www.proteomics-journal.com

Page 1

Proteomics

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

Cheng Du1,2, Hai-Fang Liu1, Yue-Zhi Lin1, Xue-Feng Wang1, Jian Ma1, Yi-Jing Li2, Xiaojun Wang1* and Jian-Hua Zhou1,3*

1

State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China

2

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150001, China

3

Hayao Pharmaceutical Group Biovaccine Co., Harbin 150069, China

* Corresponding authors: Jian-Hua Zhou, Hayao Pharmaceutical Group Biovaccine Co., Harbin 150069, China, tel.: +86-13796085512, e-mail: [email protected] Xiaojun Wang, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China, tel: +86-18946066285, e-mail: [email protected]. Keywords: Cellular protein Differential expression EIAV iTRAQ Macrophages

Received: 16-Jun-2014; Revised: 06-Jan-2015; Accepted: 05-Feb-2015. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/pmic.201400279. This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 2

Proteomics

Abbreviations: HIV

human immunodeficiency virus

SIV EIAV eMDMs DLV34 PRM

simian immunodeficiency virus equine infectious anemia virus equine monocyte-derived macrophages EIAVDLV34 parallel reaction monitoring

EIA OIE dMDMs TCID hpi IFA IAM TEAB

equine infectious anemia Office International Des Epizooties donkey monocyte-derived macrophages tissue culture infective dose hours post infection immunofluorescence assay iodoacetamide tetraethylammonium bromide

Th HCD DAVID CC

Thomson higher-energy c-trap dissociation Database for Annotation, Visualization and Integrated Discovery cellular component

BP MF MAVS RER

biological processes molecular function mitochondrial antiviral signaling rough endoplasmic reticulum

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 3

Proteomics

Abstract Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV) is another member of the Lentivirus genus in the family Retroviridae. Previous studies revealed that interactions between EIAV and the host resulted in viral evolution in pathogenicity and immunogenicity, as well as adaptation to the host. Proteomic analysis has been performed to examine changes in protein expression and/or modification in host cells infected with viruses and has revealed useful information for virus-host interactions. In this study, altered protein expression in equine monocyte-derived macrophages (eMDMs, the principle target cell of EIAV in vivo) infected with the EIAV pathogenic strain EIAVDLV34 (DLV34) was examined using two-dimensional liquid chromatography−tandem mass spectrometry (2D-LC-MS/MS) coupled with the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique. The expression levels of 210 cellular proteins were identified to be significantly upregulated or downregulated by infection with DLV34. Alterations in protein expression were confirmed by examining the mRNA levels of eight selected proteins using quantitative real-time reverse-transcription PCR, and by verifying the levels of ten selected proteins using parallel reaction monitoring (PRM). Further analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)-Pathway enrichment demonstrated that these differentially expressed proteins are primarily related to the biological processes of oxidative phosphorylation, protein folding, RNA splicing and ubiquitylation. Our results can facilitate a better understanding of the host response to EIAV infection and the cellular processes required for EIAV replication and pathogenesis.

Keywords Cellular protein/ Differential expression/ EIAV/ iTRAQ/ Macrophages

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 4

Proteomics

1. Introduction Equine infectious anemia (EIA) is a major infectious disease of equids characterized by recurrent febrile episodes, thrombocytopenia, anemia, rapid weight loss and edema. EIA is defined as a class B animal infectious disease by the Office International Des Epizooties (OIE) [1]. The etiologic agent of EIA is equine infectious anemia virus (EIAV). EIAV belongs to the genus Lentivirus in the family Retroviridae, which also includes the extensively studied human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), as well as six other animal lentiviruses [2]. A better understanding of the biological characteristics of these animal lentiviruses and their interactions with hosts should improve our knowledge regarding the lentiviral pathogenicity and immunogenicity. Unlike HIV-1, which is either macrophage (M)- or CD4+ helper T cell (T)-tropic or M/T dual-tropic, depending on the strains and stages of infection in the hosts [3], EIAV principally replicates in tissue macrophages and peripheral blood monocyte-derived macrophages (MDMs), although it also infects monocytes and endothelial cells in vivo [4-6]. Therefore, the interaction between EIAV and macrophages has great relevance for the persistence of viral infection and pathogenesis. Our previous studies showed that a variety of cytokines and chemokines expressed in macrophages were significantly upregulated or down- regulated following infection with EIAV. These changes in cytokine/chemokine expression correlated with the physiological status of the target cells (i.e., cell apoptosis and death) and the replication levels of the virus [7]. Proteomic analysis of host cells responding to viral infection has been used to identify cellular proteins involved directly or indirectly in viral infection and host restriction by detecting relative changes in protein abundance stimulated by viruses such as HIV-1, herpes simplex virus, Epstein−Barr virus, African swine fever virus and infectious bursal disease virus [8-12].

