Shotgun Identification of Structural Proteome of Shrimp White Spot Syndrome. Virus and itraq Differentiation of Envelope and Nucleocapsid Subproteomes

Transcription

1 MCP Papers in Press. Published on June 2, 2007 as Manuscript M MCP200 Shotgun Identification of Structural Proteome of Shrimp White Spot Syndrome Virus and itraq Differentiation of Envelope and Nucleocapsid Subproteomes Zhengjun Li, Qingsong Lin, Jing Chen, Jin Lu Wu, Teck Kwang Lim, Siew See Loh, Xuhua Tang, and Choy-Leong Hew Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore Running Title: Shotgun Proteomics and Subproteomes of WSSV Corresponding author: Professor Choy-Leong Hew Department of Biological Sciences National University of Singapore 14 Science Drive 4 Singapore Tel: Fax: dbshewcl@nus.edu.sg or dbshead@nus.edu.sg These authors contributed equally to this paper. 1 Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

2 ABBREVIATIONS 1D, one dimensional; 2D, two dimensional; bp, base pairs; C. I., confidence interval; hpi, hours post infection; IEM, immunogold electron microscopy; itraq, isobaric tags for relative and absolute quantification; MM, molecular mass; SCX, strong cation exchange; s/n, signal to noise ratio; WSSV, white spot syndrome virus. 2

3 SUMMARY 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 gelbased proteomic studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using offline coupling of LC system with MALDI TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach has 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 proteomic strategy, was employed to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on itraq ratios, we have successfully identified 23 envelope proteins and 6 nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore, the localization of additional 12 envelope proteins and 2 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 by itraq. The localization of 42 structural proteins has been determined, 33 of which are envelope proteins and 9 as nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection. 3

4 INTRODUCTION Shrimp white spot syndrome virus (WSSV), which belongs to Nimaviridae (Whispovirus) family, is an enveloped, double-stranded DNA virus (1, 2). It can cause up to 100 % mortality in shrimp within 7 to 10 days, resulting in huge economic losses to the shrimp farming industry (3, 4). This virus infects shrimp and other freshwater and marine crustaceans, including crabs and crayfish (5). Genomes of three WSSV isolates originating from China (GenBank accession No. AF332093), Taiwan (GenBank accession No. AF440570), and Thailand (GenBank accession No. AF369029), respectively, have been completely sequenced (6-9). WSSV originated from China contains a 305-kb double-stranded circular DNA, which encompasses 181 putative ORFs with 50 or more amino acids (9). Most of these ORFs are of unknown function as 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 host s rapidly deployed antiviral defenses. Previously, 18 structural proteins from WSSV were identified by using one dimensional (1D) SDS-PAGE and MALDI-TOF or ESI Q-TOF mass spectrometers (10). Recently, 33 WSSV structural proteins resolved by 1D SDS-PAGE were identified using the online LC-ESI Q-TOF mass spectrometer and this has increased structural proteins identified to 38 by these two proteomic studies (11). 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 (12). A number of novel viral proteins were detected by using the shotgun approach. In an effort to achieve a better understanding of the 4

5 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 (10, 13-22). Recently, a more systematic study on WSSV has led to the differentiation of 7 envelope proteins, 5 tegument proteins and 4 nucleocapsid proteins by Western blot analysis and 2 additional nucleocapsid proteins by MS (23). To date, the localization of 27 ORFs in the virion has been determined among the known structural proteins (6, 24). In this study, we applied a complementary proteomic 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 LCbased quantitative proteomic approach, which allows for comparison of up to four different samples simultaneously (25). It has been successfully applied to measure the enrichment of organelle proteins (26) as an alternative approach to the localization of organelle proteins by isotope tagging (LOPIT) using cleavable ICAT (27) and the protein correlation profiling (PCP) (28). Determining the viral protein localization by itraq is based on the principle that the enrichment of envelope proteins in the detergentsolubilized fraction and nucleocapsid proteins in the pellet fraction can be quantified by 5

6 the reporter ions, while their protein identities can be determined by other MS/MS fragment ions. Using this approach, we have identified 23 envelope proteins and 6 nucleocapsid proteins, among which, 12 envelope proteins and 2 nucleocapsid proteins are reported for the first time. Our results demonstrated that itraq is a powerful approach for rapid protein localization and 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 PROCEDURES Proliferation 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 (29). 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 to 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 to 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 and 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. 6

7 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 the procedures as described previously (12). Briefly, five 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 and 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 mm trap column (3-µm C 18 PepMap, 100 Å, Dionex-LC Packings) and separated by a mm analytical column (3-µm C 18 PepMap, 100 Å, Dionex-LC Packings), at a flow rate of 0.4 µl/min. The mobile 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 % B over 3 hrs, and % 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 de-isotoped. MS/MS analyses were carried out using nitrogen at collision energy of 1 kv and a collision gas pressure of ~ torr. Two 7

