Differential Proteome Analysis of Host Cells Infected with Porcine

Aug 27, 2009 - Technology Development Center, Beijing 100045, PR China, and Chinese Institute ... for HIV-1,18 isotope-coded affinity tag (ICAT) techn...
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Differential Proteome Analysis of Host Cells Infected with Porcine Circovirus Type 2 Xin Zhang,†,‡ Jiyong Zhou,*,†,‡ Yongping Wu,†,‡ Xiaojuan Zheng,†,‡ Guangpeng Ma,§ Zhongtian Wang,¶ Yulan Jin,†,‡ Jialing He,†,‡ and Yan Yan†,‡ Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou 310029, PR China, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University, Hangzhou 310003, PR China, China Rural Technology Development Center, Beijing 100045, PR China, and Chinese Institute of Veterinary Drug Control, Beijing 100081, PR China Received June 2, 2009

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome, which is an emerging swine immunosuppressive disease. To uncover cellular protein responses in PCV2-infected PK-15 cells, the comprehensive proteome profiles were analyzed utilizing two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification. Multiple comparisons of 2-DE revealed that the majority of changes in protein expression occurred at 48-96 h after PCV2 infection. A total of 34 host-encoded proteins, including 15 up-regulated and 19 downregulated proteins, were identified by MALDI-TOF/TOF analysis. According to cellular function, the differential expression proteins could be sorted into several groups: cytoskeleton proteins, stress response, macromolecular biosynthesis, energy metabolism, ubiquitin-proteasome pathway, signal transduction, gene regulation. Western blot analysis demonstrated the changes of R tubulin, β actin, and cytokeratin 8 during infection. Colocalization and coimmunoprecipitation analyses confirmed that the cellular R tubulin interacts with the Cap protein of PCV2 in the infected PK-15 cells. These identified cellular constituents have important implications for understanding the host interactions with PCV2 and brings us a step closer to defining the cellular requirements for the underlying mechanism of PCV2 replication and pathogenesis. Keywords: porcine circovirus type 2 • proteome analysis • two-dimensional gel electrophoresis • MALDITOF/TOF • PK-15 cell

Introduction Porcine circovirus type 2 (PCV2), a member of the Circoviridae family, is the most important pathogen in PCV2-associated disease.1,2 Three viral proteins of PCV2, with a genome size of 1.7 kb, have been identified: the replication (Rep) protein that is associated with viral replication,3 the ORF3-encoding protein that is involved in cell apoptosis,4 and the capsid (Cap) protein that is responsible for immunity.5 The antigenic epitopes of PCV2-Cap protein have been finely mapped,6 and CD8+ T cells were indicated to be responsible for the protective immunity of PCV2.7 Furthermore, the cysteine residue of PCV2-Cap protein, which is surrounded by a well-conserved 12-16 aa * To whom correspondence should be addressed: Jiyong Zhou, Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, P. R. China. E-mail: [email protected]. Tel: +86-571-8697-1698. Fax: +86-571-8697-1821. † Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, Zhejiang University. ‡ State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University. § China Rural Technology Development Center. ¶ Chinese Institute of Veterinary Drug Control. 10.1021/pr900488q CCC: $40.75

 2009 American Chemical Society

region, may be responsible for interactions with other proteins.8 However, the immunosuppressive and pathogenic mechanisms have remained unclear in PCV2-infected pigs. Proteome analysis of host cellular responses to virus infection is more likely to probe potential cellular factors involved directly or indirectly in viral infection. Some previous studies have utilized proteomics methods to examine the kinetics of protein expression of the sensitive cells under investigation during viral infection.9 Among the techniques of the differentially expressed protein spot analysis, two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification has been extensively adopted, due to the simplicity and versatility of these techniques.10 With these methods, comparative proteomics of host cells has been investigated during viral infection, including African swine fever virus,11 Epstein-Barr virus,12 hepatitis B virus,13 classical swine fever virus,14 infectious bursal disease virus,15 severe acute respiratory syndromeassociated coronavirus,16 and porcine reproductive and respiratory syndrome virus.17 In addition, there are several other protein separation methods used to study virus-induced changes in the cellular proteome, such as DIGE followed by MS analysis for HIV-1,18 isotope-coded affinity tag (ICAT) technology Journal of Proteome Research 2009, 8, 5111–5119 5111 Published on Web 08/27/2009

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Zhang et al. 19

coupled with two-dimensional LC-MS/MS for SARS, and multidimensional liquid chromatographic separations coupled with mass spectrometry for hepatitis C virus (HCV).20 Proteomics analysis enables a more comprehensive characterization of virus-virus and virus-host interactions involved in infection and pathogenesis.9 Therefore, such methods adopted for viral proteomics ultimately allow for the complete analysis of the virion and infected host cell proteomes and yield insights into the induced alterations of signaling pathways to further understand viral pathogenesis. In this study, a 2-DE/MS proteomics approach was utilized, followed by Western blot analysis, colocalization and coimmunoprecipitation assay, to determine the differentially expressed protein profiles of the PK-15 cell line during PCV2 infection. A total of 37 differentially expressed protein spots corresponding to 34 proteins were identified. Further analysis of these data will allow for a greater understanding of PCV2 pathogenesis and the virus-host interactions.

