Article pubs.acs.org/jpr
Proteomics Analysis of EV71-Infected Cells Reveals the Involvement of Host Protein NEDD4L in EV71 Replication Rei-Lin Kuo,*,†,‡ Ya-Han Lin,†,‡ Robert Yung-Liang Wang,‡,§ Chia-Wei Hsu,∥ Yi-Ting Chiu,† Hsing-I Huang,†,‡ Li-Ting Kao,†,‡ Jau-Song Yu,∥ Shin-Ru Shih,†,‡,⊥ and Chih-Ching Wu*,†,∥ †
Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan ‡ Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan § Department of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan 333, Taiwan ∥ Molecular Medicine Research Center, Chang Gung University, Tao-Yuan 333, Taiwan ⊥ Clinical Virology Laboratory, Linkou Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan S Supporting Information *
ABSTRACT: Enterovirus 71 (EV71) is a human enterovirus that has seriously affected the Asia-Pacific area for the past two decades. EV71 infection can result in mild hand-foot-and-mouth disease and herpangina and may occasionally lead to severe neurological complications in children. However, the specific biological processes that become altered during EV71 infection remain unclear. To further explore host responses upon EV71 infection, we identified proteins differentially expressed in EV71-infected human glioblastoma SF268 cells using isobaric mass tag (iTRAQ) labeling coupled with multidimensional liquid chromatography−mass spectrometry (LC−MS/MS). Network analysis of proteins altered in cells infected with EV71 revealed that the changed biological processes are related to protein and ion transport, regulation of protein degradation, and homeostatic processes. We confirmed that the levels of NEDD4L and PSMF1 were increased and reduced, respectively, in EV71-infected cells compared to mock-infected control cells. To determine the physiological relevance of our findings, we investigated the consequences of EV71 infection in cells with NEDD4L or PSMF1 depletion. We found that the depletion of NEDD4L significantly reduced the replication of EV71, whereas PSMF1 knockdown enhanced EV71 replication. Collectively, our findings provide the first evidence of proteome-wide dysregulation by EV71 infection and suggest a novel role for the host protein NEDD4L in the replication of this virus. KEYWORDS: enterovirus 71, NEDD4L, PSMF1, IFN-β, iTRAQ
■
INTRODUCTION Enterovirus 71 (EV71) is one of the human enteroviruses that has emerged in the Asia-Pacific region in recent years. Infection with EV71 can cause mild hand-foot-and-mouth disease and herpangina and may occasionally result in severe complications that impair the function of the central nervous system in children, including aseptic meningitis, encephalitis, and neurogenic pulmonary edema.1 EV71 belongs to the family of picornaviruses, which have a single-stranded RNA genome with positive polarity. The positive-sensed viral genome serves as viral mRNA for translating a precursor polyprotein that can be processed into functional structural and nonstructural viral proteins by virus-encoded proteases 2A and 3C. The genomic EV71 RNA is also the template for the generation of viral complementary RNA, which can be further replicated to © 2015 American Chemical Society
produce genomic RNA by viral RNA-dependent RNA polymerase 3D in the cytoplasm.2 Because EV71 can be highly pathogenic in humans, it is crucial to investigate cellular responses upon EV71 infection. Induction of type I interferons (IFNs), IFN-α/β, is crucial to innate immunity against EV71 infection, and cytoplasmic melanoma differentiation-associated gene 5 (MDA5) is a critical pathogen sensor for activating type I IFN expression in response to EV71 infection. Previous studies have shown that EV71 infection can cause MDA5 degradation via caspase activation or EV71 protease 2A.3,4 However, several other mechanisms, including the degradation of the mitochondrial Received: November 20, 2014 Published: March 18, 2015 1818
DOI: 10.1021/pr501199h J. Proteome Res. 2015, 14, 1818−1830
Article
Journal of Proteome Research
Eagle’s medium (DMEM, Life Technologies, Inc. [Gibco], Taipei, Taiwan) containing 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin− L-glutamate (PSG) mixture (Gibco). The EV71 strains, TW2231/98 (obtained from the Clinical Virology Laboratory, Department of Pathology, Linkou Chang Gung Memorial Hospital, Taiwan) and MP4 (kindly provided by Dr. Jen-Ren Wang at the National Cheng-Kung University, Taiwan), were amplified in Vero cells that were cultured in DMEM containing 2% FBS. The titer of EV71 was determined using a plaque formation assay on a monolayer of RD cells, denoted as plaque formation units per milliliter (PFU/mL).