8 thousand to ten thousand sub-spectra were combined for each spectrum using stop conditions based on the quality of the data. The spectra were smoothed using Savitsky- Golay method with points across peak (FWHM) set to 3 and polynomial order to 4. The peaks were de-isotoped 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 (64335 entries) containing all predicted ORFs from three WSSV isolates (2013 entries) together with the International Protein Index human database version 3.16 (www. ebi.ac.uk/ipi/ipihelp.html, entries) was used in order 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 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 (30, 31). Putative signal sequences and transmembrane domains were predicted by DAS (32). 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 8

9 using an RNeasy Mini Kit (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 the supplemental data I). All procedures were performed according to the manufacturer's instruction. Briefly, cdna was reversetranscribed at 50 C for 45 min. The PCR amplification segment was started with an initial heating step at 95 C for 15 min in order 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 base pairs (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. was adopted with modification (23). Briefly, purified virus was treated with buffer A containing 20 mm Tris-HCl, 5 mm EDTA-Na 2, 1 % triton X-100, 0.5 M NaCl, and 1 protease inhibitor cocktail (Roche Diagnostics Asia Pacific Pte. Ltd., Singapore), 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, while the other one 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 9

10 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 on 12 % SDS-PAGE gels and processed for Western blot analysis. The nitrocellular 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 blot. Primary antibodies were used with the following concentrations: rabbit anti-vp28 (wsv421) polyclonal antibody (1:2,000), 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, Uppsala, Sweden). Pierce supersignal west pico chemiluminescent substrate (Pierce, Rockford, IL) was used according to the manufacturer s protocol, and the protein bands were visualized using lumi-film chemiluminescent detection film (Roche). 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 2D Clean-Up kit (GE Healthcare) and re-suspended in the dissolution buffer (0.5 M triethylammonium bicarbonate, ph 8.5, containing 0.1 % SDS). The samples were then reduced and cysteines 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 and reconstituted with 30 μl dissolution buffer, and labeled with 10

11 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 % NH 4 OH in 30 % methanol. After drying, the itraq-labeled peptides were resuspended with 20 μl 5 mm KH 2 PO 4 buffer containing 5 % ACN, ph 3.0 and separated using an Ultimate dualgradient LC system (Dionex-LC Packings). The first dimension separation used a mm SCX column (FUS-15-CP, Poros 10S, Dionex-LC Packings) and the mobile phase A and B were 5 mm KH 2 PO 4 buffer, ph 3, containing 5 % ACN and 5 mm KH 2 PO 4 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 %) were captured alternatively with two mm trap columns (3-µm C 18 PepMap, 100 Å, Dionex-LC Packings) and washed with 0.05 % TFA to remove salts. The second dimension separation was performed with a 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, respectively, 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). 11

12 MS analysis was performed as described above and the MS/MS analysis settings were the same as the shotgun analysis except that a collision gas pressure was changed to ~ Torr. For the precursor ions with s/n 100, six thousand shots were combined for each spectrum. For the precursors with s/n between 50 and 100, ten thousand shots were acquired. The peak processing and detection parameters were the same as the shotgun analysis as described above. GPS Explorer software version 3.5 (Applied Biosystems) employing 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 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 6 His tag. After sequencing, the construct was transformed into E. coli strain BL21 Star (DE3) (Invitrogen, Carlsbad, CA) and the protein was expressed after isopropyl-β-d-thiogalactopyranoside induction at 18 C. The recombinant protein was purified using Ni-nitrilotriacetic column and its identity was confirmed by MS. The antibody was prepared by Bam Biotech Co., LTD (Xiamen, Fujian, P. R. China) using the purified fusion protein to immunize the rabbits. Proteins from the virion, the 12

13 envelope and the nucleocapsid were resolved on 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 seconds. 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, while pre-immune rabbit serum was included in parallel as a negative control. The second antibody was goat anti-rabbit IgG conjugated with 15 nm of gold (Electron Microscopy Sciences, Hatfield, PA). The subsequent immunogold labeling was carried out according to Leu et al (33). The specimens were examined under the transmission electron microscope. RESULTS Identification of Virion-associated Proteins by Shotgun Proteomics The purity of isolated WSSV virions using sucrose gradient was confirmed by electron microscopy (Fig. 1A). Most of purified virions were intact. The structural proteome of WSSV was analyzed by shotgun proteomics, employing offline LC-MALDI workflow. In total, 45 viral proteins were detected with amino acid sequences that matched WSSV ORFs (Table I). Except for wsv198 and wsv419, all other identified proteins contained at least one top-ranked peptide match with MASCOT expect value < 0.05 (Supplemental data II). wsv198 was included as a true identification as it had three top-ranked peptides with MASCOT scores of 26, 24 and 15, respectively (Supplemental data II). Although wsv419 only contained one top-ranked peptide with a MASCOT score of 28, the MS/MS 13

