Identification of cellular proteome using two-dimensional difference gel electrophoresis in ST cells infected with transmissible gastroenteritis coronavirus
© Zhang et al.; licensee BioMed Central Ltd. 2013
Received: 6 March 2013
Accepted: 23 June 2013
Published: 16 July 2013
Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus that causes diarrhea in pigs, which is correlated with high morbidity and mortality in suckling piglets. Information remains limited about the comparative protein expression of host cells in response to TGEV infection. In this study, cellular protein response to TGEV infection in swine testes (ST) cells was analyzed, using the proteomic method of two-dimensional difference gel electrophoresis (2D DIGE) coupled with MALDI-TOF-TOF/MS identification.
33 differentially expressed protein spots, of which 23 were up-regulated and 10 were down-regulated were identified. All the protein spots were successfully identified. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and the mitochondrial pathway. Western blot analysis was used to validate the changes of alpha tubulin, keratin 19, and prohibitin during TGEV infection.
To our knowledge, we have performed the first analysis of the proteomic changes in host cell during TGEV infection. 17 altered cellular proteins that differentially expressed in TGEV infection were identified. The present study provides protein-related information that should be useful for understanding the host cell response to TGEV infection and the underlying mechanism of TGEV replication and pathogenicity.
KeywordsProteome ST cell DIGE TGEV
Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family . The infection of TGEV causes severe diarrhea in suckling piglets (about 2 weeks old) and its lethality approaches 100%, which results in enormous economic loss in swine-producing areas in the world . TGEV is an enveloped virus with a positive-sense RNA genome of 28.5 kb. About two-thirds of the TGEV genome encodes the replicase proteins (rep) at the 5′ end, and one-third of the genome encodes other viral genes at the 3′ end in an order of 5′-S-3a-3b-E-M-N-7-3′ . The genome of TGEV encodes four structural proteins: spike (S), membrane (M,), minor envelope (E), and nucleocapsid (N) proteins. The surface protein S, a large type I transmembrane glycoprotein that forms peplomers, is responsible for cell receptor binding and membrane fusion . The M protein spans the membrane and interacts with the N protein to form core of the virus during assembly [5, 6]. The small E protein, a transmembrane protein detected as a minor structural component, is essential for TGEV replication . The N protein, an internal phosphoprotein , interacts with the TGEV genomic RNA to form viral nucleocapsid [5, 9], and may disrupt host cell division . To date, there is limited information about host cell responses to TGEV infection.
Proteomics analysis enables a more comprehensive characterization of virus-virus and virus-host interactions involved in infection and pathogenesis [11, 12]. The development of proteomic approaches have greatly facilitated detection of proteins induced in virus infected cells. Among those techniques for differentially expressed protein spot analysis, two-dimensional difference gel electrophoresis (2D DIGE) is reproducible and sensitive . 2D DIGE has greatly facilitated the comparison of two samples by removing gel-to-gel variability and by using dyes (CyDye) with a greater dynamic range than traditionally used silver or Coomassie stains . Using 2D DIGE method followed by mass spectrometry (MS) identification, comparative proteomics of host cells have been investigated during virus infection, including human influenza A virus , human immunodeficiency virus type 1 (HIV-1) , hepatitis B virus , hepatitis C virus (HCV) , Epstein-Barr virus (EBV) , and Dengue virus (DENV) , and porcine reproductive and respiratory syndrome (PRRSV) . Proteomics method analyzing host cellular responses to TGEV infection can be used to identify important cellular factors involved in viral pathogenesis.
In present study, to determine protein profiles of swine testes (ST) cell line that expressed differentially after TGEV infection, fluorescent 2D DIGE coupled with a MALDI-TOF-TOF/MS identification proteomics approach was utilized. A total of 33 differentially expressed protein spots were identified, and those proteins of interest were confirmed by Western blot. This study can provide useful clues for better understanding of TGEV replication and pathogenesis.
Confirmation of TGEV propagation in ST cells
Analysis of differentially expressed proteins after TGEV infection
Mass spectral identification of differentially expressed proteins
Proteins identified from the differential 2D DIGE analysis after TGEV infection
Spot number a
Protein name (Abbr.)