antiviral signaling (MAVS) protein, inhibition of retinoic acidinducible gene I (RIG-I) activation, and cleavage of interferon regulatory factor 7 (IRF7), have been identified as events regulating the type I IFN response during EV71 infection.5−7 Moreover, it has been reported that EV71 infection can alter host protein expression through the cleavage of the translational initiation factor eIF4G and polyadenylation factor CstF64 and through interaction with the splicing factor Prp8.8−10 These results suggest that characterizing the cellular response to viral infection requires further investigation of the biological processes that are affected by EV71 infection. Since the determination of host responses that are induced directly or indirectly by virus infection is extremely important in understanding of virus-mediated pathogenesis, proteomic approaches have been widely applied for exploring global host protein expression during viral infection.11,12 Several studies have utilized these strategies to demonstrate changes in host proteins during EV71 infection. The molecular changes in EV71-infected RD cells were probed by 2D-PAGE and MALDI-TOF mass spectrometry analysis,13 an analysis that highlighted, but did not validate, differences in the expression of five cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2, and STMN1) following EV71 infection.13 The cellular response of host cells infected with EV71 and Coxsackievirus A16 (CA16) was also characterized using the same gel-based strategies,14 with a total of 16 proteins identified as downregulated in EV71-infected RD cells, which suggests that EV71 rapidly shuts down the host translation machinery. These findings provide insight into the nature of the highly virulent EV71 infection compared to CA16 infection.14 In these studies, the comparative proteome profiling was conducted using twodimensional gels followed by in-gel tryptic digestion and analysis via MALDI-TOF MS. Alternatively, the proteome could be analyzed with an in-solution trypsin digestion combined with LC−MS/MS, a technique that generally resolves many more proteins than MALDI-TOF MS.15,16 Indeed, advances in protein separation and identification technologies have made it possible to identify more proteins, which thus facilitates the discovery of dysregulated proteins in EV71-infected cells. In the present study, we investigated the host response by profiling the changes in cellular protein expression in EV71infected cells using isobaric tags for relative and absolute quantification (iTRAQ) combined with LC−MS/MS. We discovered a panel of dysregulated proteins in EV71-infected cells that participate in protein transport, homeostatic processes, and protein metabolic processes. Among the proteins identified, the E3 ubiquitin−protein ligase NEDD4like (NEDD4L) and the proteasome inhibitor PI31 subunit (PSMF1) are upregulated and downregulated, respectively, upon EV71 infection and were validated by Western blotting. Furthermore, the roles of these two proteins in EV71 replication were verified using gene knockdown experiments.
