analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to classify and group the identified proteins. The protein-protein interaction network was analyzed using the STRING 10 database (http://string.embl.de/).

To determine the morphology of ST cells and the optimal time-point for proteomic analysis following BTV infection, the growth kinetics of BTV replication in primary ST cells was determined by monitoring CPE and viral titers at 0, 12, 24, 48 and 72 hpi. Compared to mock-infected cells, CPE was minimal at 24 hpi and significant at 48 and 72 hpi, characterized by rounding, swelling, granular degeneration and 

detachment of cells. At 72 hpi, almost all the cells had detached (Fig. 1A) . The one-step growth curve revealed that viral titer reached 5.1 × 10 7 PFU/mL at 48 hpi, followed by a gradual decline (Fig. 1B) . Generally, the time-point at which viral replication remains high with no significant host cell cytoskeleton or membrane rearrangement is taken as the optimal window for proteomic analysis. To ensure a higher percentage of infected cells and avoid excessive CPE, BTV-and mock-infected cells were harvested at 24 and 48 hpi for further proteomic analysis. Virus infection at different time-points was additionally confirmed via RT-PCR using a total of 23 cycles. Abundance of viral S7 RNAs increased gradually over time until 48 hpi (Fig. 1C) . Sequencing analysis of PCR products further validated BTV infection (data not shown).

The host response to BTV-1 infection at 24 and 48 hpi was analyzed by examining differences in protein expression. To identify the proteins differentially expressed between BTVinfected and mock-infected ST cells, we conducted iTRAQbased quantitative proteomic analyses. iTRAQ-labeled samples were subsequently analyzed via LC−MS/MS. Using this approach, a total of 17 471 peptides and 4455 proteins were detected and quantified, among which 101 and 479 proteins displayed significant differences in expression between BTVand mock-infected cells at 24 and 48 hpi, respectively, identified by at least two high confidence score (95%) peptides with p-values <0.05, calculated using ProQUANT. At 24 hpi, 83 and 18 proteins were significantly up-and down-regulated (Table 2) , while 345 and 134 proteins were significantly upand down-regulated, respectively, at 48 hpi (Supporting Information Table S1 ), according to the criteria (p-values < 0.05, and fold changes >1.5 or <0.667). Furthermore, 48 proteins (43 upregulated and five downregulated) were significantly dysregulated between the two time-points. We observed a greater number of significantly expressed immune-related proteins at 24 than 48 hpi. In view of the growing research interest on antiviral proteins, here we focused on proteins that were differentially expressed during the earlier stages of infection. Figure 2 depicts the correlation of uninfected ST cells between the two time-points (24 and 48 hpi). Proteins showing lower than 50% variation accounted for 94% of the total proteins, indicating significant correlation. Since the current sheep genome database is poorly annotated compared to the human genome database, 55 proteins remained unassigned or uncharacterized, resulting in a large number of predicted proteins in our analysis. Further research focusing on the functions of these proteins is therefore warranted.

To validate the differentially expressed proteins identified via iTRAQ-labeled LC-MS/MS analysis, transcriptional profiles of ten selected cellular proteins of ST cells with and without BTV infection were analyzed at 24 and 48 hpi. Real-time RT-PCR results showed that the at 24 hpi, mRNA levels of CASP8, DDX58 (also known as RIG-I), eIF4a1, HSP27, IFIH1 (also known as MDA5), IFIT3, ISG20, OAS1, RSAD2 (also known as viperin), and STAT1 were increased by 3. shown in Fig. 3B , the three representative proteins were upregulated in BTV-infected ST cells at 24 and 48 hpi, in keeping with the alterations in protein expression during BTV infection identified using iTRAQ analysis (Fig. 3C ).