Experimental Procedures Cell Culture, Virus Infection, and Sample Preparation. The PCV2 isolate TZ0601 strain (accession no. EU257511), which was isolated from a pig farm in the Zhejiang province of China,6,21 was propagated on the PK-15 cell monolayer. The protein was extracted as previously described.15 Briefly, the PK15 cells were infected with TZ0601 at a multiplication of infection (MOI) of 1, and the cells were harvested using a cell scraper at 12, 48, and 96 h post-infection (p.i.), and centrifuged at 10 000g 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 3/10 and 1 mM PMSF) containing DNase I (20 units/mL) /RNase A (0.25 mg/mL) in the final concentration and were vertically vibrated for 1 min on ice at 2 min intervals until the cells were completely lysed. The supernatant was collected after centrifuging at 12 000g at 4 °C for 60 min. The protein concentration was determined by the Bradford method.22 Mock-infected PK-15 cells were run in parallel and used as control. 2-DE and Image Analysis. 2-DE was performed using the 24 cm ReadyStrip IPG strips (Linear, pI 5-8, Bio-Rad, Hercules, CA) in the PROTEAN IEF Cell and PROTEAN plus Dodeca Cell (Bio-Rad), as previously described15,23 with some modifications. The IPG strips were rehydrated for 14 h with 450 µL of rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 65 mM DTT, 0.2% Pharmalyte 3/10), which contained 250 µg of proteins. The rehydration and separation programs were automatically processed using the following parameters: 50 V, 14 h; 250 V, linear, 1 h; 1000 V, linear, 1 h; 10 000 V, linear, 5 h; 10 000 V, rapid, 90 000 Vh. The isoelectric-focused proteins in strips were incubated for 15 min in 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 equilibration buffer containing 2.5% iodoacetamide. The equilibrated IPG strips were further resolved with 12% SDSPAGE gels at 80 V for 45 min and then 200 V until the dye front reached the bottom of gels. Gels were stained by the modified silver staining method compatible with MS24 and scanned at 500 dpi resolution using the Uniscan D3000 scanner (Tsinghua, China). Spot detection, spot matching, and quantitative intensity analysis were performed using PDQuest 2-D analysis software (Bio-Rad). The gel images were normalized according to the total quantity in the analysis set. Relative comparison 5112

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of spot intensity between PCV2-infected groups and the mockinfected groups at three time points, including, respectively, three biological and technical replicates for each group, were performed using the Student’s t test. Expression intensity ratios of infected/uninfected values greater than 2.0 (p e 0.05) or less than 0.5 (p e 0.05) were set as the thresholds to indicate significant changes. Protein Identification by MALDI-TOF/TOF Mass Spectrometry and Database Search. The protein spots of interest were manually excised from the silver-stained gels and then transferred to V-bottom 96-well microplate loaded with 100 µL of 50% acetonitrile (ACN)/25 mM ammonium bicarbonate solution per well. The in-gel tryptic digest was performed as previously described.15,23 Subsequently, the peptides mixtures were redissolved in 0.8 µL matrix solution (R-cyano-4-hydroxycinnamic acid (Sigma) in 0.1% TFA, 50% ACN) and, then, spotted on the MALDI plate. Samples were allowed to air-dry and analyzed by a 4700 MALDI-TOF/TOF Proteomics Analyzed (Applied Biosystems, Foster City, CA). Trypsin-digested peptides of myoglobin were added to the six calibration spots on the MALDI plate to calibrate the mass instrument with internal calibration mode. The UV laser was operated at a 200-Hz repetition rate at a wavelength of 355 nm. The accelerated voltage was operated at 20 kv. All acquired spectra of samples were processed using 4700 Explore software (Applied Biosystems) in a default mode. Parent mass peaks with mass range of 700-3200 Da and minimum signal-to-noise ratio of 20 were chosen for tandem TOF/TOF analysis. Combined MS and MS/ MS spectra were subjected to MASCOT (Version 2.1, Matrix Science, London, U.K.) by GPS Explorer software (version3.6, Applied Biosystems) and searched with the following parameters: National Center for Biotechnology information nonredundant (NCBInr) database (release date, June 27, 2007), taxonomy of bony vertebrates or viruses, trypsin digest with one missing cleavage, no fixed modifications, MS tolerance of 0.2 Da, MS/MS tolerance of 0.6 Da, and possible oxidation of methionine. Known contaminant ions (tryptic autodigest peptides) were excluded. A total of 4 182 491 sequences and 1 439 956 234 residues in the database were actually searched. MASCOT protein scores (based on combined MS and MS/MS spectra) greater than 69 were considered statistically significant (p e 0.05). The individual MS/MS spectrum, with a statistically significant (confidence interval g95%) ion score (based on MS/ MS spectra), was accepted. To eliminate the redundancy of proteins that appeared in the database under different names and accession numbers, the single protein member belonging to the species Sus scrofa or with the highest protein score (top rank) was separated from the multiprotein family. Western Blot. Samples of PCV2-infected and uninfected PK15 cells were lysed at 12, 48, and 96 h p.i., and the protein concentration was determined. Equivalent amounts of cell lysates (80 µg) were subjected to 15% SDS-PAGE gels and then transferred to 0.22 µm nitrocellulose membranes (Hybond-C extra, Amersham Biosciences). After blotting, the membranes were incubated, respectively, with mouse monoclonal antibodies (mAbs) to R tubulin (TUBA1A, Abcam, Cambridge, U.K.), β actin (ACTB, Abcam, Cambridge, U.K.), cytokeratin 8 (KRT8, Acris Antibodies GmbH, Hiddenhausen, Germany), and rabbit polyclonal antibody to proteasome activator complex subunit 1 (PSME1, Abcam, Cambridge, U.K.) at 37 °C for 60 min. After washing three times with 0.05% PBST, the membranes were inoculated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaith-