■
EV71 Infection
The procedure of EV71 infection was described previously.4 Briefly, cells were seeded and cultured overnight in 100 mm or six-well plates. The multiplicity of infection (MOI) was determined by dividing PFU (titrated in RD cells) by the cell number. After the treatments indicated below, viruses were inoculated into the treated cells at the indicated MOI. After 1 h of adsorption, the infected cells were maintained in DMEM with 2% FBS and 1% PSG. Immunoblotting, Immunofluorescence, and Antibodies
Total cellular proteins were extracted, separated by SDS-PAGE, and transferred to PVDF membranes as described previously.4 The membranes were probed individually with anti-EV71 3C,17 anti-EV71 3D,8 anti-NEDD4L (Cat. No. A302−512A, Bethyl Laboratories, Montgomery, TX, USA), anti-PSMF1 (Cat. No. GTX-104760, GeneTex, Irvine, CA, USA), anti-c-Myc (Clone 9E10, Cat. No. M4439, Sigma-Aldrich, St. Louis, MO, USA), anti-FLAG (Clone M2, Cat. No. F1804, Sigma-Aldrich), and anti-β-actin (Clone AC-15, Cat. No. A5441, Sigma-Aldrich) primary antibodies. The membranes were then incubated with HRP-conjugated secondary antibodies (GE Healthcare, Taipei, Taiwan) and then with a chemiluminescent HRP substrate (Merck Millipore, Taipei, Taiwan) for detection. For detecting EV71 infection by immunofluorescence, cells cultivated on glass slides were fixed with 3% paraformaldehyde, treated with 0.2% Triton X-100, and then blocked with 5% bovine serum albumin for 30 min. The treated cells were incubated with anti-EV71 monoclonal antibody (Clone 422−8D-4C-4D, Cat. No. MAB979, Millipore, USA) at 37 °C for 30 min and then with antimouse IgG conjugated with Alexa Fluor 488 dye (Life Technologies, USA). After they were washed with phosphatebuffered saline (PBS), the cells were mounted and observed under fluorescence microscopy. Preparation of Cell Extracts and Digestion of Protein Mixtures for Proteome Analysis
SF268 cells were lysed in buffer containing 100 mM triethylammonium bicarbonate (TEABC, Sigma-Aldrich) and 0.1% RapiGest SF (Waters Corporation, Milford, MA, USA) on ice for 15 min. The cell lysate was collected and sonicated on ice, followed by centrifugation at 10 000g for 25 min at 4 °C. The resulting supernatant was used as the cell extract. For tryptic in-solution digestion, protein mixtures were denatured with 8 M urea containing 50 mM TEABC, reduced with 10 mM tris(2-carboxyethyl)-phosphine (TCEP, Sigma-Aldrich) at 37 °C for 90 min, and then alkylated with 10 mM methylmethanethiosulfonate (MMTS, Sigma-Aldrich) at room temperature for 20 min. After desalting, the protein mixtures were in-solution digested with modified, sequencing-grade trypsin (Promega, Madison, WI, USA) at 37 °C overnight.
MATERIALS AND METHODS
Cells and Viruses
Vero (ATCC, CCL81), RD (human rhabdomyosarcoma cell line, ATCC, CCL-136), SF268 (human glioblastoma cell line, provided by Dr. Jim-Tong Horng at Chang Gung University, Taiwan), and HeLa (provided by Dr. Szu-Hao Kung at National Yang-Ming University, Taiwan) cells were incubated at 37 °C with 5% CO2 and maintained in Dulbecco’s modified 1819
DOI: 10.1021/pr501199h J. Proteome Res. 2015, 14, 1818−1830
Article
Journal of Proteome Research iTRAQ Reagent Labeling and Fractionation by SCX Chromatography
analyses, respectively. Automatic gain control was used to prevent overfilling of the ion trap, and 5 × 104 ions were accumulated in the ion trap for generation of PQD spectra. For MS scans, the m/z scan range was 350−2000 Da.
The peptides were labeled with iTRAQ reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Briefly, one unit of label (defined as the amount of reagent required to label 100 μg of protein) was thawed and reconstituted in ethanol (70 μL). The peptide mixtures were reconstituted with 25 μL of iTRAQ dissolution buffer. The aliquots of iTRAQ 114 and 115 were combined with peptide mixtures from the mock-infected control cells and EV71-infected cells, respectively. For biological replication, the second set of peptide mixtures from the control cells and the EV71-infected cells were prepared in different batches and labeled with iTRAQ 116 and 117, respectively. After incubation at room temperature for 1 h, the peptide mixtures were then pooled and dried by vacuum centrifugation. The dried peptide mixtures were reconstituted with 0.5 mL of buffer A (10 mM NH4HCO3, pH 10) for fractionation by reversed-phase chromatography using the Ettan MDLC system (GE Healthcare). For peptide fractionation, the iTRAQ-labeled peptides were loaded onto a 4.6 mm × 100 mm Gemini column containing 3μm particles with 110-Å pore size (Phenomenex, Torrance, CA, USA). The peptides were eluted at a flow rate of 100 μL/min with a gradient of 2% buffer B (10 mM NH4HCO3 in acetonitrile, pH 10) for 5 min, 2−25% buffer B for 35 min, 25− 50% buffer B for 25 min, 50−75% buffer B for 10 min, 75− 100% buffer B for 2.5 min, and 100% buffer B for 7.5 min. The elution was monitored by absorbance at 220 nm, and fractions were collected every 1 min. Each fraction was vacuum-dried and resuspended in 0.1% formic acid (20 μL) for further desalting and concentration using a ziptip home-packed with C18 resin (5−20 μm, LiChroprep RP-18, Merck Millipore).
