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Proteomic Analysis of Shrimp White Spot Syndrome Viral Proteins and Characterization of a Novel Envelope Protein VP466

2002; Elsevier BV; Volume: 1; Issue: 3 Linguagem: Inglês

10.1074/mcp.m100035-mcp200

1535-9484

Canhua Huang, Xiaobo Zhang, Qingsong Lin, Xun Xu, Zhìhóng Hú, C. L. Hew,

Insect symbiosis and bacterial influences

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV. White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV. White spot syndrome virus (WSSV), 1The 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. 1The 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. considered to be a new virus (1.Chen X.F. Chen P. Wu D.H. Study on a new bacilliform virus in cultured shrimps.Sci. China Ser. C. 1997; 27: 415-420Google Scholar), is one of the major pathogens in cultured penaeid shrimp. First appearing in the 1990s in Taiwan, WSSV has spread rapidly to shrimp-farming areas around the world causing large economic losses. The virus has a broad host range, including other invertebrate aquatic organisms, such as crab and crayfish (2.Lo C.F. Ho C.H. Peng S.E. Chen C.H. Hsu H.C. Chiu Y.L. Chang C.F. Liu K.F. Su M.S. Wang C.H. Kou G.H. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods.Dis. Aquat. Org. 1996; 27: 215-225Google Scholar, 3.Huang C. Shi Z. Zhang J. Zhang L. Chen D. Bonami J.R. Establishment of a model for proliferating white spot syndrome virus in vivo.Virol. Sin. 1999; 14: 358-364Google Scholar). The WSSV particles are non-occluded and bacilliform in shape with double envelopes (4.Chou H.Y. Huang C.Y. Wang C.H. Chiang H.C. Lo C.F. Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan.Dis. Aquat. Org. 1995; 23: 165-173Google Scholar). In 1997, the WSSV genomic DNA was purified successfully from Penaeus japonicus shrimp in China (5.Yang F. Wang W. Chen R.Z. Xu X. A simple and efficient method for purification of prawn baculovirus DNA.J. Virol. Methods. 1997; 67: 1-4Google Scholar), and the genomic DNA and cDNA libraries were constructed. WSSV genome contains a 305-kb double-stranded circular DNA (6.Yang 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: 11811-11820Google Scholar), which is longer than the 293-kb genome isolated from Penaeus monodon shrimps in Thailand (7.van Hulten M.C. Witteveldt J. Peters S. Kloosterboer N. Tarchini R. Fiers M. Sandbrink H. Lankhorst R.K. Vlak J.M. The white spot syndrome virus DNA genome sequence.Virology. 2001; 286: 7-22Google Scholar). Approximately 180 open reading frames (ORFs) of 50 amino acids or more were revealed by the analysis of the WSSV genomic DNA sequences (6.Yang 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: 11811-11820Google Scholar). However, in contrast to the insect baculovirus, one of the best studied viruses, only a few genes from WSSV have been reported (8.Zhang X. Xu X. Hew C.-L. The structure and function of a gene encoding a basic peptide from prawn white spot syndrome virus.Virus Res. 2001; 79: 137-144Google Scholar).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.Naaby-Hansen S. Waterfield M.D. Cramer R. Proteomics-post-genomic cartography to understand gene function.Trends Pharmacol. Sci. 2001; 22: 376-384Google Scholar). 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.Mann M. Pandey A. Use of mass spectrometry-derived data to annotate nucleotide and protein sequence databases.Trends Biochem. Sci. 2001; 26: 54-61Google Scholar).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.EXPERIMENTAL PROCEDURESProliferation and Purification of Shrimp WSSVProliferation and Purification of WSSV Virion—The infected tissues from penaeid shrimp P. monodon (e.g. gill, stomach, midgut, etc.) were homogenized in TN buffer (20 mm Tris-HCl and 400 mm NaCl, pH 7.4) at 0.1 g/ml. After centrifugation at 2000 × g for 10 min, the supernatant was filtered (0.22-μm filter) and injected (1:100 dilution in 0.9% NaCl) intramuscularly into healthy crayfish Cambarus clarkii from Hainan province, China in the lateral area of the fourth abdominal segment. Four days later, hemolymph freshly extracted from infected crayfish was layered on the top of the 10–40% (w/v) continuous sodium bromide gradient and centrifuged at 110,000 × g using a SW41-Ti rotor in a Beckman ultracentrifuge (XL-90; Beckman Coulter) for 2 h at 4°C. Virus bands were collected by side puncture, diluted 1:10 with TNE buffer (50 mm Tris-HCl, 100 mm NaCl, and 1 mm EDTA, pH 7.4), and pelleted at 119,000 × g for 1 h at 4°C. The resulting pellets were resuspended in TNE. Virus samples were examined under a transmission electron microscope (JEOL 100 CXII) for purity (11.Huang C. Zhang L. Zhang J. Xiao L. Wu Q. Chen D. Li J.K. Purification and characterization of white spot syndrome virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii.Virus Res. 2001; 76: 115-125Google Scholar).Purification of WSSV Nucleocapsid—Purified WSSV virion was treated with Triton X-100 for 15 min at room temperature, subjected to 20–50% continuous CsCl gradient, and centrifuged for 24–48 h at 110,000 × g using a SW 41-Ti. Viral capsid band was collected by side puncture and then diluted with 1× TN buffer (1:10), subsequently sedimented at 120,000 × g for 45 min. The pellet was resuspended in 1× TN buffer, pH 7.4.Computer Analysis of the ORFs of WSSV GenomeThe 305,107-bp DNA sequence of the WSSV genome (6.Yang 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: 11811-11820Google Scholar) 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.Yang 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: 11811-11820Google Scholar). 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.Hofmann K. Bucher P. Falquet L. Bairoch A. The PROSITE database, its status in 1999.Nucleic Acids Res. 1999; 27: 215-219Google Scholar).Mass Spectrometric Analysis of Viral ProteinsIn-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 × 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.Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of protein silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar).MALDI-TOF MS—The matrix used was a saturated solution of α-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 GenesShrimp 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 × 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 manufacturer's 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 GeneRapid 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 × 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 × g, the supernatant was mixed with 1× 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 1× 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 × 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, 3–4 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 manufacture's 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, 1× 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 2–3 h. After washing three to four times with 1× 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 1× 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.RESULTSIdentification of WSSV Proteins by MSWSSV was proliferated in an alternate host crayfish C. clarkii. The virus purification was performed according to an improved protocol (11.Huang C. Zhang L. Zhang J. Xiao L. Wu Q. Chen D. Li J.K. Purification and characterization of white spot syndrome virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii.Virus Res. 2001; 76: 115-125Google Scholar) (Fig. 1a). After being treated with Triton X-100 and CsCl gradient ultracentrifugation, the purified viral capsid was obtained (Fig. 1b).The proteins of the WSSV virion were separated by SDS-PAGE. More than 20 bands ranging from 5 to 190 kDa were visible with Coomassie Blue staining (Fig. 1c). Twenty-four of them were excised from the gel. Following trypsin digestion of the reduced and alkylated WSSV proteins, the peptides were first analyzed by MALDI-TOF MS. Peak lists of tryptic peptide masses of each protein were generated and were subjected to the WSSV ORF database search using the MS-Fit search engine to identify the proteins and corresponding genes. Reliable matches with the genes from the theoretical WSSV ORF database, covering 7 to 72% of amino acid sequences, were obtained for 16 of the 24 gel-excised bands (Table I). Subsequently the tryptic peptides of each band were sequenced using nano-ESI-MS/MS. To identify the proteins, the peptide sequences derived from the MS/MS data were searched against the WSSV ORF database using a global server. 14 of the 24 protein bands were identified with 3–48% coverage of amino acid sequences, respectively (Table I).Table IWSSV genes and their productsBand No.GenePosition in the WSSV genomeSizeaSize of ORFs in amino acids (aa) and predicted molecular mass (kDa).Estimated size from SDS-PAGEGenBank™ accession numberCharacteristics of deduced proteinsGene transcription p.i.Mass spectrometrySequence coveragestart codonstop codonaamassMALDIESIkDah%1vp682281962279936877AF411464Not known18–36Q-TOF192vp9526722388951112AF402996Not known18MALDI724vp1212416372412751211317AF402997Not known6–24MALDI, Q-TOF28195vp1841731781737291842122AF402998Not known30MALDI76vp20811184952082324AF402999vp24 of WSSV (Ref. 14)18–30MALDI, Q-TOF61337p221800361794252042225AF227911vp26 of WSSV (Ref. 14)18–36MALDI, Q-TOF44488p2042442422448532042227.5AF308164vp28 of WSSV (Ref. 14)18–36MALDI, Q-TOF42459p20428MALDI, Q-TOF423610p20429Q-TOF1911vp2811416961425382813235AF411634Not known30MALDI, Q-TOF27712p20437Q-TOF1213vp3001329941338933003438AF403003Not