14 spectrum was manually inspected and was considered as a reliable assignment. One protein was identified in WSSV (China isolate), yet the predicted ORF can only be found in the Taiwan (wssv349) and Thailand (ORF144) isolates, but not in the China isolate. Thus, we used wssv349 to name it. Although a comprehensive study on the WSSV proteome was carried out by the gel-based approach earlier, our shotgun method has successfully identified additional 13 structural proteins in WSSV. Potential functions of these 13 ORFs were predicted by InterProScan. It is not surprising to find that putative functions could only be assigned for 2 of these proteins because most of WSSV ORFs are functionally unknown. wsv143 is homologous to SOX transcription factors, while wsv161 has 5 sequence regions that are homologous to prichextensn, which represents a signature for proline-rich extensins of plant cell-wall proteins. Post-translational Modification of WSSV Proteins From the MS/MS data, 7 identified proteins contained acetylated N termini (Table II and supplemental data III). wsv134, wsv198 and wsv360 were acetylated at the N-terminal methionine residues. wsv131, wsv289, wsv414 and wsv432 were acetylated at the second amino acids, indicating that the N-terminal methionines of these proteins were removed after translation. RT-PCR Confirmation of the Gene Expression To confirm the expression of the genes of those 13 newly identified viral proteins by shotgun proteomics, RT-PCR was conducted to detect the existence of mrnas for these ORFs. Our results showed that all these genes were expressed (Fig. 2). The sizes of the RT-PCR products of each gene are provided in the supplemental data I. 14

15 Separation of WSSV Envelope Proteins and Nucleocapsid Proteins Viral structural proteins were separated into envelope and nucleocapsid fractions after triton X- 100 and 0.5 M NaCl treatment (Fig. 1B) (23). In the present investigation, we could not distinguish the tegument proteins from envelope proteins, so we classified viral structural proteins as envelope proteins (including tegument proteins) and nucleocapsid proteins. Western blot was performed using antibodies against VP28, VP24 and VP466 to check the separation of viral proteins (Fig. 3). Our results confirmed that VP466 is a nucleocapsid protein (23). As expected, envelope proteins and nucleocapsid proteins were enriched in their respective fractions after separation. Localization of Structural Proteins in WSSV by itraq A quantitative proteomics experiment was carried out using itraq reagents and 2D LC-MALDI MS to localize various structural proteins in WSSV. The workflow of this technique is outlined in Fig. 4. After separation, total viral proteins, envelope proteins and nucleocapsid proteins were labeled with itraq reagents 114, 115 and 116, respectively. Because of their enrichment through the separation process, we expected that envelope proteins would have 115/114 ratios higher than 116/114 ratios, whereas nucleocapsid proteins would instead have lower 115/114 ratios compared with 116/114 ratios. Fig. 5 shows the representative MS/MS spectra of itraq ratios from an envelope protein wsv009 and a nucelocapsid protein wsv289 determined in this study (for the rest of the spectra, see supplemental data IV). Based on the itraq reporter ion ratios, we observed that 23 structural proteins are envelope proteins and 6 as nucleocapsid proteins (Table III). These included 7 proteins with best ion scores 30 (wsv198, wsv216, wsv230, wsv238, wsv242, wsv256 and wsv289). Since these proteins have been confidently identified as 15

16 viral structural proteins by the shotgun proteomic study, and the peptides matched were all top-ranked in the itraq study, these protein assignments should be reliable. The MS/MS spectra were manually inspected for further validation (Supplemental data IV). Using the itraq approach, we confirmed 11 envelope proteins and 4 nucleocapsid proteins that were previously identified, among which VP24 and VP26 are envelope proteins, while VP466 is a nucleocapsid protein (23, 34). Moreover, we have identified additional 12 envelope proteins and 2 nucleocapsid proteins, demonstrating the effectiveness of this approach in the study of subproteomes of viruses. Five newly identified structural proteins by shotgun proteomics and 23 known proteins were further verified in the itraq study. One envelope protein wsv295 was identified as a viral structural protein for the first time in our itraq study. Expression and Localization of the Structural Protein wsv432 in WSSV Recombinant wsv432, a protein newly identified by shotgun proteomics, was expressed in E. coli and purified (Fig. 6A). The MS analysis confirmed the identity of this protein (data not shown). Western blot analysis indicated that this protein was present on the virus envelope, not the nucleocapsid (Fig. 6B). IEM was carried out to further validate the presence of wsv432 in WSSV. To verify the localization of wsv432 on virus particles, the virus was treated with Tween 20 to partially separate the envelope from the nucleocapsid. Subsequent immunogold-labeling demonstrated that gold particles were localized on the envelopes but not on the naked nucleocapsids, while no gold particles were observed on the negative control (Fig. 7). The result revealed that wsv432 is an envelope protein, which is consistent with our Western blot result. 16