Sequence Database Searching and Quantitative Data Analysis
The data analysis was carried out using Proteome Discoverer software (version 1.3, Thermo Fisher Scientific) including the reporter ions quantifier node for iTRAQ quantification. The MS/MS spectra were searched against the Swiss-Prot human sequence database (released Jun 15, 2010, selected for Homo sapiens, 20367 entries) using the Mascot search engine (Matrix Science, London, UK; version 2.2.04). For protein identification, 10 ppm mass tolerance was permitted for intact peptide masses and 0.5 Da for PQD fragment ions, with allowance for two missed cleavages made from the trypsin digest: oxidized methionine (+16 Da) as a potential variable modification, and iTRAQ (N terminal, +144 Da), iTRAQ (K, +144 Da), and MMTS (C, +46 Da) as the fixed modifications. Data were then filtered based on medium confidence of peptide identification to ensure an overall false discovery rate below 0.01. The identification of epithelial keratins was excluded. Proteins with single peptide hits were removed, and quantitative data were exported from Proteome Discoverer and manually normalized such that the log2 of iTRAQ ratios displayed a median value of zero for all peptides in a given protein. This was performed across an entire labeling experiment to correct for variation in protein abundance. The cutoff value for determining whether a protein is considered dysregulated was selected according to the analysis using comparison of protein ratios between equal amounts of the same cell extract.18,20 On the basis of this, proteins with log2 ratios below the mean of all log2 ratios minus the standard deviation (SD) of all log2 ratios were considered to be underexpressed. Proteins above the mean plus one SD were considered to be overexpressed.
LC−ESI−MS/MS Analysis by LTQ-Orbitrap PQD
To analyze the iTRAQ-labeled peptide mixtures, each peptide fraction was reconstituted in buffer C (0.1% formic acid), loaded across a trap column (Zorbax 300SB-C18, 0.3 × 5 mm, Agilent Technologies, Wilmington, DE, USA) at a flow rate of 0.2 μL/min in buffer C, and separated on a resolving 10 cm analytical C18 column (inner diameter, 75 μm) with a 15-μm tip (New Objective, Woburn, MA, USA). The peptides were eluted using a linear gradient of 2−30% buffer D (acetonitrile containing 0.1% formic acid) for 63 min, 30−45% buffer D for 5 min, and 45−95% buffer D for 2 min with a flow rate of 0.25 μL/min. The LC apparatus was coupled with a two-dimensional linear ion trap mass spectrometer (LTQ-Orbitrap Discovery, Thermo Fisher Scientific, CA, USA) operated using the Xcalibur 2.0 software package (Thermo Fisher Scientific). Intact peptides were detected in the Orbitrap at a resolution of 30000. Internal calibration was performed using the ion signal of polycyclodimethylsiloxane at m/z 445.120025 as the lock-mass ion. Peptides were selected for MS/MS using the PQD operating mode with a normalized collision energy setting of 27%, and fragment ions were detected in the LTQ.18,19 The datadependent procedure that alternated between one MS scan followed by three MS/MS scans for the three most abundant precursor ions in the MS survey scan was applied. The m/z values selected for MS/MS were dynamically excluded for 180 s. The electrospray voltage applied was 1.8 kV. Both MS and MS/MS spectra were acquired using the four microscans with a maximum fill-time of 1000 and 100 ms for MS and MS/MS
Bioinformatics Analysis