Differential Proteome Analysis of Host Cells Infected with PCV2 ersburg, MD) or goat anti-rabbit IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) at 37 °C for 60 min and visualized using 3,3′,5,5′-tetramethylbenzidine-stabilized substrate (TMB) (Promega, Madison, WI). Immunofluorescence Assay (IFA). PK-15 cells inoculated with PCV2 were cultured for 72 h. The cells were washed twice with PBS and fixed with cold acetone/methanol (1/1) for 20 min at -20 °C, and then allowed to air-dry. After membranes were blotted with 5% skimmed milk powder, the fixed cells were incubated, respectively, with a swine IgG to PCV2, mAb to Cap protein of PCV2, or mAb to Rep protein of PCV2, which were prepared in our lab,6,25,26 for 60 min at 37 °C in a humidified chamber. After three washes with 0.05% PBST, the fixed cells were incubated, respectively, with FITC-labeled goat anti-swine IgG (1:200, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) or FITC-labeled goat anti-mouse IgG (1:400, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). After three washes with 0.05% PBST, the fixed cells were incubated, respectively, with the mAbs to R tubulin, β actin, cytokeratin 8 or rabbit IgG to R tubulin (Abcam, Cambridge, U.K.). After three washes with 0.05% PBST, the cells were incubated for 60 min at 37 °C with a corresponding PE-conjugated goat anti-mouse IgG (1:400, Southern Biotechnology Associates, Inc.) or TRITCconjugated goat anti-rabbit IgG (1:400, Sigma, Saint Louis, MO). The additional nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was performed as described previously.27 The triple-stained cells were washed three times with 0.05% PBST and subsequently examined under a Zeiss LSM510 laser confocal microscopy. Coimmunoprecipitation Assay. The lysate of PK-15 cells infected with PCV2 for 72 h was prepared in 1 mLof lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) containing a protease inhibitor PMSF (1 mM). After centrifugation at 12 000g for 15 min, lysate supernatant was pretreated with protein A/G PLUS-agarose (Santa Cruz Biotechnology) for 30 min at 4 °C to eliminate nonspecific binding to the agarose gel. The lysate supernatant (500 µg) was incubated with 1 µg of mAb to R tubulin or mAb to Cap protein of PCV2 overnight at 4 °C. Then, 20 µL of resuspended Protein A/G PLUS-Agarose was added to this mixture and incubated at 4 °C on a rocker platform for overnight. After washing four times with lysis buffer, the isolated immunoprecipitated proteins were then analyzed by Western blot analysis using the mAb to Cap protein6 and mAb to R tubulin. The lysate of mockinfected PK-15 cells was used as the control.

Results Confirmation of PCV2 Propagation in PK-15 Cells by IFA and Western Blot. To obtain a detailed comparison of differences in protein expression profile, the cellular proteins were extracted for 2-DE analysis at 12, 48, and 96 h p.i. from PCV2-infected and mock-infected PK-15 cells and were identified by IFA using swine IgG to PCV226 and Western blot analysis using mAb to Rep protein of PCV225 as the primary antibody. IFA analysis revealed that the PK-15 cells infected with PCV2 could be recognized with swine IgG to PCV2 at 12, 48, and 96 h p.i. (Figure 1A), although in Western blot analysis the Rep protein of PCV2 was only detected at 48 and 96 h p.i. from PCV2- infected PK-15 cells (Figure 1B), indicating that PCV2 virions begin to replicate in the inoculated PK-15 cells at 12 h p.i. 2-DE Profiles of PCV2-infect PK-15 Cells. In the 2-DE analysis, the detectable protein spots ranged from 1450 to 1600

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Figure 1. Identification of the PCV2 infected and noninfected PK15 cells. Identification of PCV2 infected and mock-infected PK15 cells using the swine IgG to PCV2 by the method of IFA (A). Check of the Rep protein in PCV2-infected and mock-infected PK15 cells using the mAb to Rep protein of PCV2 by the method of Western blot (B). P+ and P- represent the PCV2 infected and uninfected PK-15 cells, respectively.