Accession number b
Peptide matched c
Sequence coverage (%)
Protein score d
Average volume ratio e
p value e
beta actin (ACTB)
beta actin (ACTB)
beta actin (ACTB)
beta actin (ACTB)
beta actin (ACTB)
beta actin (ACTB)
alpha tropomyosin (TPM1)
beta actin (ACTB)
alpha Tubulin (TUBA1B)
beta Tubulin (TUBB)
alpha Tubulin (TUBA1B)
Intermediate filament proteins
keratin 19 (KRT19)
heterogeneous nuclear ribonucleoprotein U (HNRNPU)
thiopurine S-methyltransferase (TPMT)
thiopurine S-methyltransferase (TPMT)
Protein biosynthesis and modification
protein phosphatase 2A (PP2A)
eukaryotic initiation factor 4A-I (EIF4A1)
eukaryotic initiation factor 4A-I (EIF4A1)
acidic ribosomal protein P0 (RPLP0)
annexin A8 (ANXA8)
annexin A4 (ANXA4)
Mitochondrion associated protein
cytochrome b-c1 complex subunit 1 (UQCRC1)
14-3-3 protein eta (YWHAH)
L-lactate dehydrogenase B chain (LDHB)
Thirty-three differential protein spots corresponded to 17 proteins, including cytoskeleton-associated proteins (35.3%), protein biosynthesis and modification proteins (17.6%), RNA processing proteins (11.8%), vesicle transport proteins (11.8%), mitochondria-associated proteins (5.9%), signal transduction-associated proteins (5.9%), and 5.9% metabolism-associated proteins. These proteins (Figure 3) were mainly located in the cytosol (64.7%), membrane (11.8%), nucleus (11.8%), and mitochondrion (11.8%). In addition, some different spots were identified to be products of the same gene, including beta actin, alpha tubulin, vimentin, eukaryotic initiation factor 4A-I, and thiopurine S-methyltransferase. In particular, 7 protein spots (6 up-regulated spots and 1 down-regulated spot) were identified as beta actin, and 8 protein spots (5 up-regulated spots and 3 down-regulated spots) were identified as vimentin.
Analysis of identified proteins at the transcriptional level
Protein validation by western blot
Proteomics is a novel methodology to detect components of cellular protein interactions as well as host cellular pathophysiological processes that occur during virus infection . Until present investigation, no results have been reported for performing analysis of differential proteome of host cells infected with TGEV. In this study, 2D DIGE coupled with MALDI-TOF/TOF was used to analyze the differential proteome of ST cells infected with TGEV. The 33 differential protein spots were successfully identified as 17 proteins, of which function in diverse biological processes.
Cytoskeletal protein expression was altered in TGEV-infected ST cells. The cytoskeleton filaments are dynamic and divided into three types: microfilaments (actin filament), microtubules, and intermediate filaments . The intermediate filaments can provide mechanical stability to cells, while actin and microtubule cytoskeletons are responsible for trafficking of numerous endogenous cargos as well as intracellular microorganisms throughout the cells . Many viruses use the cytoskeleton for infection and replication, such as HIV-1 . In present study, differentially expressed microfilament-associated proteins beta actin and alpha tropomyosin, microtubule-associated alpha tubulin and beta tubulin, as well as the intermediate filament-associated vimentin and keratin 19 were identified (Table 1), indicating that TGEV infection and replication involves cellular skeleton.
The actin and microtubule cytoskeleton play important roles in the life cycle of viruses . Numerous viral proteins interact with actin-binding proteins or directly with actin . Microtubules and microtubule-associated proteins are known to play important roles in intracellular trafficking of viral components as well as virions in the infected host cell . In this study, the up-regulated microfilament-associated proteins beta actin and microtubule-associated alpha tubulin and beta tubulin were identified, which was believed to facilitate the transport of viral proteins of TGEV from rough endoplasmic reticulum (ER) and Golgi apparatus to the reservoir for viral replication.
Vimentin is a major component of type III intermediate filaments found in many cell lines . This protein serves to maintain cell shape and is involved in attachment, migration and cell signaling . Major changes in the distribution of vimentin are observed when the cell moves and divides , but this protein is also redistributed in cells expressing misfolded proteins and during virus infection . Previous studies have shown that intermediate filament protein vimentin was cleaved by human immunodeficiency virus type 1 protease (HIV-1 PR)  and that vimentin networks collapsed and was dispersed in IBDV-infected cells . In present study, 8 differential protein spots were identified as vimentin including 5 up-regulated protein spots and 3 down-regulated protein spots. Further study is required to determine whether TGEV papain-like protease 1 (PL1pro)  cleaves vimentin into different isoform or subunit, using a similar strategy as HIV.