To annotate the potential biological functions of the 101 differentially regulated proteins upon BTV infection, proteins were analyzed via gene ontology (GO) enrichment using the online DAVID and UniProt databases. Three major annotation types were obtained from the GO consortium website: cellular components, molecular functions, and biological processes. The cellular component annotation (GO-CC) revealed that the differentially expressed proteins were well distributed within different cell components, with intrinsic to membrane and organelle lumen being two highly distributed components (12 and 11 proteins, respectively) (Fig. 4A) . The molecular function (GO-MF) annotation demonstrated that proteins related to cation binding, nucleotide binding and enzyme binding were most commonly affected by viral infection (Fig. 4B ). The biological process (GO-BP) annotation revealed that differentially regulated proteins are involved in nucleocytoplasmic transport, immune response, signal transduction, transcription, apoptosis, stress response, and antigen processing and presentation (Fig. 4C) . To further investigate the pathway involvement of these proteins, KEGG pathway analysis based on the DAVID program was performed. Although most of the grouped pathways of the KEGG analysis were not statistically significant, these data sorted and grouped differentially expressed proteins based on function. According to the results, differentially expressed proteins were mainly involved in the RIG-I-like receptor signaling pathway and endocytosis (Fig. 4D ).

To clarify the mechanisms underlying BTV interactions with proteins of ST cells and consequent effects on cell function, 101 differentially expressed proteins were further analyzed by searching the STRING database and protein-protein interaction networks. As demonstrated in Fig. 5 , we identified two groups of strongly interacting proteins that were significantly regulated by BTV, including the ISG family regulated by IFN, STAT1-ISG15-ISG20-OAS1-OAS2-Mx1-Mx2-DDX58-IFIT1-IFIT2-IFIT3-IFIT5-IFI44-IFIH1-ADAR-RSAD2-HERC6-PML, and nuclear pore complex (NPC) proteins, RanBP2-Nup5-IPO4-IPO7-XPO5. These seed proteins have important functions in innate immune response and nuclear pore transport. For instance, IFIH1 (MDA5) is an innate immune receptor that acts as a cytoplasmic sensor of viral nucleic acids with a major role in sensing viral infection and activation of a cascade of antiviral responses, including induction of type I interferons and proinflammatory cytokines [36] . RanBP2 (originally named Nup358) is a large cyclophilin-related nuclear pore protein involved in the Ran-GTPase cycle that orchestrates the majority of nuclear import and export and also required for nuclear import or viral DNA integration of human immunodeficiency virus [37] . These findings suggest that entirely different sets of host proteins, interactions and processes, including the immune response, are perturbed during BTV infection. The interaction networks that appear to be regulated by BTV should provide clues for further clarification of the impact of viral infection on the physiological functions of target cells and host response.

Virus-cell interactions are highly complex, and gene expression, signaling and immune response pathways are commonly altered during viral infection [17, 18] . To date, no research has focused on differential proteome analysis of host cells in response to BTV infection. In the present study, iTRAQ coupled with LC-MS/MS was applied to analyze the differential proteome of ST cells infected with BTV. We obtained relative quantitative information for 4455 proteins in BTV-and mock-infected ST cells. At 24 hpi, the expression patterns of 101 (83 upregulated and 18 downregulated) Figure 5 . Interaction network of differentially expressed proteins generated using the STRING database. Network analysis was set at medium confidence (STRING score = 0.4). The edges represent predicted functional associations. An edge was drawn with up to seven different colored lines representing the existence of seven types of evidence used in predicting the associations. The red line indicates the presence of fusion evidence, the green line neighborhood evidence, the blue line co-occurrence evidence, the purple line experimental evidence, the yellow line textmining evidence, the light-blue line database evidence, and the black line co-expression evidence. different host proteins were significantly changed while 479 proteins (345 upregulated and 134 downregulated) displayed significant alterations in expression at 48 hpi in BTV-infected cells, relative to mock-infected cells. Our data revealed a larger proteomic shift at 48 hpi, compared with 24 hpi, in BTV-infected ST cells. However, a greater number of innate immune proteins were differentially expressed at 24 than 48 hpi, and we identified numerous proteins related to the cell cycle, ribosome and chromosome maintenance at 48 hpi, indicating that cell morphological changes at the later stages of infection initiate the mechanism of cell maintenance. With antiviral response in mind, we focused on the differentially expressed proteins during the early stage of infection (24 hpi) . Differential expression of three representative proteins, eIF4a1, STAT1 and HSP27, was validated via western blot. Since no specific antibodies against ovine CASP8, DDX58, IFIH1, IFIT3, ISG20, OAS1, and RSAD2 are available and no cross-reaction was detected with antibodies against the homologous human proteins (data not shown), real-time RT-PCR was used to analyze expression patterns of the ten selected proteins. Our data provide a comprehensive insight into the cellular response to BTV infection and the mechanisms involved in viral pathogenesis.