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 5

Proteomics

Isobaric tags for relative and absolute quantification (iTRAQ) is an isotope-labeled proteomics quantification technique that was developed in 2004 [13]. This system enables the analysis of up to eight samples simultaneously in one measurement with favorable accuracy and repeatability and is one of the most sensitive techniques currently used in comparative proteomics [14]. Parallel reaction monitoring (PRM) represents a new generation of mass spectrometry (MS) that contains a quadrupole-equipped Orbitrap (Q Exactive). PRM is more specific and sensitive than the traditional method (selected reaction monitoring or SRM) and has begun to be widely used to quantify and detect target proteins [15-17]. In the present study, the types and expression levels of differentially expressed proteins in equine eMDMs infected with a pathogenic EIAV strain were analyzed by iTRAQ to investigate host cell responses to infection. Alterations in protein expression levels were verified using quantitative real-time reverse-transcription (RT)-PCR and PRM. The possible biological significances of 210 differentially expressed proteins in the host response to EIAV infection were further evaluated using various bioinformatics programs.

2. Materials and Methods 2.1 Cells and viruses Preparations of eMDMs were obtained from equine peripheral blood mononuclear cells (PBMCs) as described previously [18]. Briefly, PBMCs were isolated from 200-300 ml of heparinized horse peripheral blood by centrifugation through a HybriMax Histopaque cushion (d=1.077 g/cm3) (Sigma, USA). Isolated PBMCs were washed with PBS three times and resuspended in RPMI-1640 medium supplemented with 10% horse serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate and 0.25 mM sodium HEPES. These cells were seeded into tissue culture flasks (Corning, USA) at 5×106 cells/cm2 and incubated at 37°C, 5% CO2 for approximately 12 hours. Nonadherent and loosely adherent cells were removed by mildly shaking the flasks before changing the medium, and the remaining adherent cells were further incubated for 3 days to allow differentiation to eMDMs. This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 6

Proteomics

Wild-type pathogenic EIAV strains isolated in China did not replicate well in cultivated cells, including primary target cells such as donkey monocyte-derived macrophages (dMDM) and donkey fetal dermal cells. EIAVDLV34 (DLV34) is a dMDMs-adapted EIAV strain that was derived from the pathogenic EIAV strain EIAVDV117 by 34 passages in dMDMs. Experimental infection of horses with DLV34 resulted in the appearance of the typical symptoms of equine infectious anemia in all of the inoculated animals [19].

2.2 Virus titration The infectious titer of the EIAV strain was measured using the median tissue culture infective dose method (TCID50) as reported previously [20]. Three days post plating, 5105 eMDMs were infected with 5×103 TCID50 of DLV34 in 24-well plates. Cell culture medium was used to mock-infect eMDMs as a negative control. Media samples were collected from 0 h to 168 hours post infection (hpi) at 24 h intervals and stored at −80 °C for further titration of infectious virus. Cells were fixed in 80% cold acetone in PBS and examined for viral antigen by immunofluorescence assay (IFA) using a 1:200 dilution of EIAV-positive horse serum, followed by a TRITC-conjugated rabbit anti-horse IgG (Abcam, USA). Supernatants of DLV34-infected eMDMs were examined for the viral reverse transcriptase (RT) activity, which is only found in retroviruses, such as HIV-1 and SIV [21]. The RT activity in EIAV-infected cells was quantified using the Reverse Transcriptase Assay Colorimetric Kit (Roche, Germany) following the manufacture’s instructions [22, 23]. These experiments were performed in triplicate.

