Shotgun Identification of the Structural Proteome of Shrimp White Spot Syndrome Virus and iTRAQ Differentiation of Envelope and Nucleocapsid Subproteomes
2007; Elsevier BV; Volume: 6; Issue: 9 Linguagem: Inglês
10.1074/mcp.m600327-mcp200
ISSN1535-9484
AutoresZhengjun Li, Qingsong Lin, Jing Chen, Jinlu Wu, Teck Kwang Lim, Siew See Loh, Xuhua Tang, Choy‐Leong Hew,
Tópico(s)Insect symbiosis and bacterial influences
ResumoWhite spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection. White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection. Shrimp white spot syndrome virus (WSSV), 1The abbreviations used are: WSSV, white spot syndrome virus; 1D, one-dimensional; 2D, two-dimensional; C.I., confidence interval; hpi, hours postinfection; IEM, immunogold electron microscopy; iTRAQ, isobaric tags for relative and absolute quantification; SCX, strong cation exchange; s/n, signal to noise ratio. 1The abbreviations used are: WSSV, white spot syndrome virus; 1D, one-dimensional; 2D, two-dimensional; C.I., confidence interval; hpi, hours postinfection; IEM, immunogold electron microscopy; iTRAQ, isobaric tags for relative and absolute quantification; SCX, strong cation exchange; s/n, signal to noise ratio. which belongs to Nimaviridae (Whispovirus) family, is an enveloped, double-stranded DNA virus (1Wang C.H. Lo C.F. Leu J.H. Chou C.M. Yeh P.Y. Chou H.Y. Tung M.C. Chang C.F. Su M.S. Kou G.H. Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon.Dis. Aquat. 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WSSV originating from China contains a 305-kb double-stranded circular DNA, which encompasses 181 putative ORFs with 50 or more amino acids (9Yang F. He J. Lin X. Li Q. Pan D. Zhang X. Xu X. Complete genome sequence of the shrimp white spot bacilliform virus.J. Virol. 2001; 75: 11811-11820Crossref PubMed Scopus (460) Google Scholar). Most of these ORFs are of unknown function because no homologues to known genes can be found in public databases.Viral structural proteins have critical functions not only in viral structure and assembly but also in the early stages of infection, cell adhesion, signal transduction, and evasion of the rapidly deployed antiviral defenses of the host. Previously 18 structural proteins from WSSV were identified by using one-dimensional (1D) SDS-PAGE and MALDI-TOF or ESI Q-TOF mass spectrometers (10Huang C. Zhang X. Lin Q. Xu X. Hu Z. Hew C.L. Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.Mol. Cell. Proteomics. 2002; 1: 223-231Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Recently 33 WSSV structural proteins resolved by 1D SDS-PAGE were identified using the on-line LC-ESI Q-TOF mass spectrometer, increasing the number of structural proteins identified to 38 by these two proteomics studies (11Tsai J.M. Wang H.C. Leu J.H. Hsiao H.H. Wang A.H. Kou G.H. Lo C.F. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus.J. Virol. 2004; 78: 11360-11370Crossref PubMed Scopus (199) Google Scholar). Our previous study on Singapore grouper iridovirus suggested that the 1D gel-based approach and the LC-based shotgun approach are equally effective and complementary to each other (12Song W. Lin Q. Joshi S.B. Lim T.K. Hew C.L. Proteomic studies of the Singapore grouper iridovirus.Mol. Cell. Proteomics. 2006; 5: 256-264Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A number of novel viral proteins were detected by using the shotgun approach. In an effort to achieve a better understanding of the structural proteome of WSSV, shotgun proteomics, which involves direct digestion of total proteins to complex peptide mixtures, followed by the automated identification of the peptides by LC-MS/MS was initiated. In total, 45 viral structural proteins were identified from the purified WSSV, including 32 proteins previously identified and 13 proteins reported for the first time.Determining the localization of structural proteins in the virion is important to elucidate their roles in both virus assembly and infection. Western blot analysis and immunogold electron microscopy (IEM) are two conventional approaches to localize the viral proteins. For WSSV, IEM has been used to detect 13 envelope proteins and one nucleocapsid protein (10Huang C. Zhang X. Lin Q. Xu X. 