Since BTV infection usually causes severe symptoms in sheep, sheep primary testis cells were used to investigate the host response during BTV infection. Prior to proteomic analysis, we determined which time-points to investigate following infection by observing morphological changes and analyzing viral growth dynamics in BTV-infected cells. The results indicate that BTV induces CPE from 24 to 72 hpi in infected cells, compared to mock-infected cells. Production of mature particles was exponential at 8 and 24 h post-infection [38] . Furthermore, virus titer was continuously increased in infected cells until 48 h, at which time we observed the highest viral load. At 72 hpi, all infected cells showed rounding and granular degeneration. BTV infection can terminate host protein expression in the late stage. Thus, to avoid detecting changes in host protein expression caused by interference with translation, proteomic analyses were performed on cells at early infection time-points (24 and 48 hpi). Some viruses, if passaged at MOI>1, accumulate spontaneously deleted defective particles that are maintained during passage by the presence of complementing wild-type helper virus [39] . For proteome profiling, although most cells could be infected under high MOI (such as MOI of 1), the levels of most host proteins would be nonspecifically reduced by translational repression or strongly affected by BTV-induced apoptosis. To avoid the changes in protein expression profiles masked by BTV-induced translational shutoff and apoptotic effects, ST cells were infected with BTV at a MOI of 0.1, rather than higher MOI. Therefore, in this study, we examined differentially expressed proteins regulated by BTV infection at MOI of 0.1 at 24 and 48 hpi.

The innate immune response is the first line of defence against viruses, involving production of type I IFN (IFN-␣/␤) and other pro-inflammatory cytokines that control infection. It also shapes the adaptive immune response generated by both T and B cells. Production of IFN in response to virus infection is triggered by the recognition of pathogen-associated molecular patterns by the infected cell. Activation of innate cellular responses during viral infection requires recognition of pathogen-associated molecular patterns by pattern recognition receptors [36, 40, 41] . The genome of BTV, composed of dsRNA molecules, has been described as a potent inducer of type I IFN, both in vivo and in vitro. Recent studies have shown that BTV triggers type I IFN production in plasmacytoid dendritic cells via a MYD88-dependent/TLR7/8independent sensing and signaling pathway whereas cytoplasmic helicases (RIG-1, MDA5) mediate sensing and signaling in BTV-infected epithelial cells [42, 43] . However, pattern recognition receptor signaling pathways and the mechanisms responsible for production of IFN in response to BTV are still not completely understood. Type I IFN binds to a common IFN-␣/␤ receptor (IFNAR), initiating a signaling cascade that results in expression of hundreds of interferonstimulated genes (ISG). ISGs are key components of the host innate immune response and serve as the first line of defense against viral infection. In our study, 18 upregulated ISG proteins were identified. Among these, interferon-inducible ISG15, ISG20, Mx1, Mx2, RSAD2, OAS1 and OAS2, have been documented as critical antiviral proteins in cells and animals [44, 45] . IFIT proteins have also been shown to inhibit virus replication by binding and regulating the functions of cellular and viral proteins and RNAs [46] . DDX58 (RIG-1) plays an important role in the recognition of RNA viruses in various cells, and has been identified as a candidate cytoplasmic viral dsRNA receptor [47] . In the current study, we demonstrated that BTV infection induces overexpression of a number of antiviral proteins, several of which were identified for the first time, including ISG15, ISG20, Mx1, Mx2, RSAD2, OAS1, OAS2, IFI44 and IFIT [36, 48] .