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 7

Proteomics

2.3 Protein sample preparation and labeling with iTRAQ reagents After isolation and cultivation, eMDMs in 25 cm2 flasks (1×107 cells/flask) were infected with 1×105 TCID50 of DLV34 for 48 h. The same amount of eMDMs were mock-infected with cell culture supernatant as an uninfected control. The culture supernatant was then removed, and the monolayers were washed with cold PBS three times, lysed with 500 μl lysis buffer (7 M urea, 2 M Thiourea, 4﹪ CHAPS, 40 mM Tris-HCl, pH8.5), 1 mM PMSF, 2 mM EDTA and incubated on ice for 5 min. After adding DTT to a final concentration of 10 mM, the lysate was sonicated on ice at 200 watts for 5 min and centrifuged at 25,000 ×g for 20 min. The supernatant was collected and incubated with 10 mM DTT at 56 °C for 1 h to reduce disulfide bonds in proteins then treated with 55 mM iodoacetamide (IAM) in the dark for 45 min to block alkylation of cysteine residues. The samples were then combined with 1 ml of cold acetone, incubated at -20 °C overnight and centrifuged at 25,000×g for 20 min to collect the pellet, which was then suspended in 300 μl 0.5 M tetraethylammonium bromide (TEAB) and sonicated at 200 watts on ice for 5 min. The resulting protein preparations were centrifuged at 25,000×g for 20 min, and the pellets were removed. The protein concentration of the supernatant was analyzed with the 2D Quant Kit (Amersham, USA), according to the manufacturer's instructions. Finally, the protein samples were digested with trypsin at an enzyme:protein ratio of 1:50 at 37 °C overnight. The proteins prepared from eMDMs infected with DLV34 were labeled with iTRAQ tag 114, and the proteins from uninfected eMDMs were labeled with iTRAQ tag 116. The labeled samples were then mixed together prior to online 2D liquid chromatograph (LC)-mass spectrum (MS)/MS analysis. This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 8

Proteomics

2.4 Pre-fractionation of the peptide mixture The labeled peptide mixtures were pre-fractionated by high pH reverse-phase chromatography. After lyophilization, the peptide mixtures were redissolved in Buffer A (20 mM ammonium formate in water, pH10.0, adjusted with ammonium hydroxide) and then fractionated by high pH separation using a Shimadzu UFLC system (Shimadzu, Japan) connected to a reverse phase column (Venusil XBP C18 column, Bonna-Agela Technologies, USA). High pH separation was performed using a four-step linear gradient: starting at 5% B, increased to 8% B in 1 min (Buffer B: 20 mM ammonium formate in 90% acetonitrile, pH 10.0, adjusted with ammonium hydroxide), increased to 32% B in 64 min, and then increased to 95% B in 2 min. The column was re-equilibrated at initial conditions for 1 min. The column flow rate was maintained at 700 μl/min, and the column temperature was maintained at 45 °C. Fractions were monitored by measuring the optical absorbance at 214 nm and collected at 1 tube/min fractionated. A total of 64 fractions were collected from the 8%-32% B gradients and were lyophilized for further analysis.

2.5 LC-MS/MS online analysis of the peptide mixture The 64 fractions of sequentially eluted peptides were redissolved in 2.0% acetonitrile with 0.1% formic acid and combined into 16 samples by pooling every four sequentially adjacent fractions. Then, these samples were centrifuged at 12,000 rpm for 3 min. The supernatants were harvested and analyzed by Eksigent Nano LC 2D plus coupled with MS, which

consisted

of

a

C18

concentration

column

(5

μm,

100

μm×20

mm,

Microm BioResources Inc., Auburn, USA) and a C18 separation column (3 μm, 75 μm×120 mm, Microm BioResources Inc., Auburn, USA). MS data acquisition was performed with a This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 9