17 DISCUSSION For the past few years, gel-based techniques have been successfully employed to study the WSSV proteome and 38 viral structural proteins were identified (10, 11). To further characterize the protein components of this virus, shotgun proteomics was applied and this has led to the identification of 45 structural proteins in our study, including 13 proteins that have not been identified before. The reliability of our findings was verified by ORF-specific RT-PCR for those newly identified proteins. Surprisingly, wsv230, which was defined as a non-structural protein VP9 (wsv230) in previous study (35), was detected in the virion by both shotgun and itraq approaches. The postulation that wsv230 is a non-structural protein was based on the Western blot and IEM results. However, the inability to detect wsv230 by antibody-based approaches might be due to the low abundance of this protein in the virion. Both Western blot analysis and IEM confirmed that wsv432 identified by shotgun proteomics, not by 1D gel MS, is an envelope protein. Moreover, this protein contains an RGD motif for cell adhesion. It was reported that the threonine residue right after RGD is important for its interaction with integrin (36). As for wsv432, the threonine residue is replaced with a serine, implying that this protein might also have cell adhesion activity important for virus entry. Genome-wide transcription profile of WSSV ORFs using DNA microarrays to understand the regulation of WSSV gene expression has been reported (37-39). For the 13 previously unknown proteins, five of these genes started expression at 2 hours post infection (hpi), one at 12 hpi, and six at 24 hpi, respectively, based on the DNA microarray data (Table I)(11, 37). 17

18 Fifty-five WSSV structural proteins identified by three proteomic studies are summarized in Fig. 8. It is noted that 17 proteins were uniquely identified by the LCbased approach, 10 were uniquely identified by the gel-based approach and 28 were identified by both, confirming the complementary nature of these two approaches. Among the 17 ORFs identified by shotgun proteomics, 4 structural proteins, including 2 envelope proteins VP187 (wsv209) and VP124 (wsv216), and 2 nucleocapsid proteins, VP160B (wsv037) and VP160A (wsv289), have been identified by gel-based MS analyses in three recent publications (19, 23, 40). However, four structural proteins, VP1684 (wsv001), VP357 (wsv129), VP184 (part of wsv303) and VP448 (wsv526), were only identified from 1D gel by MALDI-TOF peptide mass finger printing and could not be confirmed by MS/MS data, suggesting that these protein identifications are of low reliability and further evidences are needed to support these findings (10). Since only visible bands were excised from the gel, it is possible that those low abundant proteins might not be included for the identification. Therefore, LC-based approach has an advantage in identifying low abundance proteins such as wsv230. Moreover, several highly basic proteins, such as wsv021 (pi 9.35) and wsv324 (pi 9.3), were also identified by the LC method. Altogether, total 58 structural proteins have been identified in WSSV, including 55 proteins identified by shotgun and previous proteomic studies, one envelope protein wsv295 identified by our itraq study and two nucleocapsid proteins VP35 (wsv493) and VP15 (wsv214) reported previously (6, 11). Traditionally, Western blot analysis and IEM have been used to determine the localization of viral proteins. These approaches would require substantial amount of work such as the generation and characterization of the antibodies. In addition, some of 18

19 these results were controversial. For example, VP466 was determined by IEM as an envelope protein, while Western blot analysis suggested that it is a nucleocapsid protein (10, 23). The precise localization of proteins in WSSV by non-quantitative MS analysis has been hindered by the difficulties in the successful separation of envelope proteins and nucleocapsid proteins. Western blot analysis could detect the traces of envelope proteins VP28 and VP24 in the nucleocapsid fraction. In the present study, quantitative MS analysis was introduced to discriminate viral envelope proteins and nucleocapsid proteins according to their distributions and hence localization. As we expected, two distinct types of itraq reporter ion spectra were achieved for envelope proteins and nucleocapsid proteins, respectively (Fig. 5). However, some itraq ratios (116/114) of nucleocapsid proteins were only slightly higher than that of 115/114 because of the presence of solubilized nucleocapsid proteins in the envelope fraction. This is most likely due to partial dissociation of nucleocapsid proteins caused by osmotic shock during the high salt treatment and the separation process (23, 41). As a verification of our approach, the localization results of structural proteins in the virion based on itraq ratios are in good agreement with the previous investigations, including 11 envelope proteins and 4 nucleocapsid proteins (19, 23, 40). Seven of the newly characterized proteins contain predicted transmembrane domains, supporting their classification as envelope proteins (Table IV). Our data demonstrated the feasibility of accurate localization of viral structural proteins using the itraq approach, even though contaminant-free fractions could not be obtained. The ability to distinguish envelope and nucleocapsid proteins without the need of complete separation represents a significant advance in the localization of viral structural proteins. 19