Gene Ontology (GO) information for the differentially expressed proteins was acquired using the Web site UniProt (http://www.uniprot.org/). The Database for Annotation, Visualization, and Integrated Discovery (DAVID, v6.7, http://david.abcc.ncifcrf.gov/) was used to test for the enrichment of certain biological processes. Any GO biological process with a false-discovery rate (FDR) < 0.05 was considered to be significant in the enrichment analysis. Knockdown of NEDD4L and PSMF1, Virus Replication, and MTT Assay
To knockdown the expression of NEDD4L or PSMF1, small interfering RNA (siRNA) targeting NEDD4L (sequence: 5′CCUAAUUCAGGCCUCUGUA-3′ and 5′UACAGAGGCCUGAAUUAGG-3′) or PSMF1 (sequence: 5′-CCUGUAUGUCCUCCGGUAU-3′ and 5′AUACCGGAGGACAUACAGG-3′) was transfected into HeLa cells using Lipofectamine 2000 (Life Technologies, Inc. [Invitrogen], Taipei, Taiwan) for 24 h. To monitor EV71 replication, siRNA-transfected HeLa cells were infected with either the TW2231/98 or MP4 strain at a MOI of 0.001. At the indicated time points after infection, virus culture media and infected cells were collected and subjected to three cycles of freezing and thawing. The supernatant of the mixtures was collected after centrifuging and titrated using the plaque 1820
DOI: 10.1021/pr501199h J. Proteome Res. 2015, 14, 1818−1830
Article
Journal of Proteome Research
Figure 1. Identification of differentially expressed proteins in EV71-infected cells. (A) Cytopathic effect of EV71 infection. SF268 cells were infected with EV71 at a MOI of 2. At 12 h postinfection, the cytopathic effect was observed. (B) Cell extracts of SF268 cells in panel A were subjected to immunoblot analysis for detecting the expression of the EV71 viral proteins 3C and 3D. (C) Schematic diagram showing the workflow designed for profiling proteins affected by EV71 infection with iTRAQ-based analysis. The cell extracts were individually harvested from two mock-infected control (Mock) and two EV71-infected (EV71 infection) SF268 cell extracts. These protein extracts were trypsin-digested, and the resulting peptides from each of the four samples were labeled with the corresponding iTRAQ reporters in parallel. The iTRAQ-labeled peptides were then pooled and applied to strong cation exchange (SCX) chromatography for fractionation followed by reverse-phase liquid chromatography (RPLC) for further separation. The peptide identities and intensities were analyzed by LTQ-Orbitrap MS in PQD mode. Data analyses were performed with the Proteome Discoverer programs using the MASCOT algorithm as the search engine. As indicated, the EV71 infection experiment was conducted in duplicate (shown as Exp 1 and Exp 2). (D) Number of proteins identified as up-regulated or down-regulated in the two EV71 infection experiments. Venn diagrams show the overlap of proteins up-regulated or down-regulated in the two experiments. The total number of proteins up-regulated or down-regulated in each experiment is listed in brackets.
IFN-β Promoter and ISRE Reporter Assays
formation assay. The MTT assay was used to determine cell viability after the indicated treatments. MTT (Thiazolyl Blue Tetrazolium Blue, Sigma-Aldrich) was diluted in serum-free DMEM to the concentration of 1 mg/mL and added to cell culture washed with PBS. After 3 h of incubation at 37 °C, the MTT−medium mixture was removed from cell culture, and 0.04 N HCl in isopropanol was added to solubilize the converted dye. The absorbance of converted dye was determined by measuring at 570 nm, subtracting the background at 630 nm.