Among those differentially expressed host proteins, some are known to participate in viral replication and translation (Table 1). Positive-strand RNA viruses must recruit normal components of host cellular RNA processing or translation machineries for viral RNA synthesis and protein synthesis . Heterogeneous nuclear ribonucleoprotein U (HNRNPU) is an abundant, strictly nuclear phosphoprotein that interacts directly with RNA through a carboxy-terminal RGG sequence . HNRNPU is known to influence pre-mRNA processing, mRNA transportation to cytoplasm, intracellular localization, translation, and turnover of mRNAs . Previous studies have shown that the levels of HIV-1 viral transcripts are dramatically down-regulated in cytoplasm of infected cells by HNRNPU . In this study, the down-regulated HHRNPU was found after TGEV infection, which may facilitate the replication of viruses. Another RNA processing protein, thiopurine S-methyltransferase (TPMT), was found to be more abundant in TGEV-infected cells. TPMT is a cytoplasmic transmethylase present in prokaryotes and eukaryotes, which has a molecular mass of 28 kDa and comprises 245 amino acids . TPMT is a drug-metabolizing enzyme widely expressed in mammalian and non-mammalian cells . Previous works have reported that TPMT plays a role in BVDV virus replication and thiopurines inhibit bovine viral diarrhea virus production in a TPMT-dependent manner . Based on these data, an up-regulation of TPMT in TGEV-infected ST cells suggests that this host protein plays an important role in TGEV biology, making it possible target for future drug development.
Viruses rely on the cellular translation machinery to translate their own proteins, which facilitates the rapid production of viral proteins and renders an inhibitory effect on the production of host proteins, including host anti-viral proteins . Translation factors have been well documented as playing crucial roles in viral RNA and protein synthesis . In herpes simplex virus type 1 (HSV-1) infected HeLa cells, the synthesis of several ribosomal proteins and their assembly into ribosomes continue in spite of a general inhibition of cellular protein synthesis . Acidic ribosomal protein P0 (RPLP0) is located in the active part of the ribosome particle, at which mRNAs, tRNAs and translation factors interact during protein synthesis . The cellular RPLP0 was observed as up-regulated after virus infection, such as pseudorabies virus (PrV) infected bovine kidney cells , and Epstein-Barr virus (EBV) infected primary B cells . In this study, the up-regulated 60S RPLP0 was also found after TGEV infection, suggesting the ribosomal protein plays an important role in the translation of TGEV viral proteins.
Viruses may inhibit host protein synthesis by targeting multiple steps in the gene expression process via various pathways, for instance, the vesicular stomatitis virus (VSV) M protein inhibits the initiation of the transcription of host genes  and the SARS-CoV spike protein inhibits host cell translation by interaction with eIF3f . In this study, the up-regulated eukaryotic initiation factor 4A-I (EIF4A1) was identified after TGEV infection. The translation initiation proteins observed in TGEV infected ST cells may be a reflection of translation regulation mechanisms exploited by TGEV virus, interfering with cellular protein synthesis and translation initiation of the host cell for beneficial reasons, which need to be further studied.
Protein phosphatase 2A (PP2A) is an evolutionarily conserved enzyme that represents a major portion of serine/threonine phosphatase activity in cell extracts . PP2A enzymes have been clearly involved in regulation of cell transcription, cell cycle and viral transformation . Up-regulation of PP2A scaffold subunit A and subsequent dephosphorylation of Tyr-307 in the catalytic subunit was found, suggesting PP2A activation in Huh7 infected cells [51, 52]. Activation of serine-threonine PP2A was found in Huh7 cells upon HSV-1 infection, and PP2A activation paralleled dephosphorylation and inactivation of the downstream mitogen-activated protein (MAP) kinase pathway . In this study, the up-regulation of PP2A was found in TGEV infected ST cells, suggesting PP2A plays an important role in the dephosphorylation of cellular and viral protein during TGEV infection.
Several proteomics analysis about coronavirus had been done including SARS-CoV , IBV [55, 56], and MHV . By comparing the finding proteins in this study to previous findings, there is no common gene of target related to coronavirus. The findings in TGEV infected ST cell might not reflect the interaction between the virus and pig intestine epithelial cell. It is surprising that the proteome responses observed did not reveal any immune responses related proteins in TGEV-infected ST cells. It may be related to the host cells chosen or the fact that attenuated TGEV strain was used. Identification of cellular proteome in pig intestine epithelial cell or immune cells infected with TGEV need to be further studied.