20 wsv143 and wsv271 were two newly identified nucleocapsid proteins. DNA microarray data demonstrated that wsv143 is an early gene, which is consistent with its potential function as a transcription factor (37). Elucidation of the function of this protein should be useful to uncover the mechanism of virus replication. Although both wsv143 and wsv271 contains KGD or RGD motif for cell adhesion, it is less likely that they may have such a function as they are localized in the nucleocapsid. Moreover, we also found that all nine nucleocapsid proteins are predicted to target to nucleus by PSORT search, and do not contain any transmembrane domains predicted by DAS, implying that these two criteria are required for most nucleocapsid proteins. All known localization of structural proteins of WSSV is summarized in Table IV. In the present investigation, more comprehensive proteomic analyses of the WSSV components and their localization in the virion were achieved using shotgun and itraq technologies. Our results demonstrated that itraq is an effective MS-based approach to distinguish the envelope and nucleocapsid proteins. This approach should also be applicable to study the protein localization of other enveloped viruses. In summary, WSSV is composed of at least 58 structural proteins, including 13 viral proteins identified by shotgun proteomics and 1 by itraq in this study. Among these structural proteins, the localization of 33 envelope proteins and 9 nucleocapsid proteins were determined, including 12 envelope proteins and 2 nucleocapsid proteins identified by our itraq study. Our findings could provide new information to investigate the molecular mechanisms of virus assembly and virus entry. 20

21 Acknowledgements We thank all the staffs in the Protein and Proteomics Centre of Department of Biological Sciences, National University of Singapore, for their technical supports and Ms. Ying Zhuang for providing the anti-vp24 antibody. This work was supported by a research grant R from the Academic Research Council of National University of Singapore to C. L. H. REFERENCES 1. Wang, 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., and Kou, G. H. (1995) Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis Aquat Organ. 23, Wongteerasupaya, C., Vickers, J. E., Sriurairatana, S., Nash, G. L., Akarajamorn, A., Boonsaeng, V., Panyim, S., Tassanakajon, A., Withyachumnarnkul, B., and Flegel, T. W. (1995) A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis Aquat Organ. 21, Nakano, H., Koube, H., Umezawa, S., Momoyama, K., Hiraoka, M., Inouye, K., and Oseko, N. (1994) Mass mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993: Epizootiological survey and infection trails. Fish Pathol. 29, Inouye, K., Yamano, K., Ikeda, N., Kimura, T., Nakano, H., Momoyama, K., Kobayashi, J., and Miyajima, S. (1996) The penaeid rod-shaped DNA virus (PRDV), which causes penaeid acute viremia (PAV). Fish Pathol. 31,

22 5. Lo, C. F., Ho, C. H., Peng, S. E., Chen, C. H., Hsu, H. E., Chiu, Y. L., Chang, C. F., Liu, K. F., Su, M. S., Wang, C. H., and Kou, G. H. (1996) White spot syndrome associated virus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis Aquat Organ. 27, Chen, L. L., Leu, J. H., Huang, C. J., Chou, C. M., Chen, S. M., Wang, C. H., Lo, C. F., and Kou, G. H. (2002) 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. 293, Chen, L. L., Wang, H. C., Huang, C. J., Peng, S. E., Chen, Y. G., Lin, S. J., Chen, W. Y., Dai, C. F., Yu, H. T., Wang, C. H., Lo, C. F., and Kou, G. H. (2002) Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology. 301, van Hulten, M. C., Witteveldt, J., Peters, S., Kloosterboer, N., Tarchini, R., Fiers, M., Sandbrink, H., Lankhorst, R. K., and Vlak, J. M. (2001) The white spot syndrome virus DNA genome sequence. Virology. 286, Yang, F., He, J., Lin, X., Li, Q., Pan, D., Zhang, X., and Xu, X. (2001) Complete genome sequence of the shrimp white spot bacilliform virus. J Virol. 75, Huang, C., Zhang, X., Lin, Q., Xu, X., Hu, Z., and Hew, C. L. (2002) Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. Mol Cell Proteomics. 1, Tsai, J. M., Wang, H. C., Leu, J. H., Hsiao, H. H., Wang, A. H., Kou, G. H., and Lo, C. F. (2004) Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol. 78,