The 293T cells were transfected with a firefly luciferase reporter plasmid driven by the IFN-β promoter (provided by Dr. Michael Gale Jr.), a Renilla luciferase reporter plasmid (pRLTK, Promega), and either a Myc-tagged NEDD4L expressing plasmid, which contains the cDNA of NEDD4L isoform 2 in pCMV-Myc vector, or an empty vector. The IFN-β promoter was activated by cotransfection with a FLAG-tagged MDA5 (provided by Dr. Michael Gale Jr.) At 24 h post-transfection, 1821
DOI: 10.1021/pr501199h J. Proteome Res. 2015, 14, 1818−1830
Article
Journal of Proteome Research Table 1. List of Differentially Expressed Proteins in EV71-Infected SF268 Cells Identified Both in Exp 1 and Exp 2 Acc. No.
a
Q9Y512 O75955 Q16795 Q9H061 Q5SRE5 Q9H0H5 Q15758 P53007 Q92581 Q99447 Q6P1J9 O14497 P46977 O96008 Q86XP3 P56945 Q96S97 Q96A49 O75436 Q9H1I8 Q8N2K0 P48449 Q9NUQ8 Q9Y277 Q9BWM7 O95747 P35610 O14908 P49721 P11388 Q969H8 P21359 Q9NS86 Q92530 Q8TEX9 O75122 Q8IXI2 O95487 P46939 Q8TCT9 P52948 Q08AM6 Q9BV36 Q9HD20 O75153 Q96HC4
protein name (gene name)
sorting and assembly machinery component 50 homolog (SAMM50) flotillin-1 (FLOT1) NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial (NDUFA9) transmembrane protein 126A (TMEM126A) nucleoporin NUP188 homolog (NUP188) Rac GTPase-activating protein 1 (RACGAP1) neutral amino acid transporter B(0) (SLC1A5) tricarboxylate transport protein, mitochondrial (SLC25A1) sodium/hydrogen exchanger 6 (SLC9A6) ethanolamine-phosphate cytidylyltransferase (PCYT2) parafibromin (CDC73) AT-rich interactive domain-containing protein 1A (ARID1A) dolichyl-diphosphooligosaccharide−protein glycosyltransferase subunit STT3A (STT3A) mitochondrial import receptor subunit TOM40 homolog (TOMM40) ATP-dependent RNA helicase DDX42 (DDX42) breast cancer antiestrogen resistance protein 1 (BCAR1) myeloid-associated differentiation marker (MYADM) synapse-associated protein 1 (SYAP1) vacuolar protein sorting-associated protein 26A (VPS26A) activating signal cointegrator 1 complex subunit 2 (ASCC2) monoacylglycerol lipase ABHD12 (ABHD12) lanosterol synthase (LSS) ATP-binding cassette subfamily F member 3 (ABCF3) voltage-dependent anion-selective channel protein 3 (VDAC3) sideroflexin-3 (SFXN3) serine/threonine-protein kinase OSR1 (OXSR1) sterol O-acyltransferase 1 (SOAT1) PDZ domain-containing protein GIPC1 (GIPC1) proteasome