Proteomics

Triple TOF 5600 System (AB SCIEX, USA). The mobile phases were composed of 0.1% formic acid, 1.9% acetonitrile in water (A) and 0.1% formic acid, 98% acetonitrile in water (B). The flow rate was 330 nl/min. The gradient run was from 5%−90% B over 36 min, and then to 5% B over 4 min. The atomization voltage was 2.3 KV, the capillary tube temperature was 23.92°C and the range of the recorded mass was 350-1,250 Daltons (Da). The collision energy was 45%, the precursor mass tolerance was 10 parts per million (ppm) and the allowed fragment mass tolerance was 20 ppm.

2.6 Data analysis and interpretation Relative abundance quantification and peptide and protein identification were performed using the ProteinPilotTM Software 4.2 Revision 50861 (Applied Biosystems, USA). Each MS/MS

spectrum

was

EquCab2.66.pep.all.fa.gz

searched

for

database

species

of

downloaded

Equus

caballus

from

the

against

the

website

(ftp://ftp.ensembl.org/pub/mnt2/). The search parameters allowed for cysteine modification by methyl methanethiosulfonate and biological modifications programmed in the algorithm (i.e., amidations, phosphorylations, and semitryptic fragments). The protein confidence threshold cutoff was set to 1.3 (unused), with at least more than two peptides above the 95% confidence level. Analysis of the iTRAQ data was performed using ProQUANT 1.0 software. The cut-off value for the confidence setting was 75, and the tolerance settings for peptide identification in the ProQUANT searches were 0.15 Da for MS and 0.1 Da for MS/MS. Relative quantification of proteins using the iTRAQ tags was performed on the MS/MS scans and was presented as the ratio of the area under the peaks at 114 and 116 Da, which were the This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 10

Proteomics

masses of the tags corresponding to the iTRAQ reagents. The relative amount of a peptide in each sample was calculated by dividing the peak areas observed at m/z 116.1 by that observed at m/z 114.1. For proteins with two or more qualified peptide matches, three average peak area ratios (designated as 116/114) were calculated using the peak area ratios of the peptides originating from the same protein. To account for small differences in protein loading, these ratios were normalized using the overall ratios for all proteins in the sample, as recommended by Applied Biosystems. In this study, protein quantification data with relative expression of ≥1.25 or ≤0.8 and a P-value ≤0.05 were chosen for further analysis.

2.7 Real-time RT-PCR Monolayers of eMDMs were infected with DLV34 or mock-infected with culture medium as described above. Total cellular RNA was extracted with TRIzol (Life Technologies, USA) according to the manufacturer's instruction. RNA concentrations were measured spectrophotometrically at 260/280 nm (IMPLEN, Germany). Complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Japan) following the manufacturer’s instructions. Real-time PCR was performed using a Stratagene 3000 system (Agilent Technologies, Germany). The reaction volume was 20 μl, containing 2 μl cDNA template, 10 μl 2×SYBR Green I (TaKaRa) and 0.8 μM of forward and reverse primers. PCR reactions were set up in triplicate. For all amplifications, the cycle conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. All samples were tested with three independent repeats, and the average values were taken as the quantitative result. Equine β-actin was used as an internal control. The primers for amplifying cDNAs of APOOL, β-actin, MAP2K2, MX1, S100A9, STAU1, TMEM111, UBE2G1 and WNK1 are presented in Table 1. Relative fold changes in gene expression were determined by the 2−ΔΔCt method [24].