Figure 2. Protein spot numbers detected in infected and mockinfected PK-15 cells at different intervals after PCV2 infection.

on the 24-cm two-dimensional gels (pI 5-8) loaded with 250 µg of total cellular proteins per gel. In the PCV2-infected PK-15 cells, no obvious changes were observed in the number of detectable protein spots at 12 h p.i., but the number of detectable protein spots decreased gradually from 48 to 96 h p.i. in PCV2-infected PK-15 cells compared with the mock-infected PK-15 cells (Figure 2). From the average intensity ratios of detectable protein spots, a total of 61 protein spots were found to dynamically change in PCV2-infected PK-15 cells (Tables S1 and S2), including 24 significantly down-regulated protein spots (ratioinfection/mock e 0.5, p e 0.05) and 37 significantly up-regulated protein spots (ratioinfection/mock g 2, p e 0.05). In the up-regulated protein spots, 8 protein spots were up-regulated at 48 h p.i., and 29 protein spots were up-regulated at 96 h p.i. However, in comparison with mockinfected PK-15 cells, 4 of the 8 protein spots that were upregulated in the PCV2-infected PK-15 cells at 48 h p.i. did not exhibit such changes at 96 h p.i., and 5 of 29 up-regulated protein spots at 96 h p.i. were novel. For all down-regulated protein spots, 1 protein spot was down-regulated at 12 h p.i., 5 protein spots exhibited down-regulation at 48 h p.i., and 18 protein spots appeared to be down-regulated at 96 h p.i. Therefore, the majority of differential expression in PCV2-infected PK-15 cell proteins occurred at 48-96 h p.i. Mass Spectral Identification of Differentially Expressed Proteins. To identify the differentially expressed proteins in PCV2-infected PK-15 cells, a total of 61 protein spots with a Journal of Proteome Research • Vol. 8, No. 11, 2009 5113

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Figure 3. Dynamic 2-DE profiles of the differentially expressed proteins in PCV2-infected PK-15 cells. Circles indicate the differentially expressed protein spots. P+ and P- indicated the PCV2 infected and uninfected PK-15 cells, respectively.

threshold greater than 2-fold were excised from these 2-DE gels and subjected to in-gel trypsin digestion and subsequent MALDI-TOF/TOF identification. As shown in Figure 3 and Table S1, 37 differentially expressed protein spots, comprising 5114

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18 up-regulated and 19 down-regulated protein spots, were successfully identified (the MS and MS/MS spectra are listed in Figure S1). According to the protein function and subcellular annotations from the Swiss-Prot and TrEMBL protein database

Differential Proteome Analysis of Host Cells Infected with PCV2

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Figure 4. Western blot confirmation of representative proteins in PCV2 infected PK-15 cells. (A) The immunoblot analysis of KRT8, TUBA1A and ACTB. (B) The averaged densitometric intensity of KRT8, TUBA1A and ACTB in immunoblot analysis. P+ and P- represent the PCV2-infected and mock-infected PK-15 cells, respectively. PM, protein marker.

(us.expasy.org/sprot/) and Gene Ontology Database, the identified cellular proteins comprised 8 cytoskeletal proteins, 1 stress response protein, 8 macromolecular biosynthesis proteins, 12 energy metabolism-associated proteins, 4 ubiquitin-proteasome pathway (UPP) proteins, 3 signal transduction proteins, and 1 gene regulation proteins. The 18 up-regulated spots, which corresponded to the 15 proteins, included proteins that were cytoskeleton-associated proteins (38%), energy metabolism-associated proteins (28%), macromolecular biosynthesis proteins (11%), signal transduction proteins (11%), ubiquitin proteasome pathway proteins (6%), and gene regulation proteins (6%) (Figure S2A). These up-regulated proteins (Figure S2B) were mainly located in the cytoskeleton (34%), cytosol (18%), and mitochondrion (24%). The actin-18 (ACT18), in particular, was expressed during late stages of PCV2 infection as newly induced proteins. A total of 19 down-regulated spots were found to correspond to 19 cellular proteins. These proteins were determined to be primarily involved in energy metabolism (37%), macromolecular biosynthesis (32%), UPP (16%), intermediate filament (IF) (5%), signal transduction (5%), and other functions (Figure S2C). These down-regulated proteins were primarily distributed within the cytoplasm (40%), and they also occurred in the nucleus (20%), cytoplasm as well as the nucleus (15%), membrane (10%), ribosome (10%), and cytoskeleton (5%) (Figure S2D). In addition, some different spots were identified to be products of the same gene, including γ actin (ACTG1), ACT18 and TUBA1A. Western Blot Confirmation of the Representative Proteins in PCV2-Infected PK-15 Cells. To further validate the proteins identified by 2-DE and MALDI-TOF/TOF mass spectrometry, the proteins PSME1, ACTB, TUBA1A and KRT8 were selected