In conclusion, a total of 17 altered cellular proteins that differentially expressed in TGEV infection were identified in this study. Most of these proteins were involved in transcription and translation processes, vesicle transport, signal transduction, and alteration of the cytoskeleton networks. Western blot analysis of alpha tubulin, keratin 19, and prohibitin validated the MALDI-TOF/TOF identification of the differentially expressed proteins in the TGEV-infected ST cells. The present study provides large scale protein-related information that should be useful for understanding the pathogenesis of TGEV infection.
Materials and methods
Cell culture, virus infection and sample preparation
The TGEV strain attenuated H (Accession NO. EU074218)  was propagated on a ST cell monolayer. The proteins of TGEV infected ST cells were extracted according to the methods previously described . Briefly, the ST cells were infected with attenuated H (H167) at a multiplication of infection (MOI) of 1, and the cells were scraped using a cell scraper at 48 h postinfection (p.i.), and centrifuged at 10,000 × g for 5 min. After washing three times with ice-cold phosphate-buffered saline (PBS), the collected cells were lysed with lysis buffer (7 M urea, 2 M thiourea, 4% [w/v] CHAPS, 65 mM DTT, 0.2% pharmalyte 4/7 and 1 mM PMSF) containing 1% nuclease mix in the final concentration and were vertically vibrated until the cells were completely lysed. The supernatant was collected after centrifuging at 12,000 × g at 4°C for 60 min. Samples were treated with a 2D clean-up kit (GE Health Care) and a 2D quant kit (GE Health Care) according to the instructions of the manufacturers. Paralleled mock-infected ST cells were used as control. Three biological replicates of TGEV-infected and mock-infected ST cells were prepared.
A total of 200 pmol of CyDye DIGE Flours (GE healthcare, Germany) were used to label 50 μg of protein samples. To access biological variation, three experimental duplicates were carried out using the samples prepared above. The internal standards (equal amounts of both samples) were labeled with Cy2. Protein extracts from mock-infected ST cells, used as a reference state, were labeled with Cy5. Protein extracts from TGEV-infected ST cells were labeled with Cy3, representing the test states. IEF was performed using an IPGphor system (GE Healthcare) and commercially available 24-cm long IPG strips (Linear, pH 4–7, GE Healthcare). The settings and conditions for active rehydration of the IPG strips were used as previously described . Briefly, IEF was performed using the following parameters: 30 V, 12 h; 200 V, 1 h; 1,000 V, 1 h; 8,000 V, 2 h; and 8,000 V, 65,000 vh. The isoelectric-focused proteins in strips were incubated for 15 min in the equilibration buffer (6 M urea, 30% glycerol, 2% SDS, and 0.375 M Tris, pH 8.8) containing 1% DTT, followed by additional equilibration for 15 min in the equilibration buffer containing 2.5% iodoacetamide. The second dimension separation was performed using the Ettan Dalt II system (GE healthcare). Gels were poured between low fluorescent glass plates, of which one plate was bind-silane treated. Three parallel gels were run at 12°C (running buffer: 25 mM Tris, 192 mM glycine and 1% SDS). The equilibrated IPG strips were further resolved with 12% SDS-PAGE gels at 1 W/gel for 30 min and then 6 W/gel until the dye front reached the bottom of the gels.
Image acquisition and analysis of 2D DIGE gels
Cy2-labeled, Cy3-labeled, and Cy5-labeled protein images were scanned directly between the low fluorescent glass plates using a Typhoon Variable Mode Imager 9400 (GE Healthcare) with the CyDye-specific settings for excitation at 488 nm, 532 nm and 633 nm, and for emission at 520 nm, 590 nm and 680 nm. All gels were scanned with a resolution of 100 μm and a standard pixel volume of 60,000-80,000 for all scans. Determination of protein abundance and statistical analysis was performed using the Decyder™ software package (version 6.04.11, GE Healthcare). Inter-gel matching performed using the Biological Variation Analysis (BVA) mode. Matching between gels was performed using the in-gel standard from each image pair. A paired t test was used for the methods of statistical analysis. Only protein spots showing significance (p < 0.05) and at least a 2-fold difference in abundance (ratio of the mean of the normalized spot volume of the TGEV-infected samples versus mock-infected samples) were considered as up-regulated (ratio > 2) or down-regulated (ratio < −2).