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Wang C.H. Lo C.F. Kou G.H. Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells.Virology. 2002; 293: 44-53Crossref PubMed Scopus (90) Google Scholar, 24Huang R. Xie Y. Zhang J. Shi Z. A novel envelope protein involved in white spot syndrome virus infection.J. Gen. Virol. 2005; 86: 1357-1361Crossref PubMed Scopus (34) Google Scholar). In this study, we applied a complementary proteomics approach to examine the localization of structural proteins in the virion by iTRAQ (isobaric tags for relative and absolute quantification). iTRAQ is a newly developed LC-based quantitative proteomics approach, which allows for comparison of up to four different samples simultaneously (25Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3647) Google Scholar). It has been successfully applied to measure the enrichment of organelle proteins (26Chen X. Walker A.K. Strahler J.R. Simon E.S. Tomanicek-Volk S.L. Nelson B.B. Hurley M.C. Ernst S.A. Williams J.A. Andrews P.C. Organellar proteomics: analysis of pancreatic zymogen granule membranes.Mol. Cell. Proteomics. 2006; 5: 306-312Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) as an alternative approach to the localization of organelle proteins by isotope tagging using cleavable ICAT (27Dunkley T.P. Watson R. Griffin J.L. Dupree P. Lilley K.S. Localization of organelle proteins by isotope tagging (LOPIT).Mol. Cell. Proteomics. 2004; 3: 1128-1134Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) and protein correlation profiling (28Foster L.J. de Hoog C.L. Zhang Y. Xie X. Mootha V.K. Mann M. A mammalian organelle map by protein correlation profiling.Cell. 2006; 125: 187-199Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Determining the viral protein localization by iTRAQ is based on the principle that the enrichment of envelope proteins in the detergent-solubilized fraction and nucleocapsid proteins in the pellet fraction can be quantified by the reporter ions, whereas their protein identities can be determined by other MS/MS fragment ions. Using this approach, we identified 23 envelope proteins and six nucleocapsid proteins among which 12 envelope proteins and two nucleocapsid proteins are reported for the first time. Our results demonstrated that iTRAQ is a powerful approach for rapid protein localization and that it can also be applied to study other enveloped viruses. A better understanding of WSSV structural proteins and the localization in the virion will shed more light on virus assembly, its infection pathway, and the discovery of antiviral drugs.EXPERIMENTAL PROCEDURESProliferation and Isolation of WSSV Virions—WSSV used in this study originated from WSSV-infected Penaeus chinensis (China isolate). Virus inoculums were prepared from the hemolymph of infected red claw crayfish, Cherax quadricarinatus, as described previously (29Wu J.L. Suzuki K. Arimoto M. Nishizawa T. Muroga K. Preparation of an inoculum of white spot syndrome virus for challenge tests in Penaeu japonicus.Fish Pathol. 2002; 37: 65-69Crossref Scopus (19) Google Scholar). After centrifugation at 1,500 × g for 10 min, the supernatant was filtered with a 0.45-μm filter and injected intramuscularly into healthy crayfish. After 4–6 days, hemolymph extracted from moribund crayfish was centrifuged at 2,000 × g for 10 min. The supernatant was layered on top of a 30–60% (w/v) stepwise sucrose gradient and centrifuged at 53,000 × g for 1 h at 4 °C. The virus band was collected and then mixed with TN buffer (20 mm Tris-HCl, 400 mm NaCl, pH 7.4) and repelleted at 53,000 × g for 1 h at 4 °C. The resulting pellet was washed with TN buffer to remove sucrose and then resuspended in TN buffer. The purified virus samples were negatively stained with 2% phosphotungstic acid and examined under the transmission electron microscope to check the purity and quantity.Shotgun LC-MALDI MS Analysis of WSSV Structural Proteins—Viral protein extraction, in-solution digestion, and LC separation of tryptic peptides were carried out following procedures described previously (12Song W. Lin Q. Joshi S.B. Lim T.K. Hew C.L. Proteomic studies of the Singapore grouper iridovirus.Mol. Cell. Proteomics. 