We additionally confirmed upregulation of STAT1, HSP27 and eIF4a1 via qRT-PCR and western blot analyses. Enhanced expression of STAT1, a key regulator of interferon-responsive genes [49] , during BTV infection suggests that the virus activates canonical interferon signaling through the JAK-STAT1 pathway. HSP, also known as stress proteins, are often involved in antigen presentation and intracellular trafficking and apoptosis, and act as molecular chaperones by helping nascent polypeptides assume their proper conformations. HSP27 is linked to different signaling pathways that regulate cellular functions, such as inflammation, apoptosis, development, differentiation and cell growth. In addition, many other viruses, including avian influenza H9N2 [50] , infectious bursal disease virus [21] , and African swine fever virus [51] , induce upregulation of HSP27. eIF4A1 was initially characterized based on its requirement in translation and later identified as a component of the eIF4F translation initiation complex responsible for RNA helicase activity [52] . Among the virus-host interactions, those that recruit cellular translational machinery to viral mRNAs play a decisive role in viral replication. Viral RNAs have evolved structures to maximize their translation and efficiently compete with cellular mRNAs [53, 54] . The issue of whether upregulation of eIF4A1 expression in BTV-infected ST cells is a contributory step in virus replication will be investigated in future studies.

The ubiquitin-proteasome system (UPS), a major intracellular degradation pathway for foreign proteins, plays a critical role in a variety of cellular functions, such as antigen processing, cell cycle regulation, apoptosis, signal transduction, transcriptional regulation, and DNA repair. Ubiquitination is a crucial cellular event in pathways for type I IFN activation [55, 56] . In addition to activation, signaling molecules in these pathways may be ubiquitinated for proteasomal degradation, consequently downregulating the IFN response. Viruses have evolved different strategies to utilize the UPS to their advantage. For example, activation of the UPS pathway is necessary for hepatitis E virus replication [57] , and also required by other viruses, such as influenza virus [58] , vaccinia virus [59] , porcine reproductive and respiratory syndrome virus [60] and rotavirus [61] . In our study, nine UPS proteins, including HERC-6, UBE2N, ISG15, VPRBP, OTUB1, NUB1, RANBP2, PSMD5, and PSME2, were differentially upregulated at 24 hpi, indicating a relatively significant influence of the virus on the UPS pathway. The finding that the cellular UPS pathway is disrupted upon BTV infection supports the potential utility of an anti-UPS-based strategy in preventing infection. However, the particular UPS pathway employed by BTV to evade host immune surveillance remains to be established.

NPCs are embedded in pores of the nuclear envelope and constitute large aqueous transport channels that mediate and regulate the bidirectional exchange of macromolecules between the nucleus and cytoplasm [37, 62] . Our iTRAQ analysis for BTV-infected ST cells revealed that many proteins involved in intracellular protein transport are significantly upregulated during BTV infection, including components of the NPC, such as RanBP2, Nup54, IPO4, IPO7 and XPO5, suggesting that infection by BTV enhances molecular transport between the nucleolus and cytoplasm and biological functions are possibly activated in the latter. Previous studies disclosed that NS4 of BTV starts to accumulate and shuttle between the nucleolus and cytoplasm as early as 4 hours postinfection and possibly interacts with the IFN pathway [6, 7] . Further research is needed to determine whether the upregulation of nuclear pore proteins is associated with trafficking of NS4 between the nucleolus and cytoplasm.

Apoptosis of host cells plays a major role in regulating the pathogenesis of many infectious diseases. Apoptosis triggered by virus infection directly leads to viral pathogenesis. However, blocking apoptosis can avoid premature death of infected cells, permitting a high titer of virus replication or persistent infection [63] . In our analysis, four apoptosisrelated genes (Casp8, K1967, PML and CIKS) were identified following BTV infection. Caspase-8 is an initiator caspase activated by interactions between external apoptotic elements and cell surface molecules. BTV infection led to significant upregulation of caspase-8 in virus-infected ST cells, as verified with qRT-PCR. Regulation of cell death is known to be important for replication and pathogenesis in various Orbiviruses [64] [65] [66] , and therefore, we propose that further investigation of these proteins should facilitate clarification of the mechanisms underlying cell death regulation during BTV infection.

In conclusion, the current study has provided a global overview of the protein alterations in BTV-infected ST cells. The identification of significantly altered proteins in BTVinfected ST cells reflects a comprehensive BTV-host cell interaction network. To our knowledge, many of the immune response-related proteins differentially expressed upon BTV infection are novel and have not been detected in previous studies [36, 48] , thus providing new protein targets for evaluation in the future. Elucidation of the functions of these proteins in virus-host cell interactions may additionally uncover new therapeutic strategies targeting BTV.