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 11

Proteomics

2.8 PRM-MS analysis To verify the protein expression levels obtained by iTRAQ analysis, the expression levels of 10 selected proteins were further quantified by PRM-MS analysis at the Beijing Proteome Research Center (Beijing 100850, China) [15]. Briefly, signature peptides for the target proteins were defined based on iTRAQ data, and their uniqueness was examined with a BLAST search against the EquCab2.66.pep.all.fa.gz database. Only unique peptide sequences were selected for the PRM assays (Table 3). The proteins (100 μg) were prepared, reduced, alkylated, and digested with trypsin according to the iTRAQ protocol. The obtained peptide mixtures (1 μg) were introduced into the mass spectrometer via an in-house packed C18 column (3 µm, 75 µm × 150 mm, C18-AQ, Bonna-Angela Technologies, USA) with an integrated electrospray emitter (New Objective, USA) operated at 2.0-2.2 kV and coupled to a

custom nano-Electrospray ionization

(ESI)

source.

A Q Exactive

bench-top

quadrupole-Orbitrap MS (QqOrbi)-coupled measurement was performed using a quadrupole mass filter-equipped bench-top Orbitrap mass spectrometer (Q Exactive, Thermo Scientific, Germany). The PRM analysis was performed with an isolation width of ±1 Thomson (Th). The full mass spectrum was conducted at 70,000 resolution relative to m/z 200, followed by up to 25 PRM scans at 17,500 resolution. Ion activation/dissociation was performed at 25% normalized collision energy in a higher-energy c-trap dissociation (HCD) collision cell. The raw data obtained were then analyzed with the analytic program Proteome Discoverer 1.3 (Thermo Electron, Germany). The false discovery rate (FDR) was set to 0.01 for proteins and peptides. Skyline 2.6 software (downloaded from ftp://ftp.ensembl.org/pub/mnt2/) was applied for quantitative data processing and proteomic analysis.

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 12

Proteomics

2.9 Bioinformatic analysis Differentially expressed proteins identified in this study were converted to human orthologous

proteins,

and

for Annotation, Visualization

the and

lists Integrated

were

submitted

to

Discovery (DAVID)

the Database web

server

(http://david.abcc.ncifcrf.gov) for enrichment analysis of the significant overrepresentation of gene ontology for cellular component (GO-CC), biological processes (GO-BP) and molecular function (GO-MF) terminologies and analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG)-pathway category [25]. In all tests, known genes were used as background, and P-values (i.e., EASE score), indicating significance of the overlap between various gene sets were calculated using a Benjamini-corrected modified Fisher’s exact test. Only GO-CC, GO-BP or GO-MF with an EASE score ≤0.05 were considered as significant and listed. It is important to note that EASE scores for the KEGG-pathway provide only the relative significance among terms for the aim of comparison. The protein-protein interaction network was analyzed by using String software version 9.1 (http://string.embl.de/) [26]. 3. Results 3.1 Determination of the DLV34 replication curve HIV-1 viral RNA is reverse transcribed and integrated into the host cell genome at approximately 24 hpi. At 48 h, all viral mRNAs, as well as the structural and regulatory proteins, are synthesized, and new infectious viral particles are released. During this period, the number of host cell proteins that respond to the infection or interact with the virus begin to increase, accompanied by an increase in viral protein expression [27, 28] . Therefore, it is reasonable to examine the differentially expressed host proteins regulated by EIAV infection This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 13

Proteomics

at the period at which the virus starts to replicate rapidly. To examine the replication curve of DLV34 in eMDMs, the virions released to the culture supernatant were titrated by detecting the activity of the viral reverse transcriptase (RT). As presented in Fig. 1A, DLV34 effectively replicated during 24-96 hpi, and at 48 hpi, DLV34 was on the early curve of replication. Therefore, eMDMs infected with DLV34 and mock-infected eMDMs were collected at 48 hpi for iTRAQ analysis. In addition, the IFA examination of DLV34-infected eMDMs demonstrated that up to 99% of the cells were infected, as determined by incubating the cells with an anti-EIAV serum and staining with TRITC-conjugated rabbit anti-horse antibody (Fig. 1B).