for Western blot analysis during PCV2 infection. Equal amounts of cell lysates from PCV2-infected and mock-infected PK-15 cells at 12, 48, and 96 h p.i. were examined with antibodies to PSME1, ACTB, TUBA1A, and KRT8, respectively. Data shown in Figure 4 indicate that ACTB, TUBA1A, and KRT8 were recognized with the respective mAbs, whereas mAb to PSME1 was not reactive to the PSME1 of PK-15 cells (data not shown). From the Figure 4 we can see that KRT8 was down-regulated (48 and 96 h p.i.) and TUBA1A was up-regulated (48 and 96 h p.i.), which were consistent with the 2-DE analysis. These data validate the MALDI-TOF/TOF identification of the differentially expressed proteins in the PCV2-infected PK-15 cells. However, the expression of ACTB did not exhibit the differences in the Western blot analysis compared to the kinetic changes determined in the 2-DE analysis. Colocalization of Cellular and PCV2-Encoded Proteins. To visualize the virus-host interactions in PCV2-infected cells, the double staining IFA analysis was performed using the mAbs to R tubulin, β actin, and cytokeratin 8, rabbit serum to R tubulin, swine anit-PCV2 serum and the mAbs to Cap and Rep proteins of PCV2. As shown in Figure S3, PCV2-encoding proteins were colocalized with the cellular R tubulin in PCV2-infected cells, but not with the cellular proteins β actin and KRT8. To further identify the colocalized PCV2-encoding protein with the cellular R tubulin, the mAbs to Cap and Rep proteins of PCV2, rabbit serum to R tubulin were used for the double staining IFA. As shown in Figure 5, the cellular R tubulin is only colocalized with the Cap protein of PCV2, but not with the Rep protein of PCV2, indicating that the Cap protein of PCV2 maybe interacts with the cellular R tubulin. Immunoprecipitation Results. The immunoprecipitation assay was utilized to further elucidate whether viral protein Journal of Proteome Research • Vol. 8, No. 11, 2009 5115

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Figure 5. Colocalization of cellular R tubulin and viral proteins. In the PCV2-infected PK-15 cells, the R tubulin reacted with the TRITC-conjugated (Red) rabbit IgG to R tubulin can overlap with the Cap protein recognized with the FITC-labeled (green) mAb to Cap protein of PCV2 (A), but can not be covered by Rep protein recognized with the FITC-labeled mAb to Rep protein of PCV2 (B). The nucleus was stained with DAPI (blue). The triple-stained cells were observed by a Zeiss LSM510 laser confocal microscopy. The arrows represent PCV2-infected PK15 cells. Bar is 10 µm.

Figure 6. Coimmunoprecipitation of viral Cap protein and cellular R tubulin. Panel A shows that Cap protein of PCV2 was precipitated by mAb to R tubulin in PCV2-infected PK-15 cells but not in mock-infected PK-15 cells. Panel B demonstrates that cellular R tubulin was precipitated by Cap protein of PCV2 in PCV2infected PK-15 cell but not in mock-infected PK-15 cells. P+ and P- represent the PCV2-infected and mock-infected PK-15 cells, respectively. PM, protein marker.

interacted with the cellular R tubulin in the PCV2-infected PK15 cells. From the immunoprecipitation results (Figure 6A), we can see that the Cap protein of PCV2 can be precipitated by the mAb to cellular R tubulin in PCV2-infected PK-15 cells. Correspondingly, the cellular R tubulin can be also precipitated by the mAb to Cap protein of PCV2 in PCV2-infected PK-15 cells (Figure 6B). Similar results were not observed in the mockinfected PK-15 cells. These results demonstrate that the cellular R tubulin interacts with the Cap protein of PCV2.

Discussion Proteomics is a novel methodology to detect the components of cellular protein interactions as well as host cellular pathophysiological processes that occur during virus infection.9,15 5116