subunit beta type-2 (PSMB2) DNA topoisomerase 2-alpha (TOP2A) UPF0556 protein C19orf10 (C19orf10) neurofibromin (NF1) LanC-like protein 2 (LANCL2) proteasome inhibitor PI31 subunit (PSMF1) importin-4 (IPO4) CLIP-associating protein 2 (CLASP2) mitochondrial Rho GTPase 1 (RHOT1) protein transport protein Sec24B (SEC24B) utrophin (UTRN) minor histocompatibility antigen H13 (HM13) nuclear pore complex protein Nup98−Nup96 (NUP98) protein VAC14 homolog (VAC14) melanophilin (MLPH) probable cation-transporting ATPase 13A1 (ATP13A1) protein KIAA0664 (KIAA0664) PDZ and LIM domain protein 5 (PDLIM5)
protein scoreb
no. of identified peptides
Down-Regulated Proteins 41.5 2
no. of matched spectra
percent coverage (%)
iTRAQ ratio (EV71 infection/Ctrl)
no. of spectra for quantification
Exp 1
Exp 2
Exp 1
Exp 2
3
2.99
0.788
0.239
3
3
77.0 347.4
2 2
3 6
7.26 8.49
0.551 0.729
0.491 0.392
2 3
2 2
203.6 228.9 94.7 279.5 429.8
2 4 3 2 3
6 9 4 6 15
18.46 2.23 5.38 4.62 9.97
0.789 0.800 0.844 0.777 0.784
0.391 0.380 0.354 0.427 0.436
2 4 4 6 15
2 3 4 6 15
209.5 72.8 364.6 532.5
2 2 2 4
4 4 13 8
3.74 9.51 5.65 3.54
0.745 0.723 0.649 0.576
0.507 0.531 0.606 0.701
4 4 9 2
4 4 10 2
228.0
2
6
4.54
0.716
0.579
3
3
72.8
2
4
4.16
0.808
0.496
4
4
194.4 57.0
2 2
4 2
4.05 3.91
0.814 0.704
0.492 0.615
2 2
3 2
389.8 484.3 107.8
2 3 2
12 14 3
10.25 13.64 6.73
0.711 0.612 0.692
0.619 0.721 0.641
6 2 3
6 4 2
64.4
2
3
4.49
0.828
0.511
2
2
91.4 232.0 192.4 247.7
2 3 2 2
4 6 7 8
4.27 4.51 2.96 8.13
0.795 0.839 0.717 0.739
0.560 0.520 0.647 0.644
4 6 7 3
4 6 7 3
312.5 233.9 82.3 414.0 220.3 537.8 126.7 98.3 40.7 84.9 1331.5 128.8 229.0 478.3 191.0 208.7 358.1
4 4 2 3 2 6 2 3 2 2 9 2 2 3 3 3 2
10 9 2 8 9 16 7 3 2 5 33 3 7 9 8 9 6
17.45 6.83 4.73 18.02 10.45 4.77 8.67 1.34 3.56 6.64 12.67 2.09 3.07 3.47 0.87 8.75 1.85
0.773 0.824 0.821 0.717 0.812 0.812 0.784 0.761 0.836 0.726 0.845 0.804 0.836 0.820 0.825 0.779 0.813
0.613 0.568 0.580 0.693 0.598 0.599 0.660 0.693 0.618 0.729 0.610 0.655 0.629 0.649 0.645 0.702 0.705
6 9 2 5 7 15 5 2 2 5 22 3 7 3 8 9 6
5 9 2 4 7 14 5 2 2 5 23 3 7 3 8 9 6
175.9 116.5 164.0
2 2 3
4 4 5
3.58 3.5 2.16
0.827 0.824 0.844
0.696 0.702 0.702
4 2 4
4 4 4
208.4 253.3
2 3
8 7
1.91 4.7
0.829 0.825
0.719 0.729
8 7
8 7
1822
DOI: 10.1021/pr501199h J. Proteome Res. 2015, 14, 1818−1830
Article
Journal of Proteome Research Table 1. continued Acc. No.