3.2 Differentially expressed proteins between EIAV-infected and mock-infected eMDMs identified by iTRAQ analysis Three batches of eMDMs were prepared from three horses, and each batch of cells was infected with DLV34 or mock-infected for 48 hours. Protein extracts were prepared separately from infected or mock-infected eMDM cells. Equal amounts of protein from each of the three batches of infected cells and three batches of mock-infected cells were mixed and subjected to iTRAQ-coupled 2D LC–MS/MS analysis. A total of 6,012 proteins were detected. Among these proteins, 210 displayed significant differences in expression levels between EIAV-infected and uninfected cells, identified by at least two high confidence (95%) peptides, with P-values ≤0.05, as calculated by ProQUANT (Table 2). As shown in Table 2, 12 proteins were downregulated and 198 proteins were upregulated.

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 14

Proteomics

3.3 Validation of differentially expressed proteins by real-time RT-PCR To validate the differentially expressed proteins identified by iTRAQ-labeled LC−MS/MS analysis, total RNA was extracted from eMDMs either infected with DLV34 or treated with the same volume of culture medium as the negative control for 48 hours. The mRNA levels of eight proteins with different biological functions and cellular locations (upregulated: APOOL, MAP2K2, STAU1, TMEM111, UBE2G1 and WNK1; downregulated: MX1 and S100A9) were selected for further analysis by real-time RT-PCR. The mRNA levels of APOOL, MAP2K2, STAU1, TMEM111, UBE2G1 and WNK1 were observed to be increased by 1.35-fold, 1.72-fold, 1.29-fold, 1.32-fold, 2.07-fold and 1.60-fold, respectively (Fig. 2). The mRNA levels of MX1 and S100A9 were observed to be decreased by 0.50-fold and 0.27-fold, respectively. These data confirmed the differentially expressed proteins identified by iTRAQ analysis. 3.4 Validation of differentially expressed proteins by PRM The PRM assay was also applied to verify a subset of the differentially expressed proteins obtained in the iTRAQ analysis. Because the signature peptides for target proteins must exhibit uniqueness, only proteins that possessed unique peptide sequences were selected for PRM analysis. Three of the eight proteins used in the real-time RT-PCR analysis (MX1, TMEM111 and UBE2G1) met the unique sequence requirement and were further verified. The ratios of these three proteins were found to be in agreement with those obtained by iTRAQ analysis and real-time RT-PCR (Table 3). Next, another seven proteins (C1QBP, COX5B, CSF1, HSPA4L, IL1A, NDUFA9 and TRIM25) were selected for PRM analysis based on an analysis of their biological functions and their possible roles during virus infections. The protein levels of C1QBP, COX5B, CSF1, HSPA4L, IL1A, NDUFA9 and TRIM25 were observed to be increased 1.26-fold, 1.25-fold, 1.45-fold, 1.68-fold, 1.29-fold, 1.30-fold and 1.47-fold, respectively (Table 3). These data further confirmed the results of the iTRAQ-coupled LC−MS/MS analysis.

This article is protected by copyright. All rights reserved.

www.proteomics-journal.com

Page 15

Proteomics

3.5 Evaluation of differentially expressed proteins by GO and pathway enrichment analysis To evaluate the potential biological significances of the 210 proteins differentially regulated by EIAV, the proteins were analyzed by GO enrichment using the online tool DAVID. Three major types of annotation were obtained from the GO consortium website: cellular components (GO-CC), biological processes (GO-BP) and molecular functions (GO-MF). GO-CC identified differentially expressed proteins that were well-distributed in different cell components, with membrane-enclosed lumen and organelle lumen being the two most distributed components (40 and 39 proteins, respectively). Differentially expressed proteins localized in the endoplasmic reticulum (ER) had the lowest EASE score (Fig. 3A). Enrichment analysis using GO-BP showed that intracellular protein distribution/translocation were the most heavily interfered (as shown by involved protein numbers) and RNA splicing were the most significantly affected (as shown by its EASE score) biological processes by EIAV infection (Fig. 3B). GO-MF demonstrated that proteins related to enzyme binding and RNA binding were most commonly affected by viral infection (Fig. 3C). Pathway analysis based on the DAVID program was also performed. Although most of the grouped pathways of KEGG-pathway analysis shown in Fig. 3D were not statistically significant (EASE score

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

Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV...
685KB Sizes 0 Downloads 6 Views