Department of Biological Sciences and Tropical Marine Science Institute, National University of Singapore, Singapore 119260
¶ Key Laboratory of Marine Biotechnology, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, Peoples Republic of China
|| Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, Peoples Republic of China
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ABSTRACT |
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With the completion of the WSSV genomic DNA sequence, research has now focused on the functional analysis of the gene products. Essential to the functional analysis is to identify the proteins expressed in WSSV. To this end, a proteomic approach using mass spectrometry has been proven to be the most effective technology for the identification of proteins (9). Recently, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) utilizing a quadrupole time-of-flight (Q-TOF) mass spectrometer have been used as tools for the characterization of proteins because of their high sensitivity and throughput (10).
In this communication, the WSSV proteins separated by SDS-PAGE were analyzed using MALDI-TOF MS and ESI-MS/MS (Q-TOF), respectively. The resulting mass spectrometric data were searched against the theoretical ORF database of WSSV. One of the newly retrieved genes, vp466 gene, was further characterized.
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EXPERIMENTAL PROCEDURES |
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Purification of WSSV Nucleocapsid
Purified WSSV virion was treated with Triton X-100 for 15 min at room temperature, subjected to 2050% continuous CsCl gradient, and centrifuged for 2448 h at 110,000 x g using a SW 41-Ti. Viral capsid band was collected by side puncture and then diluted with 1x TN buffer (1:10), subsequently sedimented at 120,000 x g for 45 min. The pellet was resuspended in 1x TN buffer, pH 7.4.
Computer Analysis of the ORFs of WSSV Genome
The 305,107-bp DNA sequence of the WSSV genome (6) was analyzed with DNAMAN (Lynnon BioSoft, Vaudreuil, Canada) to identify ORFs. In total, 4443 ORFs starting with an ATG start codon and with lengths of 50 amino acids or larger were located on both strands of the WSSV genome. From these ORFs, 181 ORFs ranging from 61 to 6077 amino acids in size were likely to encode functional proteins (6). These 181 ORFs were designated as putative genes and assigned to the WSSV ORF database. Homology searches were performed with the BLAST and BLAST2 programs. Protein motifs were analyzed by PROSITE release 16 database (12).
Mass Spectrometric Analysis of Viral Proteins
In-gel Digestion
The proteins from purified WSSV were separated by 12% SDS-PAGE and stained with Coomassie Blue R 250. Protein bands were excised and dehydrated several times with acetonitrile. After vacuum drying, the gel bands were incubated with 10 mM dithiothreitol in 100 mM ammonium bicarbonate (ABC buffer) at 57°C for 60 min and subsequently with 55 mM iodoacetamide (Sigma) in 100 mM ABC buffer at room temperature for 60 min. Then the gels were washed with 100 mM ABC buffer and dried. All in-gel protein digestions were performed using sequencing grade modified porcine trypsin (Promega, Madison, WI) in 50 mM ABC buffer at 37°C for 15 h. Digests were centrifuged at 6000 x g. The supernatants were separated, and the gel pieces were extracted further first with 50% acetonitrile, 5% formic acid and then with acetonitrile. The extracts were combined with the original digesting supernatants, vacuum-dried, and redissolved in 0.5% trifluoroacetic acid and 50% acetonitrile (13).
MALDI-TOF MS
The matrix used was a saturated solution of -cyano-4-hydroxycinnamic acid in 0.5% trifluoroacetic acid and 50% acetonitrile. The sample and the matrix (1:1, v/v) were loaded on the target plate. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry work station mass spectrometer (PerSeptive Biosystems, Inc., Framingham, MA). The analyses were performed in positive ion reflector mode with an accelerating voltage of 20 kV and a delayed extraction of 150 ns. Typically 180 scans were averaged. The trypsin autoproteolysis products were used as internal calibrants. Data mining was performed using MS-Fit software against the WSSV ORF database. A mass deviation of 100 ppm was usually allowed in the database searches.