a
Q14934 O75718 Q9H1A4 Q03426 Q9H3F6 Q14195 Q96PU5 P50583 Q7L775 Q969T9 Q9H2G2 P82930 P46976 Q14156 O15498 Q01780 P35613 P49458 O76003 O00273 P49959 Q9BQE3 P20073 Q9UNH7 Q9Y2B0 P43307 O15013
protein name (gene name)
nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4) cartilage-associated protein (CRTAP) anaphase-promoting complex subunit 1 (ANAPC1) mevalonate kinase (MVK) BTB/POZ domain-containing protein KCTD10 (KCTD10) dihydropyrimidinase-related protein 3 (DPYSL3) E3 ubiquitin−protein ligase NEDD4-like (NEDD4L) bis(5′-nucleosyl)-tetraphosphatase [asymmetrical] (NUDT2) EPM2A-interacting protein 1 (EPM2AIP1) WW domain-binding protein 2 (WBP2) STE20-like serine/threonine-protein kinase (SLK) 28S ribosomal protein S34, mitochondrial (MRPS34) glycogenin-1 (GYG1) protein EFR3 homolog A (EFR3A) synaptobrevin homolog YKT6 (YKT6) exosome component 10 (EXOSC10) basigin (BSG) signal recognition particle 9 kDa protein (SRP9) glutaredoxin-3 (GLRX3) DNA fragmentation factor subunit alpha (DFFA) double-strand break repair protein MRE11A (MRE11A) tubulin alpha-1C chain (TUBA1C) annexin A7 (ANXA7) sorting nexin-6 (SNX6) protein canopy homolog 2 (CNPY2) translocon-associated protein subunit alpha (SSR1) Rho guanine nucleotide exchange factor 10 (ARHGEF10)
protein scoreb
no. of identified peptides
no. of matched spectra
Up-Regulated Proteins 42.6 2
percent coverage (%)
iTRAQ ratio (EV71 infection/Ctrl)
no. of spectra for quantification
Exp 1
Exp 2
Exp 1
Exp 2
4
1.44
1.463
6.194
4
4
41.2 53.5 224.9 35.3
2 2 2 2
3 4 3 4
3.49 0.77 10.61 4.79
2.099 2.045 2.110 1.257
4.585 1.834 1.685 2.393
3 4 3 4
3 4 3 4
160.4 111.1 155.0
3 2 2
9 4 4
4.56 2.97 26.53
1.323 1.339 1.619
1.980 1.926 1.587
6 4 4
6 4 4
37.4 78.5 70.2 116.0 60.4 176.1 120.9 87.5 89.6 66.9 303.3 87.0 133.4
2 2 2 2 2 2 2 2 3 2 3 2 2
3 4 3 7 3 5 3 2 4 5 7 3 3
1.98 6.13 2.27 6.88 4.86 3.9 15.15 2.26 6.23 17.44 15.52 7.25 5.23
1.220 1.384 1.542 1.214 1.514 1.473 1.506 1.449 1.520 1.287 1.310 1.304 1.342
1.956 1.769 1.601 1.917 1.554 1.582 1.518 1.540 1.404 1.621 1.524 1.507 1.457
2 4 2 7 3 5 3 2 4 5 4 3 3
2 4 2 7 3 4 3 2 4 5 4 3 3
13442.9 145.5 121.0 379.6 206.5 91.8
19 2 4 3 2 2
312 4 4 14 6 4
56.57 4.71 12.81 21.43 8.04 1.68
1.210 1.239 1.272 1.320 1.351 1.217
1.581 1.529 1.460 1.355 1.324 1.364
7 4 2 8 6 4
7 4 2 8 6 4
a Accession number (Acc. No.) of protein in the UniProt/Swiss-Prot database (http://www.uniprot.org/). bProtein scores are obtained from the Proteome Discoverer software (version 1.3).
GGGTCATCTTCTC-3′). The relative expression of IFN-β was analyzed using the method described previously.21 The protein levels of IFN-β in culture supernatant were determined with an ELISA kit (PBL Assay Science, USA) following the manufacturer’s instructions.
the transfected cells were collected and lysed with passive lysis buffer. The cell lysates were then subjected to a dual-luciferase reporter assay (Promega) following the manufacturer’s instructions. To evaluate the activity of the IFN-stimulated response element (ISRE), a pISRE-TA-Luc reporter plasmid (Clontech, USA) was used instead of the IFN-β promoter reporter for the cotransfection experiment described above. Relative IFN-β promoter and ISRE activity were determined by normalizing the firefly luciferase activity to the Renilla luciferase activity.
Statistical Analysis
All data were processed using SPSS 12.0 (SPSS Inc., Chicago, IL, USA). All continuous variables are expressed as the mean ± SD. The between-group differences were evaluated using the nonparametric Mann−Whitney U-test; p values