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Animal models of highly pathogenic RNA viral infections: hemorrhagic fever viruses

Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparison to other European strains

Recombinant vaccines against bluetongue virus

Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8

Performance of clinical signs to detect bluetongue virus serotype 8 outbreaks in cattle and sheep during the 2006-epidemic in The Netherlands

The molecular biology of Bluetongue virus replication

Trafficking of bluetongue virus visualized by recovery of tetracysteinetagged virion particles

Virus-host interactomes and global models of virus-infected cells

New horizons for antiviral drug discovery from virus-host protein interaction networks

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses

Proteomic alteration of PK-15 cells after infection by classical swine fever virus

Proteomics analysis of host cells infected with infectious bursal disease virus

Plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry

Differential proteome analysis of host cells infected with porcine circovirus type 2

Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus type 2 infected PK-15 cells

Quantitative proteomics using SILAC coupled to LC-MS/MS reveals changes in the nucleolar proteome in influenza A virus-infected cells

Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals changes in the cytoplasmic, nuclear, and nucleolar proteomes in Vero cells infected with the coronavirus infectious bronchitis virus

Identification of host proteins involved in Japanese encephalitis virus infection by quantitative proteomics analysis

HIV induces both a down-regulation of IRAK-4 that impairs TLR signalling and an up-regulation of the antibiotic peptide dermcidin in monocytic cells

Quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the JAK-STAT1 signaling pathway

Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine reproductive and respiratory syndrome virus

Two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2

Proteome analysis of porcine epidemic diarrhea virus (PEDV)-infected Vero cells

Proteomic alteration of equine monocyte-derived macrophages infected with equine infectious anemia virus

The sheep genome illuminates biology of the rumen and lipid metabolism

Analyzing real-time PCR data by the comparative C(T) method

Induction and control of the type I interferon pathway by Bluetongue virus

Global analysis of host-pathogen interactions that regulate earlystage HIV-1 replication

Bluetongue virus: virology, pathogenesis and immunity

Incomplete forms of influenza virus

Pathogen recognition and innate immunity

RNA recognition and signal transduction by RIG-I-like receptors

NS3 of bluetongue virus interferes with the induction of type I interferon

The double-stranded RNA bluetongue virus induces type I interferon in plasmacytoid dendritic cells via a MYD88-dependent TLR7/8-independent signaling pathway

A diverse range of gene products are effectors of the type I interferon antiviral response

Interferon-inducible antiviral effectors

Interferon-induced Ifit proteins: their role in viral pathogenesis

Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses

The immune response of ruminant livestock to bluetongue virus: from type I interferon to antibody

Regulation of JAK-STAT signalling in the immune system

Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells

Identification of cellular proteins modified in response to African swine fever virus infection by proteomics

The diverse roles of the eIF4A family: you are the company you keep

Requirements for eIF4A and eIF2 during translation of Sindbis virus subgenomic mRNA in vertebrate and invertebrate host cells

Active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein

The ubiquitin-proteasome pathway in viral infections

The ubiquitinproteasome system in positive-strand RNA virus infection

Hepatitis E virus replication requires an active ubiquitin-proteasome system

Inhibition of the ubiquitin-proteasome system affects influenza A virus infection at a postfusion step

Inhibition of the ubiquitin-proteasome system prevents vaccinia virus DNA replication and expression of intermediate and late genes

Identification of differentially expressed proteins in porcine alveolar macrophages infected with virulent/attenuated strains of porcine reproductive and respiratory syndrome virus

Replication of the rotavirus genome requires an active ubiquitinproteasome system

Towards understanding nuclear pore complex architecture and dynamics in the age of integrative structural analysis

Viral apoptotic mimicry

Role of cellular caspases, nuclear factor-kappa B and interferon regulatory factors in Bluetongue virus infection and cell fate

Bluetongue virus induces apoptosis in cultured mammalian cells by both caspase-dependent extrinsic and intrinsic apoptotic pathways

Epizootic hemorrhagic disease virus induces and benefits from cell stress, autophagy, and apoptosis