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Zhang et al. Until the present investigation, no research had been reported for performing analysis of the differential proteome of host cells infected with PCV2. In this study, 2-DE coupled with MALDITOF/TOF was used to analyze the differential proteome of PK15 cells infected with PCV2. The 34 identified cellular proteins function in gene regulation, cytoskeleton organization, signal transduction, the stress response, the ubiquitin-proteasome pathway, energy metabolism, and macromolecular biosynthesis. Also, the interplay between Cap protein of PCV2 and cellular R tubulin was further demonstrated via IFA and immunoprecipitation assay. These results provide critical clues for further analysis of PCV2 pathogenesis, and this is the dynamic overview reported of the altered protein expression of host cells responding to PCV2 infection. In our experiment, the cell culture condition, cell collection time and 2-DE protein concentration were isochronous and identical in PCV2-infected and mock-infected cells. However, an interesting phenomenon is that more protein spots appeared in mock-infected cells over time in comparison with PCV2-infected cells, especially during 96 h of mock-infected cells. In comparison with mock infection, PCV2 did not induce visible cytopathic effects; furthermore, the full cell monolayer still appeared in the PCV2-infected cells. Therefore, the most possible explanation of this phenomenon is that PCV2 infection changed and inhibited the profile of the cellular protein expressions. In addition, we also find that the proteins KRT8, GALM, PAGM, and NCK1 (Figures 3 and 4) are constantly expressed during PCV-2 infection, but up-regulated in mockinfected cells. Actually, this implies that the expression of four proteins, KRT8, GALM, PAGM, and NCK1, was down-regulated in PCV2 replication. Functional Disorders of the Ubiquitin-Proteasome Pathway (UPP) in PCV2-Infected PK-15 Cells. UPP, a major intracellular system for protein degradation, plays an important role in a wide variety of cellular functions, such as antigen processing, cell cycle regulation, apoptosis, signal transduction, transcriptional regulation, and DNA repair.28 Some viruses have been reported to evolve different strategies to utilize the UPP for beneficial reasons, including the indication that ubiquitinproteasome system is required for avoidance of host immune surveillance during HIV-129 and is necessary for transcriptional regulation of the DNA virus of herpes simplex virus (HSV).30 Zheng et al.15 determined that there were dynamic changes of 9 ubiquitin-proteasome-associated proteins [polyubiquitin, proteasome subunit beta-type, proteasome subunit alpha type 2, proteasomal ATPase, prosomal P27K protein, proteasome activator subunit 3, ubiquitin carboxyl-terminal esterase L1, huntingtin interacting protein 2, and carboxyl-terminal hydrolase L5] in IBDV-infected host cells. Leong and Chow31 also indicated the down-regulation of proteasome subunit alpha type 2 and ubiquitin carboxyl-terminal esterase L3 in enterovirus 71-infected cells. In this study, PCV2 infection induced expression of the proteasome subunit p40 protein (MOV40), and the down-regulation of the proteins proteasome beta 3 subunit (PSMB3), PSME1, and suppressor of G2 allele of SKP1 proteins (SGT1) (Table S1, Figure 3). These results demonstrate that the cellular UPP system is disordered in function after PCV2 infection and that PCV2 differs from the reported viruses in the disturbance of cellular UPP. PCV2 Infection Decreased the Host Macromolecular Biosynthesis. Eukaryotic elongation factor-2 (eEF2) mediates the translocation step during the elongation phase of protein synthesis in eukaryotic cells.32 The replication protein A2

Differential Proteome Analysis of Host Cells Infected with PCV2 (RPA2) is a specific component of the preinitiation complex for replication.33 Acidic ribosomal phosphoprotein P0 (ARPP0) is located in the active part of the ribosome particle, at which mRNAs, tRNAs and translation factors interact during protein synthesis.34 Tyrosyl-tRNA synthetase (YARS2) catalyze specific attachment of amino acids on the respective cognate tRNAs and, therefore, are essential factors for correct expression of the genetic code at the translational level.35 In this study, eEF2 and RPA2 were down-regulated in PCV2-infected PK-15 cells (Table S1, Figure 3), which implied that the PCV2 interfered with the cellular protein synthesis and translation elongation of the host cell for beneficial reasons. In contrast, the cellular proteins YARS2 and ARPP0, which are associated with macromolecular biosynthesis, were up-regulated in PCV2-infected PK-15 cells (Table S1, Figure 3). Therefore, up-regulation of YARS2 and ARPP0 may be required for PCV2 propagation in host cells. Up-regulation of the macromolecular biosynthesisassociated proteins were also indicated during other viral infection, such as YARS2 in HIV-1-infected cells36 and ARPP0 in EBV-infected and IBDV-infected cells.15,37 PCV2 Infection Hijacks Cellular RNA Processing and Energy Metabolism. There was decreased expression of cellular proteins associated with RNA processing and biosynthesis (Table S1, Figure 3), which included purine nucleoside phosphorylase (PNP), a ubiquitous enzyme of purine metabolism that functions in the salvage pathway;38 GMP synthetase (GMPS), which catalyzes the ATP-dependent formation of GMP from xanthosine 5′-phosphate and glutamine;39 Hypoxanthineguanine phosphoribosyltransferase (HGPRT), a key enzyme in the salvage pathways for purine nucleotide synthesis that catalyzes the reversible transfer of the 5-phosphoribosyl group to form nucleotide IMP or GMP;40 and histidine triad protein member 5 (HINT5), mRNA decapping protein.41 Furthermore, the proteomics data revealed that the key proteins associated with energy metabolism were down-regulated (Table S1, Figure 3), including galactose mutarotase (GALM), an enzyme required for rapid lactose metabolism;42 transaldolase (TALDO), an enzyme in the oxidative pentose phosphate pathway; peptidyl prolyl isomerase D (PPID), also known as cyclophilin D, which is one of the peptidyl prolyl isomerases that catalyze protein conformational changes;43 alpha enolase (ENO1), which catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate and plays an important role in various pathophysiological processes;44 6-phosphogluconolactonase (6PGL), an enzyme that hydrolyzes 6-phosphogluconolactone to 6-phosphogluconate in the pentose-phosphate pathway;45 and phosphoglucomutase 3 (PAGM), a member of the phosphoglucomutases, which provide the glycolytic intermediate glucose 6-phosphate from glucose 1-phosphate.46 On the basis of the differential proteome analysis, we speculate that PCV2 replication hijacks the host cellular RNA processing and energy metabolism, although some expression of other energy metabolism-associated proteins, including isocitrate dehydrogenase (IDH3A) and pyruvate dehydrogenase E1 component subunit beta (PDHB), was induced in PCV2-infected PK-15 cells. The heat shock protein beta-1 (HSP27), which belongs to a family of small heat shock proteins, can affect protein assembly and may also participate in protein degradation.47 In addition, HSP27 can prevent caspase-independent apoptosis.48 In this study, HSP27 was down-regulated in PCV2-infected cells (Table S1, Figure 3), which was similar to previous results with adenovirus49 and hepatitis B virus.50 In contrast, the HSP27