Protein identification by MALDI-TOF-TOF mass spectrometry (MS) and a database search
For identification of protein spots a preparative gel containing 800 μg of protein was run as described above and stained with PhastGel™ Blue R (GE Healthcare). The protein spots of interest were manually excised from the gels and plated into 96-well microplates. Excised spots were firstly destained twice with 60 μl of 50 mM NH4HCO3 and 50% acetonitrile (ACN) and then dried twice with 60 μl of ACN. Afterwards, the dried pieces of gels were incubated in ice-cold digestion solution (trypsin 12.5 ng/μl and 20 mM NH4HCO3) for 20 min and then transferred into a 37°C incubator for digestion overnight. Finally, peptides in the supernatant were collected after extraction twice with 60 μl extract solution (0.1% TFA in 50% ACN). The peptide solution was dried under the protection of N2, and 0.8 μl matrix solution (5 mg/ml α-cyano-4-hydroxy-cinnamic acid diluted in 0.1%TFA, 50%ACN) was pipetted to dissolve it. Then the mixture was spotted on a MALDI target plate (Applied Biosystems). MS analysis of peptides was performed on an AB SCIEX 5800 MALDI-TOF/TOF. The UV laser was operated at a 400 Hz repetition rate with wavelength of 355 nm. The accelerated voltage was operated at 20 kV and mass resolution was maximized at 1,600 Da. Myoglobin digested with trypsin was used to calibrate the mass instrument with an internal calibration mode. All acquired spectra from samples were processed using TOF/TOF Explore™ Software in a default mode. The data were searched by GPS Explorer (V3.6) with the search engine MASCOT (2.1). The following parameters were used in the search: National Center for Biotechnology information non-redundant (NCBInr) database (release date, July, 2011), Sus scrofa, protein molecular mass ranged from 700 to 3,600 Da, trypsin digest with one missing cleavage, peptide tolerance of 100 ppm,MS/MS tolerance of 0.8 Da and possible oxidation of methionine. Known contaminant ions (tryptic autodigest peptides) were excluded. A total of 41,373 sequences and 16,019,616 residues in the database were actually searched. All identified proteins had a protein score greater than 59, corresponding to a statistically significant (p < 0.05) confident identification. Besides protein score, at least one ion score with p < 0.05 was recommended to increase the reliability of identifications.
Samples of TGEV-infected and mock-infected ST cells were lysed at 48 h p.i., and the protein concentration was determined. Equivalent amounts of cell lysates (60 μg) were subjected to 12% SDS-PAGE gels and then transferred to 0.22 μm nitrocellulose membranes (Hybond-C extra, Amersham Biosciences). After blotting, the membranes were incubated with mouse monoclonal antibody (mAb) to alpha tubulin (1:1000, Abcam), mAb to prohibitin (1:500, II-14-10, Santa Cruz), and rabbit polyclonal antibody to keratin19 (1:500, Bioss) at 37°C for 60 min. After washing three times with PBST, the membranes were inoculated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Inc.) or goat anti-rabbit IgG (Kirkegaard & Perry Laboratories, Inc.) at 37°C for 60 min and visualized using 3,3′,5,5′-tetramethylbenzidine-stabilized substrate (TMB, Promega).
Real time RT-PCR
Primers used for real time RT-PCR
Forward primers (5′-3′)
Reverse primers (5′-3′)
Gene accession no.
- 2D DIGE:
Two-dimensional difference gel electrophoresis
Transmissible gastroenteritis coronavirus
Immobilized pH gradient
Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry
Reverse transcriptase-polymerase chain reaction
Peptide mass fingerprinting
Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
This work was supported by the National Natural Science Foundation of China (Grant No.31172350), Heilongjiang Provincial Natural Science Foundation (Grant No.JC201118) and Research Team Program on Scientific and Technological Innovation in Heilongjiang Provincial University (Grant No.2011TD001). We thank Mr. Wang Shao (Institute of Animal Husbandry and Veterinary Science, Fujian Academy of Agricultural Science, China) for the donation of mAb to N protein of TGEV. We thank the studies about proteomics analysis of other coronavirus because these studies about coronavirus are very close to this work.
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