23 12. Song, W., Lin, Q., Joshi, S. B., Lim, T. K., and Hew, C. L. (2006) Proteomic studies of the Singapore grouper iridovirus. Mol Cell Proteomics. 5, Huang, C., Zhang, X., Lin, Q., Xu, X., and Hew, C. L. (2002) Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry. J Gen Virol. 83, Zhang, X., Huang, C., Xu, X., and Hew, C. L. (2002) Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein. J Gen Virol. 83, Zhang, X., Huang, C., Xu, X., and Hew, C. L. (2002) Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus. J Gen Virol. 83, Zhang, X., Huang, C., Tang, X., Zhuang, Y., and Hew, C. L. (2004) Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE-MS. Proteins. 55, Li, Q., Chen, Y., and Yang, F. (2004) Identification of a collagen-like protein gene from white spot syndrome virus. Arch Virol. 149, Li, L., Xie, X., and Yang, F. (2005) Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31. Virology. 340, Zhu, Y., Xie, X., and Yang, F. (2005) Transcription and identification of a novel envelope protein (VP124) gene of shrimp white spot syndrome virus. Virus Res. 113,

24 20. Zhu, Y. B., Li, H. Y., and Yang, F. (2006) Identification of an envelope protein (VP39) gene from shrimp white spot syndrome virus. Arch Virol. 151, Xie, X., and Yang, F. (2006) White spot syndrome virus VP24 interacts with VP28 and is involved in virus infection. J Gen Virol. 87, Li, L., Lin, S., and Yang, F. (2006) Characterization of an envelope protein (VP110) of White spot syndrome virus. J Gen Virol. 87, Tsai, J. M., Wang, H. C., Leu, J. H., Wang, A. H., Zhuang, Y., Walker, P. J., Kou, G. H., and Lo, C. F. (2006) Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion. J Virol. 80, Huang, R., Xie, Y., Zhang, J., and Shi, Z. (2005) A novel envelope protein involved in White spot syndrome virus infection. J Gen Virol. 86, Ross, 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., and Pappin, D. J. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 3, Chen, 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., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol Cell Proteomics. 5, Dunkley, T. P., Watson, R., Griffin, J. L., Dupree, P., and Lilley, K. S. (2004) Localization of organelle proteins by isotope tagging (LOPIT). Mol Cell Proteomics. 3,

25 28. Foster, L. J., de Hoog, C. L., Zhang, Y., Xie, X., Mootha, V. K., and Mann, M. (2006) A mammalian organelle map by protein correlation profiling. Cell. 125, Wu, J. L., Suzuki, K., Arimoto, M., Nishizawa, T., and Muroga, K. (2002) Preparation of an inoculum of white spot syndrome virus for challenge tests in Penaeu japonicus. Fish Pathol. 37, Zdobnov, E. M., and Apweiler, R. (2001) InterProScan--an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 17, Quevillon, E., Silventoinen, V., Pillai, S., Harte, N., Mulder, N., Apweiler, R., and Lopez, R. (2005) InterProScan: protein domains identifier. Nucleic Acids Res. 33, W Cserzo, M., Eisenhaber, F., Eisenhaber, B., and Simon, I. (2002) On filtering false positive transmembrane protein predictions. Protein Eng. 15, Leu, J. H., Tsai, J. M., Wang, H. C., Wang, A. H., Wang, C. H., Kou, G. H., and Lo, C. F. (2005) The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found. J Virol. 79, van Hulten, M. C., Westenberg, M., Goodall, S. D., and Vlak, J. M. (2000) Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology. 266, Liu, Y., Wu, J., Song, J., Sivaraman, J., and Hew, C. L. (2006) Identification of a novel nonstructural protein, VP9, from white spot syndrome virus: its structure reveals a ferredoxin fold with specific metal binding sites. J Virol. 80,