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was up-regulated in swine fever virus, feline herpesvirus, enterovirus 71, and IBDV infections,11,15,31,51 indicating that the dynamic profile of HSP27 expression is different in various virus infections and is not specific for PCV2 infection. Cytoskeletal protein expression was altered in PCV2-infected PK-15 cells. The cytoskeleton consists of a filament scaffold within the cell. These filaments are dynamic and divided into three types: microfilaments (actin filament), microtubules, and intermediate filaments.52 The intermediate filaments can provide mechanical stability to cells, while the actin and microtubule cytoskeletons are responsible for trafficking of numerous endogenous cargos as well as intracellular microorganisms throughout the cells.53 The actin filament can mediate transport of organelles along the cell formed from globular actin monomers,54 and microtubules can act as tracks to move cellular components based on polarized filaments.55 Many viruses use the cytoskeleton for infection and replication, such as HIV,56 vesicular stomatitis virus,57 simian virus 40,58 and herpes simplex virus type 1.59 In this study, the microfilamentassociated proteins ACTB, ACT18 and ACTG1, microtubuleassociated TUBA1A, were up-regulated (Table S1, Figure 3), while the intermediate filament-associated protein KRT8 were down-regulated, indicating that PCV2 infection and replication involves the cellular skeleton. Inhibition of actin polymerization has been reported to block the PCV2 infection in PCV2-infected epithelial cells,60 and the microfilament-associated actin was critical during PCV2 internalization into dentritic cells.61 However, up-regulated ACTB and TUBA1A have been detected in IBDV-infected cells,15 influenza A virus-infected cells,62 and HIV-1-infected cells,36 demonstrating that the up-regulation of ACTB and TUBA1A is not specific for PCV2 infection. But, in our study, we demonstrated that the Cap protein of PCV2 interacts with the microtubule-associated R tubulin by double staining immunofluorescence and immunoprecipitation assays (Figure 5A and Figure 6). This data provides important evidence to further understand the pathogenesis and replication of PCV2. In summary, this study was the first to utilize proteomics to elucidate the cellular responses to PCV2 infection. A total of 34 altered cellular proteins, including 15 up-regulated and 19 down-regulated proteins, were identified in the PCV2-infected PK-15 cells, and the comparative proteomics approach demonstrated that most of the altered cellular proteins appear to have roles in viral pathogenesis. The interaction between the cellular protein R tubulin and Cap protein of PCV2 was confirmed in PCV2-infected PK-15 cells. Therefore, further large-scale studies are necessary to understand the roles of the differentially expressed cellular proteins in PCV2 infection. Abbreviations: DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; CHAPS, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate; ACN, acetonitrile; IEF, isoelectric focusing; IPG, immobilized pH gradient; 2-DE, two-dimensional gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; PBST, phosphate buffered saline tween-20; IFA, indirect immunofluorescence assay; FITC, fluorescein isothocyanate; PE, phycoerythrin; TRITC, tetramethyl rhodamine isothiocyanate; DAPI, 4′,6diamidino-2-phenylindole.

Acknowledgment. This work was supported by National Natural Science Foundation of China (Grant No.30625030, 30700025) and Zhejiang Provincial Bureau of Science and Technology (Grant No.2008C22041). We thank Mr. Xin-Wen Zhou (Fudan University, China) for help with Journal of Proteome Research • Vol. 8, No. 11, 2009 5117

research articles MALDI-TOF/TOF mass spectrometry and Mr. Han-Min Chen for technical help on laser confocal microscopy.