2006; 5: 256-264Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly 5 volumes of 50 mm Tris-HCl with 0.1% SDS, pH 8.5, were used to extract proteins from purified WSSV virions. The extracted proteins were reduced with triscarboxyethylphosphine, alkylated with iodoacetamide, and then digested with sequencing grade porcine trypsin (Promega, Madison, WI). The digested peptide mixture was separated using an Ultimate LC system (Dionex-LC Packings, Sunnyvale, CA) equipped with a Probot MALDI spotting device. Approximately 10 μg of peptide mixture were captured by a 0.3 × 1-mm trap column (3-μm C18 PepMap, 100 Å, Dionex-LC Packings) and separated by a 0.075 × 150-mm analytical column (3-μm C18 PepMap, 100 Å, Dionex-LC Packings) at a flow rate of 0.4 μl/min. The mobile phases A and B were 2% ACN, 0.05% TFA and 80% ACN, 0.04% TFA, respectively. The LC gradients used were 0–20% B in 10 min, then 20–60% B over 3 h, 60–100% B in 1 min, and kept at 100% B for 5 min. The LC fractions were mixed with MALDI matrix solution (7 mg/ml α-cyano-4-hydroxycinnamic acid and 130 μg/ml ammonium citrate in 75% ACN) before spotting onto MALDI target plates.An ABI 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA) was used to analyze the samples. The instrument was controlled by 4000 Series Explorer version 3.0. For MS analyses, typically 1,000 subspectra were accumulated. Peaks were detected with the minimum signal to noise ratio (s/n) set to 15, and the peaks were deisotoped. MS/MS analyses were carried out using nitrogen at a collision energy of 1 kV and a collision gas pressure of ∼9.0 × 10−7 torr. Two thousand to 10,000 subspectra were combined for each spectrum using stop conditions based on the quality of the data. The spectra were smoothed using the Savitsky-Golay method with points across peak (full-width half-maximum) set to 3, and polynomial order was set to 4. The peaks were deisotoped, and only the peaks with s/n ≥10 were picked.GPS Explorer software version 3.0 (Applied Biosystems) was used to create and search files with the MASCOT search engine version 2.0 (Matrix Science, Boston, MA) to identify viral proteins. A database (64,335 entries) containing all predicted ORFs from three WSSV isolates (2,013 entries) together with the International Protein Index human database version 3.16 (www.ebi.ac.uk/IPI/IPIhelp.html, 62,322 entries) was used to minimize false positive identifications. The search was restricted to tryptic peptides, and one missing cleavage was allowed. Cysteine carbamidomethylation, N-terminal acetylation and pyroglutamation (Glu or Gln), and methionine oxidation were selected as variable modifications. Precursor error tolerance and MS/MS fragment error tolerance were set to 60 ppm and 0.4 Da, respectively. Only the top ranked peptide matches were taken into consideration for protein identification. For peptide matches with an expect value >0.05, the MS/MS spectra were further validated manually.Bioinformatics Analysis of WSSV Structural Proteins—To characterize the previously unknown WSSV structural proteins, the homology analysis was achieved by searching InterProScan (30Zdobnov E.M. Apweiler R. InterProScan—an integration platform for the signature-recognition methods in InterPro.Bioinformatics. 2001; 17: 847-848Crossref PubMed Scopus (2119) Google Scholar, 31Quevillon E. Silventoinen V. Pillai S. Harte N. Mulder N. Apweiler R. Lopez R. InterProScan: protein domains identifier.Nucleic Acids Res. 2005; 33: W116-W120Crossref PubMed Scopus (1908) Google Scholar). Putative signal sequences and transmembrane domains were predicted by dense alignment surface (32Cserzo M. Eisenhaber F. Eisenhaber B. Simon I. On filtering false positive transmembrane protein predictions.Protein Eng. 2002; 15: 745-752Crossref PubMed Scopus (125) Google Scholar). PSORT was used for the prediction of protein cellular localization in cells based