26 36. Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., and Smith, J. W. (2000) Ligand binding to integrins. J Biol Chem. 275, Wang, H. C., Lin, A. T., Yii, D. M., Chang, Y. S., Kou, G. H., and Lo, C. F. (2004) DNA microarrays of the white spot syndrome virus genome: genes expressed in the gills of infected shrimp. Mar Biotechnol (NY). 6, S106-S Marks, H., Vorst, O., van Houwelingen, A. M., van Hulten, M. C., and Vlak, J. M. (2005) Gene-expression profiling of White spot syndrome virus in vivo. J Gen Virol. 86, Lan, Y., Xu, X., Yang, F., and Zhang, X. (2006) Transcriptional profile of shrimp white spot syndrome virus (WSSV) genes with DNA microarray. Arch Virol. 151, Li, H., Zhu, Y., Xie, X., and Yang, F. (2006) Identification of a novel envelope protein (VP187) gene from shrimp white spot syndrome virus. Virus Res. 115, Durand, S., Lightner, D. V., Redman, R. M., and Bonami, J. R. (1997) Ultrastructure and morphogenesis of White Spot Syndrome Baculovirus (WSSV ). Dis Aquat Organ. 29, van Hulten, M. C., Reijns, M., Vermeesch, A. M., Zandbergen, F., and Vlak, J. M. (2002) Identification of VP19 and VP15 of white spot syndrome virus (WSSV) and glycosylation status of the WSSV major structural proteins. J Gen Virol. 83,

27 FIGURE LEGENDS FIG. 1. Electron micrographs of negatively stained WSSV. A, intact enveloped virions. B, nucleocapsids, which appear as stacked, ringed structures. FIG. 2. RT-PCR amplification of the cdnas of 13 newly identified structural proteins of WSSV. For each protein, the left lane is the genomic DNA contamination control and the right lane is its corresponding cdna product. All protein coding sequences were completely amplified including wsv143 and wsv161. For wsv143, 5 RT- PCR products (wsv143-1 to 5) were amplified to cover the entire coding region; while for wsv161, 2 RT-PCR products (wsv161-1 and 2) were produced. FIG. 3. Western blot analysis of total proteins, envelope proteins and nucleocapsid proteins. Three antibodies used were anti-vp466, VP28 and VP24, respectively. The total protein concentrations were 0.3 μg for the left panel and 12 μg for the right panel, respectively. FIG. 4. The itraq labeling workflow and 2D LC MS for the localization of structural proteins in WSSV. The virus envelopes were separated from the nucleocapsids after detergent treatment. To quantitatively distinguish envelope proteins from nucleocapsid proteins, total viral proteins, proteins from the envelope fraction and the nucleocapsid fraction were reduced, cysteine-blocked and digested with trypsin, respectively. The tryptic peptides were labeled with 114, 115, and 116 itraq reagents, respectively. Then, the combined peptide mixture was cleaned up using SCX chromatography. The eluate was further separated by 2D LC coupling SCX with reversed phase chromatography and the column effluent was added with MALDI matrix 27

28 solution and spotted on MALDI target plates, which were later analyzed by an ABI 4700 Proteomics Analyzer MALDI TOF/TOF MS. FIG. 5. Representative itraq reporter ion spectra of an envelope protein wsv009 and a nucleocapsid protein wsv289. FIG. 6. Expression of recombinant wsv432 and Western blot analysis of its localization in WSSV. A, lane 1, molecular mass (MM) marker; lane 2 and lane 3, uninduced and induced bacteria lysates, respectively. B, Localization of wsv432 in the virion by Western blot analysis. Lane 1, total viral proteins; lane 2, envelope proteins; lane 3, nucleocapsid proteins. Rabbit anti-wsv432 antibody was used. FIG. 7. Localization of wsv432 in WSSV by IEM. A, WSSV with immunogoldlabeled anti-wsv432 antibody. Gold particles were localized to the envelope, not the nucleocapsid. Insertion (a) shows another labeled virus. B, WSSV with immunogoldlabeled control serum. No gold-labeled particles were found. FIG. 8. A summary of WSSV structural proteins identified by proteomics studies. WSSV structural proteins identified in the present study and two earlier reports were summarized. * ORFs identified in other literatures as described in the discussion (19, 23, 40). Underlined ORFs were also verified by the itraq study. ** Altogether, 58 structural proteins were identified, including 55 proteins identified by these three studies, wsv295 identified by itraq, VP35 and VP15 reported previously. Tables TABLE I Structural proteins of WSSV identified by shotgun proteomics. (Page 13 and Page 17) 28

29 TABLE II N-terminal acetylation detected in WSSV structural proteins by shotgun proteomics. (Page 14) TABLE III Envelope proteins and nucleocapsid proteins of WSSV identified by itraq. (Page 15) TABLE IV The localization of structural proteins in WSSV. (Page 19 and Page 20) 29

30 Fig. 1A, page 13, Fig. 1B, page 15 A 50 nm B 100 nm 30

31 Fig. 2, page 14 wsv006 wsv021 wsv134 wsv432 wsv136 wssv349 wsv010 wsv324 1,000 bp 300 bp wsv131 wsv143-1 wsv143-2 wsv143-3 wsv ,000 bp 1,000 bp wsv143-5 wsv161-1 wsv161-2 wsv230 wsv419 2,000 bp 1,000 bp 31