Nano-ESI-MS/MS
The in-gel digested samples were desalted using C18 ZipTip (Millipore, Bedford, MA) and dried. After dissolving in 2 µl of 50% acetonitrile and 0.5% formic acid, the sample was loaded into a metallized glass capillary. The capillary was then mounted on the nanoflow Z-spray source of a Q-TOF2 mass spectrometer (Micromass, Manchester, United Kingdom). Flow rates usually varied from 8 to 16 nL/min. Instrument operation, data acquisition, and analysis were performed using MassLynx/BioLynx 3.5 software (Micromass). The capillary voltage and collision energy were optimized for each sample. The microchannel plate voltage was set to 2200 V. Data searches against the WSSV ORF database were performed using Global Server (Micromass).
Transcriptional Analyses of Genes
Shrimp Infection with WSSV
The tissue from P. monodon shrimp with pathologically confirmed infection was homogenized in TN buffer at 0.1 g/ml. After centrifugation at 2000 x g, the supernatant was diluted to 1:100 with 0.9% NaCl and filtered (0.22-µm filter). 0.2 ml of the filtrate was injected intramuscularly into each healthy shrimp in the lateral area of the fourth abdominal segment. At various times post-infection (p.i.), four specimens were selected at random, and their hemolymphs were collected. The collected hemolymphs were immediately frozen and stored at -70°C.
RT-PCR
The total RNA was isolated from WSSV-infected shrimp hemolymph according to the manufacturers instruction (NucleoSpin RNA II; Macherey-Nagel GmbH & Co. KG, Germany). Then RT-PCR was performed with ORF-specific primers using a TITANIUM One-step RT-PCR kit (CLONTECH Laboratories, Inc.). The RT-PCR cycles were as follows: 50°C for 1 h, 94°C for 5 min, 30 cycles of 94°C for 30 s, 65°C for 30 s, 68°C for 1 min, followed by an elongation at 68°C for 2 min.
Characterization of WSSV vp466 Gene
Rapid Amplification of vp466 cDNA Ends (5' and 3' RACE)
The 5' and 3' cDNA of vp466 was revealed by RACE. For 5' RACE, a gene-specific primer SP1 and a nested SP2 were designed as 5'-GCTCTCCATCCGCTTAGTCACATTGGC-3' and 5'- GCCGAAGCTGAAGGTTTTGGAGGTGC-3', respectively. For 3' RACE, the gene-specific primer SP3 was 5'-GCAGTAGCAAATCTCACCGGACCTGTG-3'. RACE reaction was performed according to the instructions of the 5'/3' RACE kit (Roche Molecular Biochemicals).
Expression of GST-VP466 Fusion Protein in Escherichia coli
The vp466 gene was amplified using the synthesized forward primer 5'-CACGGATCCATGTCTGCATCTTTAAT-3' with BamHI site (italic and underlined) and the reverse primer 5'-AGACCCGGGTTATGACACAAACCTAT-3' with SmaI site (italic and underlined). The amplified DNA and plasmid vector pGEX-4T-2 were digested with BamHI + SmaI. After purification and ligation of DNA fragments, the vp466 gene was inserted into pGEX-4T-2 vector downstream of GST and expressed in pGEX-4T-2-pLysS as a fusion protein with GST (Amersham Biosciences Corp.). The resulted recombinant plasmid was named pGEX466. The competent cells of E. coli BL-21 pLysS were transformed with the recombinant pGEX466, and colonies containing transformants were screened by colony PCR. The identity of pGEX466 was subsequently confirmed by both restriction enzyme digestion (BamHI + SmaI) and DNA sequencing.
After overnight incubation at 37°C, pGEX466-pLysS and pGEX-4T-2-pLysS (BL-21 pLysS containing pGEX466 and pGEX-4T-2, respectively) were inoculated into new media at a ratio of 1:100. When A600 reached 0.6, the cultures were induced with 1 mM IPTG and continued to grow for 6 h. Then the bacteria were spun down (4000 x g) at 4°C. The pellets were suspended in ice-cold phosphate-buffered saline (PBS) (containing 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 4 mM benzamidine, 10 µg of leupeptin, and 10 µg of aprotinin) and sonicated for 30 s on ice. After spinning at 60,000 x g, the supernatant was mixed with 1x PBS-buffered glutathione-agarose beads (Sigma) and incubated at 4°C for 2 h on a shaking device. The beads were washed three times with ice-cold 1x PBS and incubated in reducing buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) at room temperature for 10 min. After centrifugation at 1000 x g for 5 min, the supernatant was collected and analyzed by SDS-PAGE.