on the Swiss-Prot data.Isolation of Total RNA and RT-PCR—WSSV-infected crayfish gills were treated with RNAlater (Qiagen, Hilden, Germany). Total RNA was isolated from these tissues using an RNeasy minikit (Qiagen). To remove any residual DNA, RNA solution was treated with DNA-free kit (Ambion, Austin, TX) following the protocol described. Gene-specific primers were used to amplify the target genes by the One-Step RT-PCR kit (Qiagen) (primer pairs are provided in Supplemental Data I). All procedures were performed according to the manufacturer's instruction. Briefly cDNA was reverse transcribed at 50 °C for 45 min. The PCR amplification segment was started with an initial heating step at 95 °C for 15 min to activate HotStarTaq DNA polymerase and simultaneously inactivate reverse transcriptase. After activation of DNA polymerase, PCR amplification reactions were performed (40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min). A final extension step was carried out at 72 °C for 7 min. RT-PCR products were resolved on 1.2% agarose gels. The genes of two ORFs (wsv143 and wsv161) were divided into several short fragments (less than 1,600 bp) to get complete coverage of the entire coding region. For controls, purified RNA was added after inactivating reverse transcriptase to exclude the possibility of genomic DNA contamination.Separation of Viral Envelope Proteins and Nucleocapsid Proteins—A previously validated separation procedure described by Tsai et al. (23Tsai J.M. Wang H.C. Leu J.H. Wang A.H. Zhuang Y. Walker P.J. Kou G.H. Lo C.F. Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion.J. Virol. 2006; 80: 3021-3029Crossref PubMed Scopus (169) Google Scholar) was adopted with modification. Briefly purified virus was treated with buffer A containing 20 mm Tris-HCl, 5 mm EDTA-Na2, 1% Triton X-100, 0.5 m NaCl, 1× protease inhibitor mixture (Roche Diagnostics Asia Pacific Pte. Ltd.), pH 7.4, at 4 °C for 30 min. Then the virus was divided into two equal portions. One portion was set aside as a control for total viral proteins, whereas the other portion was separated into two fractions, supernatant and pellet, by centrifugation at 200,000 × g for 1 h at 4 °C. The pellet fraction was washed one more time with buffer A and centrifuged again for 1 h. The pellet was resuspended in an equal volume of buffer A as the envelope fraction.Western Blot Analysis of Envelope and Nucleocapsid Fractions—Virion-associated proteins from each fraction were resolved by 12% SDS-PAGE and processed for Western blot analysis. The nitrocellulose membrane was blocked with 5% nonfat milk in 1× TBST (20 mm Tris base, 137 mm NaCl, 0.1% Tween 20, pH 7.6) at room temperature and then subjected to Western blotting. Primary antibodies were used with the following concentrations: rabbit anti-VP28 (wsv421) polyclonal antibody (1:2,000) and mouse anti-VP24 (wsv002) and anti-VP466 (wsv308) polyclonal antibody (1:2,000). The secondary antibodies, anti-rabbit or anti-mouse horseradish peroxidase-conjugated antiserum, were diluted 1:5,000 (GE Healthcare). Pierce SuperSignal West Pico chemiluminescent substrate (Pierce) was used according to the manufacturer's protocol, and the protein bands were visualized using Lumi-Film chemiluminescent detection film (Roche Applied Science).iTRAQ Labeling and Two-dimensional (2D) LC-MALDI MS to Determine Viral Protein Localization—One hundred micrograms of total viral proteins and the equivalent envelope and nucleocapsid fractions (separated from 100 μg of total viral proteins) were processed using a 2D Clean-Up kit (GE Healthcare) and resuspended in the dissolution buffer (0.5 m triethylammonium bicarbonate, pH 8.5, containing 0.1% SDS). The samples were then reduced, and cysteines were blocked according to the protocol of the iTRAQ kit (Applied Biosystems). Ten microliters of a 1 μg/μl trypsin (Applied Biosystems) solution were added, and the samples were digested at 37 °C overnight. The samples were vacuum-dried, reconstituted with 30 μl of dissolution buffer, and labeled with iTRAQ tags as follows: total viral proteins, iTRAQ 114 reagent; the