Supporting Information Available: MS and MS/MS spectra of the 37 identified protein spots. Classification of the differentially expressed proteins in PCV2-infected PK-15 cells according to subcellular location and biological function. Subcellular location of KRT8, R-tubulin, and β-actin in PCV2infected PK-15 cells by IFA. Proteins differentially expressed in PCV2-infected versus mock-infected PK-15 as identified by MALDI-TOF/TOF or MALDI-TOF. List of the differentially expressed protein spots unsuccessfully identified by MALDITOF. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Allan, G. M.; Ellis, J. A. Porcine circoviruses: a review. J. Vet. Diagn. Invest. 2000, 12 (1), 3–14. (2) Opriessnig, T.; Meng, X. J.; Halbur, P. G. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J. Vet. Diagn. Invest. 2007, 19 (6), 591–615. (3) Mankertz, A.; Mankertz, J.; Wolf, K.; Buhk, H. J. Identification of a protein essential for replication of porcine circovirus. J. Gen. Virol. 1998, 79 (Pt 2), 381–384. (4) Liu, J.; Chen, I.; Kwang, J. Characterization of a previously unidentified viral protein in porcine circovirus type 2-infected cells and its role in virus-induced apoptosis. J. Virol. 2005, 79 (13), 8262– 8274. (5) Nawagitgul, P.; Morozov, I.; Bolin, S. R.; Harms, P. A.; Sorden, S. D.; Paul, P. S. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J. Gen. Virol. 2000, 81 (Pt. 9), 2281– 2287. (6) Shang, S. B.; Jin, Y. L.; Jiang, X. T.; Zhou, J. Y.; Zhang, X.; Xing, G.; He, J. L.; Yan, Y. Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus Type 2. Mol. Immunol. 2009, 46 (3), 327–334. (7) Shen, H. G.; Zhou, J. Y.; Huang, Z. Y.; Guo, J. Q.; Xing, G.; He, J. L.; Yan, Y.; Gong, L. Y. Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice. J. Gen. Virol. 2008, 89 (Pt 8), 1857–1865. (8) Timmusk, S.; Fossum, C.; Berg, M. Porcine circovirus type 2 replicase binds the capsid protein and an intermediate filamentlike protein. J. Gen. Virol. 2006, 87 (Pt. 11), 3215–3223. (9) Maxwell, K. L.; Frappier, L. Viral proteomics. Microbiol. Mol. Biol. Rev. 2007, 71 (2), 398–411. (10) Viswanathan, K.; Fruh, K. Viral proteomics: global evaluation of viruses and their interaction with the host. Expert Rev. Proteomics 2007, 4 (6), 815–829. (11) Alfonso, P.; Rivera, J.; Hernaez, B.; Alonso, C.; Escribano, J. M. Identification of cellular proteins modified in response to African swine fever virus infection by proteomics. Proteomics 2004, 4 (7), 2037–2046. (12) Toda, T.; Sugimoto, M.; Omori, A.; Matsuzaki, T.; Furuichi, Y.; Kimura, N. Proteomic analysis of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines before and after immortalization. Electrophoresis 2000, 21 (9), 1814–1822. (13) Narayan, R.; Gangadharan, B.; Hantz, O.; Antrobus, R.; Garcia, A.; Dwek, R. A.; Zitzmann, N. Proteomic analysis of HepaRG cells: a novel cell line that supports hepatitis B virus infection. J. Proteome Res. 2009, 8 (1), 118–122. (14) Sun, J. F.; Jiang, Y.; Shi, Z. X.; Yan, Y. J.; Guo, H. C.; He, F. C.; Tu, C. C. Proteomic Alteration of PK-15 cells after infection by classical swine fever virus. J. Proteome Res. 2008, 7 (12), 5263–5269. (15) Zheng, X.; Hong, L. L.; Shi, L. X.; Guo, J. Q.; Sun, Z.; Zhou, J. Y. Proteomics analysis of host cells infected with infectious bursal disease virus. Mol. Cell. Proteomics 2008, 7 (3), 612–625. (16) Chen, J. H.; Chang, Y. W.; Yao, C. W.; Chiueh, T. S.; Huang, S. C.; Chien, K. Y.; Chen, A.; Chang, F. Y.; Wong, C. H.; Chen, Y. J. Plasma proteome of severe acute respiratory syndrome analyzed by twodimensional gel electrophoresis and mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (49), 17039–17044. (17) Zhang, H. M.; Guo, X.; Ge, X. N.; Chen, Y. H.; Sun, Q. X.; Yang, H. C. Changes in the cellular proteins of pulmonary alveolar macrophage infected with porcine reproductive and respiratory syndrome virus by proteomics analysis. J. Proteome Res. 2009, 8 (6), 3091–3097.

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