Antibody Preparation
The purified GST-VP466 fusion protein was used to immunize mice (Swiss Albino, 34 weeks) once every 2 weeks by intradermal injection over an eight-week period. Titers of the antisera were 1:20,000 as determined by enzyme-linked immunosorbent assay. Protein A-SepharoseTM CL-4B was used to isolate anti-GST-VP466 IgG according to the manufactures instruction (Amersham Biosciences). The optimal dilution of purified IgG, after serial dilutions, was 1:1,000 as determined by enzyme-linked immunosorbent assay. Horseradish peroxidase-conjugated goat anti-mouse IgG was obtained from Sigma. For negative control, 1x PBS buffer was used.
Western Blot
WSSV virions were subjected to 12% SDS-PAGE, followed by transferring onto nitrocellulose membrane (Bio-Rad) in electroblotting buffer (Tris 25 mM, glycine 190 mM, methanol 20%) for 3 h. The membrane was immersed in blocking buffer (3% bovine serum albumin, 20 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.2) at 4°C overnight, followed by incubation with a polyclonal mouse anti-GST-P466 IgG, pre-immune mouse serum, or mouse anti-GST IgG for 3 h. Subsequently, horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) was used as the secondary antibody, and the detection was performed with a substrate solution containing 4-chloro-1-naphthol (Sigma).
Localization of VP466 by Immunoelectron Microscopy
WSSV virion and nucleocapsid were mounted onto the Formvar-coated, carbon-stabilized nickel grids, respectively, and the grids were then blocked with 2% AURION BSA-CTM (Electron Microscopy Sciences) for 1 h, followed by incubation with the primary antibody (purified polyclonal mouse anti-VP466 antibody IgG, 1:1000 dilution in 1% AURION BSA-CTM) for 23 h. After washing three to four times with 1x PBS, the grids were incubated with goat anti-mouse IgG conjugated with 15 nm colloid gold (Electron Microscopy Sciences) for 1 h. The grids were further washed two times with 1x PBS and briefly stained with 2% phosphotungstic acid (pH 7.0) for 1 min. The specimens were examined under the transmission electron microscope (JEOL 100 CXII, Japan). In the control experiments, the primary antibody was replaced with pre-immune mouse serum and mouse anti-GST antibody, respectively, and following other experimental steps.
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RESULTS |
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Structures of Genes and Homologies with Known Proteins
A guanine residue from the beginning of the largest BamHI fragment was designated as the starting point of the physical map of the WSSV genome (6). The positions of 18 genes in the WSSV genome and their accession numbers in GenBankTM are listed in Table I. A typical TATA box sequence was found in the promoter regions of all 18 genes, indicating that this sequence may be essential in WSSV for the efficient transcription of these genes. Except for the vp184, vp300, and vp674 genes, the putative polyadenylation signal sequences (AATAAA) were present downstream of the stop codons of the remaining 15 genes. Among the 18 genes, the start codons (ATGs) were in a favorable context for efficient eukaryotic translation initiation (PuNNATGPu) (16) for 14 genes. Exceptions were the vp357, vp466, vp544, and vp800 genes. The 18 genes encoded proteins ranging from 68 to 1684 amino acids (Table I).
Based on homology searches of the 18 proteins using BLAST and BLAST2, putative functions of four of them could be assigned. Two genes contained sequence motifs based on PROSITE analysis (Table I). The remaining 12 genes showed no homology to any known proteins or sequence motifs. The vp1684 gene encoded a large protein (168 kDa) from the collagen family. This was the first time that an intact collagen gene was found in a virus. The collagen-like protein contained a typical repeat of Gly-X-Y (X was mostly proline, and Y could be any amino acid), but its function was not clear. The proteins encoded by the vp208, p22, and p204 genes showed characteristics of structural proteins (14). The p22 and p204 genes were identified further to encode envelope proteins of WSSV by immunoelectron microscopy in our earlier studies (15).