known18MALDI24vp2921305661314412923338AF411636Not known24–48MALDI, Q-TOF24514p2241Q-TOF4vp35758956600263573941AF403004Not known36MALDI2016vp4661771241785214665250AF395545Not known24–30MALDI, Q-TOF2411vp3841425451436963844350AF411635Not known24MALDI, Q-TOF12818vp4483004322990894485055AY048543Not known24MALDI919vp5442417752434065446260AY044842Not known36MALDI, Q-TOF8821vp6741190571210786747676AY048545Class I cytokine receptor24MALDI923vp8002550752574748008990AY044843ATP/GTP binding motif36MALDI, Q-TOF18324vp1684300501bSize = 300500 → 305107 + 1 → 445.445bSize = 300500 → 305107 + 1 → 445.1684186180AY048547Collagen30MALDI22a Size of ORFs in amino acids (aa) and predicted molecular mass (kDa).b Size = 300500 → 305107 + 1 → 445. Open table in a new tab In total, 18 genes from WSSV were identified by MALDI-TOF MS and Q-TOF from SDS-PAGE. Of the 18 proteins reported in this study, only three have been described previously, VP208, P22, and P204, the products encoded by the vp208, p22, and p204 genes, respectively (14.van Hulten M.C. Goldbach R.W. Vlak J.M. Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication.J. Gen. Virol. 2000; 81: 2525-2529Google Scholar, 15.Zhang X. Huang C. Xu X. Hew C.-L. The transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus (WSSV).J. Gen. Virol. 2002; 83: 471-477Google Scholar). The remaining 15 proteins were identified for the first time. The P204 protein was found in bands 8, 9, 10, and 12, and the P22 protein in bands 7 and 14, respectively. On the other hand, two genes were revealed in bands 13, 14, and 16, respectively (Table I).Structures of Genes and Homologies with Known ProteinsA 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.Yang 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: 11811-11820Google Scholar). 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.Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs.Nucleic Acids Res. 1987; 15: 8125-8148Google Scholar) 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.van Hulten M.C. Goldbach R.W. Vlak J.M. Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication.J. Gen. Virol. 2000; 81: 2525-2529Google Scholar). The p22 and p204 genes were identified further to encode envelope proteins of WSSV by immunoelectron microscopy in our earlier studies (15.Zhang X. Huang C. Xu X. Hew C.-L. The transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus (WSSV).J. Gen. Virol. 2002; 83: 471-477Google Scholar).Temporal Analyses of Gene TranscriptionsRT-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 GeneIdentification 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.Fig. 2Characterization of VP466 by mass spectrometry.a, MALDI-TOF MS spectrum of protein band 16. Peptides were produced by in-gel tryptic digestion. The peptide masses were used for the WSSV ORF database search and retrieved the vp466 gene. Tryptic peptides that map to the protein sequence encoded by the vp466 gene within a mass accuracy of 100 ppm were indicated by a dotted underline. b, nano-ESI-MS spectrum of protein band 16. Peptides were produced by in-gel tryptic digestion. The peptide solution was desalted by pipetting through C18 resin packed in a Millipore ZipTip before loading into a Micromass Q-TOF2 MS system. The band was found to be the encoding product of the vp466 gene after searching against the WSSV ORF database. Tryptic peptides that map to the protein sequence encoded by the vp466 gene within a mass accuracy of 100 ppm were indicated by a solid underline.View Large Image Figure ViewerDownload (PPT)Location of WSSV vp466 Gene—The start and stop codons of vp466 gene were present at 177,124 and 178,521 nucleotides, respectively, in the WSSV genome (6.Yang 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: 11811-11820Google Scholar). This 1398-bp ORF presumably encoded a 466-amino acid protein (hence termed vp466 gene; GenBankTM accession number AF395545; the resulting protein termed as VP466), with a theoretical molecular mass of 51.2 kDa and with an isoelectric point of 6.51 (isoelectric point computation, EMBL). The vp466 gene was found to be located at the same 6-kb BamHI fragment of WSSV genome as vp26/p22 gene but with an opposite orientation (15.Zhang X. Huang C. Xu X. Hew C.-L. The transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus (WSSV).J. Gen. Virol. 2002; 83: 471-477Google Scholar). A typical TATA box (TATAAAT) was present at 37 nucleotides upstream of the transcription initiation site, which was proved by 5′ RACE, and 111 nucleotides upstream of the translation starting site. A putative polyadenylation (poly(A)) signal (AATAAA) is present at 161 nucleotides downstream of the translational stop codon of vp466 and 24 nucleotides upstream of poly(A) revealed by 3′ RACE. The structure of vp466 gene and its deduced amino s