32 Fig. 3, page 15 Total (0.3 μg) envelope nucleocapsid Total (12 μg) envelope nucleocapsid VP466 VP28 VP24 32

33 Fig. 4, page 15 WSSV 1% Triton, 0.5 M NaCl Centrifugation Total proteins Supernatant (Envelope proteins) Pellet (Nucleocapsid proteins) Reduce/ Block Cysteines Reduce/ Block Cysteines Reduce/ Block Cysteines Trypsin digest Trypsin digest Trypsin digest React with itraq Reagent 114 React with itraq Reagent 115 React with itraq Reagent 116 Mix SCX cleanup 2D LC- MALDI TOF/TOF MS 33

34 Fig. 5, page 15 and page 19 A Envelope protein wsv B Nucleocapsid protein wsv Mass (m/z) 34

35 Fig. 6, page 16 A MM (kda) wsv432 B

36 Fig. 7, page 16 A (a) 100 nm B 100 nm 36

37 Fig.8, page proteins Huang et. al. (10) wsv001 wsv009 wsv129 wsv303 wsv526 wsv006 wsv010 wsv021 wsv037* wsv131 wsv134 wsv136 wsv143 wsv161 wsv209* wsv216* wsv230 wsv289* wsv324 wsv419 wsv432 wssv349 wsv002 wsv220 wsv237 wsv242 wsv254 wsv256 wsv308 wsv311 wsv386 wsv414 wsv415 wsv421 wsv442 Shotgun 45 proteins wsv077 wsv269 wsv332 wsv390 wsv465 wsv011 wsv035 wsv115 wsv198 wsv238 wsv259 wsv271 wsv284 wsv306 wsv321 wsv325 wsv338 wsv339 wsv340 wsv proteins Tsai, et. al. (11) Total: 55+3=58** 37

38 TABLE I Structural Proteins of WSSV Identified by Shotgun Proteomics Unique Peptide Count Total Ion Score Total Ion Score C.I. % hpi 3 TM 4 SP 5 No. Name 1 Accession No. MM (kda) 2 PI 1 wsv002 gi TM SP KGD VP24 (34) 2 wsv006 gi TM this study 3 wsv010 gi this study 4 wsv011 gi TM VP53A (11) 5 wsv021 gi TM this study 6 wsv035 gi TM RGD VP110 (11) 7 wsv037 gi KGD VP160B (23) 8 wsv115 gi TM VP53B (11) 9 wsv131 gi this study 10 wsv134 gi this study 11 wsv136 gi TM this study 12 wsv143 gi KGD this study 13 wsv161 gi this study 14 wsv209 gi R/KGD VP187 (40) 15 wsv216 gi VP124 (19) 16 wsv220 gi TM VP674,VP73,VP76 (10,11,24) 17 wsv230 gi this study, VP9 (35) 18 wsv237 gi VP292, VP41A (10,11) 19 wsv238 gi TM SP KGD VP51A (11) 20 wsv242 gi VP300, VP41B (10,11) 21 wsv254 gi RGD VP281, VP36B (10,11,13) 22 wsv256 gi TM SP VP384, VP51B (10,11) 23 wsv259 gi VP38A (11) 24 wsv271 gi RGD VP136A (11) 25 wsv284 gi TM SP VP13A (11) 26 wsv289 gi VP160A (23) 27 wsv306 gi VP39A (11) 28 wsv308 gi VP466, VP51C (10,11) 29 wsv311 gi TM VP26 (34) 30 wsv321 gi TM SP VP13B (11) 31 wsv324 gi this study 32 wsv325 gi TM VP60A (11) 33 wsv338 gi TM SP VP11 (11) 34 wsv339 gi VP39B (11) 35 wsv340 gi RGD VP31(11) 36 wsv360 gi R/KGD VP664 (11,33) 37 wsv386 gi TM SP VP68, VP12B (10,11) 38 wsv414 gi TM VP19 (42) 39 wsv415 gi KGD VP544, VP60B (10,11) 40 wsv421 gi TM SP VP28 (34) 41 wsv432 gi RGD this study 42 wsv442 gi VP800, VP95 (10,11) 43 wssv349 gi TM this study 44 wsv198 7 gi VP32 (11) 45 wsv419 gi NA this study RGD or KGD 6 Alternative Names & References 1 Based on the genome of the China isolate, except for wssv349 (Taiwan isolate). 2 Predicted molecular mass. 3 hpi, hours post infection that ORFs started expression. NA, not available. 4 TM, transmembrane domain. 5 SP, signal peptide sequence. 6 potential cell-adhension sequences. 7 WSSV proteins with the best peptide ion scores 30 are listed at the bottom of the table and seperated from other proteins with a dashed line. 38