Temporal Analyses of Gene Transcriptions
RT-PCR was used to detect the ORF-specific transcripts in the total RNAs extracted from the hemolymph of adult P. monodon at various infection stages (0, 6, 18, 24, 30, 36, and 48 h p.i) with WSSV. The transcripts of all 18 genes were detected at different post-infection stages (Table I). These results confirmed the coding fidelity of the ORFs. Based on the temporal analysis, only the vp121 gene could be detected at 6 h p.i. until 24 h p.i. The remaining 17 genes were transcribed after 6 h p.i., suggesting that these genes were expressed in the late course of WSSV infection. However no putative late promoter element, ATAAG, canonical in insect baculoviruses, was found in the promoter regions of these late genes.
Characterization of WSSV vp466 Gene
Identification of WSSV vp466 Gene by Mass Spectrometry
The putative vp466 gene was associated with a 51-kDa protein, corresponding to band 16 (one of the minor bands) in the WSSV SDS-PAGE profile. Trypsin digests of reduced and alkylated protein from band 16 were first analyzed by MALDI-TOF MS (Fig. 2a). The WSSV ORF database search with the list of tryptic peptide masses identified one of the proteins as the product of the vp466 gene (termed as the VP466 protein). Four experimentally derived peptide masses were found to match the predicted peptide masses of the VP466 protein within 100 ppm, covering 21% of its amino acid sequence. The tryptic peptides of band 16 were sequenced subsequently by mass spectrometry using nano-ESI-MS/MS. The measured and calculated masses of the tryptic peptides of the nano-ESI-MS spectrum were shown in Fig. 2b. The resulted amino sequences corresponded to the product of the vp466 gene after searching the WSSV ORF database.
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Temporal Analysis of vp466 Gene Transcription
RT-PCR was used to detect the vp466 gene-specific transcript in the total RNA extracted from the hemolymph of adult P. monodon at various WSSV infection stages (0, 12, 18, 24, 30, 36, 48, and 60 h p.i.). The transcript was first detected at 18 h p.i. and reached the highest level at 30 h p.i. (Fig. 4), indicating that vp466 is a late gene.
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DISCUSSION |
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MALDI-TOF MS and ESI-MS/MS are two complementary MS methods for proteomic analysis. Each of these MS methods can be used independently, but when the high throughput of MALDI-TOF MS analysis is combined with the sequence specificity of ESI-MS/MS analysis, identification of unknown proteins from database search is greatly facilitated. In this study, the proteins from WSSV SDS-PAGE gels were analyzed first by MALDI-TOF MS, and 16 WSSV genes with reliable matches were obtained (Table I). Despite lower sequence coverage, the peaks with stronger signals of the tryptic mass spectra of bands 5, 18, 19, and 21 were found to match the predicted peptide masses of the vp184, vp448, vp544, and vp674 genes, respectively. ESI-MS/MS analysis was used subsequently to analyze the tryptic samples because of its sequence specificity. Fourteen bands were revealed by Q-TOF (Table I). Moreover, some MALDI-TOF MS results were confirmed. Although the sequence coverage of bands 11, 13, 14, 16, 19, and 23 to the predicted proteins of the vp281, vp292, p22, vp384, vp544, and vp800 genes were lower, at least 10 successive amino acid sequences were matched. These sequence tags should be specific. Besides three bands containing two protein components, proteins encoded by the p204 and p22 genes were found to be present in different bands (Table I). This may be because of post-translation modifications such as glycosylation or lipidation.