envelope fraction, iTRAQ 115 reagent; and the nucleocapsid fraction, iTRAQ 116 reagent. The labeled samples were then pooled and purified using a strong cation exchange (SCX) column (Applied Biosystems), and the bound peptides were eluted with 5% NH4OH in 30% methanol.After drying, the iTRAQ-labeled peptides were resuspended with 20 μl of 5 mm KH2PO4 buffer containing 5% ACN, pH 3.0, and separated using an Ultimate dual gradient LC system (Dionex-LC Packings). The first dimension separation used a 0.3 × 150-mm SCX column (FUS-15-CP, Poros 10S, Dionex-LC Packings), and the mobile phases A and B were 5 mm KH2PO4 buffer, pH 3, containing 5% ACN and 5 mm KH2PO4 buffer, pH 3, containing 5% ACN and 500 mm KCl, respectively, with a flow rate of 6 μl/min. The eluants with step gradients of mobile phase B (unbound, 0–5, 5–10, 10–15, 15–20, 20–30, 30–40, 40–50, and 50–100%) were captured alternatively with two 0.3 × 1-mm trap columns (3-μm C18 PepMap, 100 Å, Dionex-LC Packings) and washed with 0.05% TFA to remove salts. The second dimension separation was performed with a 0.2 × 50-mm reverse-phase column (Monolithic PS-DVB, Dionex-LC Packings) using 2% ACN with 0.05% TFA as mobile phase A and 80% ACN with 0.04% TFA as mobile phase B with a gradient of 0–60% mobile phase B in 15 min and a flow rate of 2.7 μl/min. The LC fractions were mixed with MALDI matrix solution in a flow rate of 5.4 μl/min through a 25-nl mixing tee (Upchurch Scientific, Oak Harbor, WA) and spotted onto 192-well MALDI target plates (Applied Biosystems) with a Probot Micro Fraction collector (Dionex-LC Packings).MS analysis was performed as described above, and the MS/MS analysis settings were the same as those for the shotgun analysis except that the collision gas pressure was changed to ∼2 × 10−6 torr. For the precursor ions with s/n ≥100, 6,000 shots were combined for each spectrum. For the precursors with s/n between 50 and 100, 10,000 shots were acquired. The peak processing and detection parameters were the same as those for the shotgun analysis described above. GPS Explorer software version 3.5 (Applied Biosystems) using the MASCOT search engine (version 2.1, Matrix Science) was used for peptide and protein identifications and iTRAQ quantification. The database used was the same as mentioned above and restricted to tryptic peptides. Cysteine methanethiolation, N-terminal iTRAQ labeling, and iTRAQ-labeled lysine were selected as fixed modifications, and methionine oxidation was selected as a variable modification. One missing cleavage was allowed. Precursor error tolerance and MS/MS fragment error tolerance were set to 120 ppm and 0.4 Da, respectively. Maximum peptide rank was set to 1, and minimum ion score confidence interval (C.I.; %) for peptide was set to 0. For proteins with low ion scores (≤30), the MS/MS spectra were manually inspected.Antibody Preparation and Western Blot Analysis of wsv432—The full-length gene of wsv432 was PCR-amplified and inserted into a modified pET vector containing a C-terminal His6 tag. After sequencing, the construct was transformed into Escherichia coli strain BL21 Star (DE3) (Invitrogen), and the protein was expressed after isopropyl β-d-thiogalactopyranoside induction at 18 °C. The recombinant protein was purified using a nickel-nitrilotriacetic acid column, and its identity was confirmed by MS. The antibody was prepared by Bam Biotech Co., Ltd. (Xiamen, Fujian, China) using the purified fusion protein to immunize the rabbits. Proteins from the virion, the envelope, and the nucleocapsid were resolved by SDS-PAGE and subjected to Western blot analysis as described above. The anti-wsv432 antibody was diluted 1:1,000.Localization of wsv432 in the Virion by IEM—The purified virus was treated with 0.1% Tween 20 at room temperature for 30 s. After washing with 0.2 m phosphate buffer, pH 7.3, to remove the detergent, the virus suspension was absorbed on carbon-coated nickel grids. Rabbit anti-wsv432 antibody was used to recognize wsv432 in the viral particles, whereas preimmune rabbit serum was included
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