In this study, vp466 gene was chosen as an example of proteomic identification and was characterized further. As this gene was derived from a minor protein band (band 16), the reliability of this assignment should theoretically be lower than those of more abundant bands. Western blot analysis showed that the antibody against the expressed VP466 specifically recognized a 51-kDa protein of WSSV origin. Thus the authenticity of the proteomic results in this study was substantiated further. Immunogold labeling provided visualized evidence that VP466 is a WSSV envelope protein. This is the third WSSV envelope protein hitherto characterized. In the previous studies, VP28/P204, VP26/P22 have been identified as two major WSSV envelope proteins.2
Baculoviruses produce enveloped progeny viruses in the following two different ways: (i) budded virus obtains its envelope from the cell surface when the nucleocapsid buds through the plasma membrane as it exits the cell; (ii) occlusion-derived virus acquires its envelope with viral-induced membranes within the nucleoplasm (20). WSSV morphogenesis is exclusively intranuclear, where viral envelope is formed de novo (21, 22); this feature is closer to that of occlusion-derived virus than that of the budded virus. Although the precise mechanism of the induction of intranuclear membrane structures and protein movement to nuclear envelope is still unknown, evidence showed that some hydrophobic transmembrane domains, for example, lamin B receptor and herpes simplex virus glycoprotein B, are sufficient to direct proteins to be incorporated with nuclear membrane (20). It is thus speculated that VP466 putative transmembrane domain, formed by amino acid residues 338 to 358 (Fig. 3), may play a role in mediating the movement of this protein into the nuclear membrane and subsequently assembling as viral envelope within the nucleoplasm.
Computer analysis of VP466 predicted that there are multiple putative glycosylation, as well as phosphorylation sites. Viral glycoproteins are known to be responsible for cell tropism, spreading infection, and pathogenicity, etc., and there are accumulating evidences showing that phosphorylation of viral proteins plays important roles in initiating virus infections, e.g. it has been reported that phosphorylation of the Plodia interpunctella granulosis virus core protein may be required for the release of viral DNA from the nucleocapsid at the start of the infection process (23). There is little doubt that the putative post-translational modifications in VP466 should be important functionally in initiation of WSSV infection. Experiments on recombinant or deletion viral mutants will be needed to confirm the validity of these modification sites and to elucidate which of these subdomains are functionally important.
This study showed a gene-protein band pattern of WSSV based on one-dimensional PAGE. The samples used in this study were purified WSSV particles, which contain mainly structural proteins. However, many viral non-structural genes are usually transcribed at the early p.i. stages (i.e. 06 h p.i.). Because of the lack of a suitable cell line for culturing shrimp viruses, the hemolymph from various infection stages with WSSV, instead of purified WSSV, should be used to reveal more genes, especially the early genes, in future studies. Two-dimensional PAGE has the capacity to separate thousands of proteins in a single analysis. The combination of two-dimensional PAGE and MS would reveal more new genes for the proteomic analysis of WSSV.
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ACKNOWLEDGMENTS |
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Note Added in ProofVP19, which is identical to VP121 in our present paper, was characterized recently as a WSSV envelope protein (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.(2002) 83,257265).
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FOOTNOTES |
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Published, MCP Papers in Press, February 15, 2002, DOI 10.1074/mcp.M100035-MCP200
1 The abbreviations used are: WSSV, white spot syndrome virus; ABC, ammonium bicarbonate; ESI-MS/MS, electrospray ionization tandem mass spectrometry; GST, glutathione S-transferase; p.i., post-infection; MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight; MS, mass spectrometry; ORF, open reading frame; Q-TOF, quadrupole time-of-flight; RACE, rapid amplification of cDNA ends; RT, reverse transcription; PBS, phosphate-buffered saline.
2 VP26 was identified originally as a WSSV nucleocapsid protein (14). However, a recent study (15) confirmed that VP26/P22 is an envelope protein by immunogold labeling.
* This investigation is supported by the National Science Technology Board, Singapore, under Grant "Establishment of a Laboratory of Excellence in Aquatic and Marine Biotechnology (LEAMB)."
Contributed equally to this paper.
** To whom correspondence should be addressed: Dept. of Biological Sciences, National University of Singapore, Singapore 119260. Tel.: 65-8742692; Fax: 65-7792486; E-mail: dbshead{at}nus.edu.sg.
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REFERENCES |
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