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

• DOI: 10.1074/mcp.m100035-mcp200
• Publication Date: 2003-04-02T21:49:56Z
• Authors: Canhua Huang; Xiaobo Zhang; Qingsong Lin; Xun Xu; Zhihong Hu; Choy-L. Hew
• Keywords: White spot syndrome; Envelope (radar); White (mutation)

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome this has been sequenced recently. To identify structural and functional proteins WSSV, purified virions were separated by SDS-PAGE. Twenty-four protein bands excised, in-gel digested with trypsin, subjected to matrix-assisted laser desorption ionization-time flight mass spectrometry electrospray ionization tandem spectrometry, respectively. Eighteen matching open reading frames WSSV identified. Except for three known collagen, functions remaining 14 unknown. Temporal analysis revealed that all genes transcribed late stage infection except vp121. Of newly identified proteins, VP466 (derived from band 16) was further characterized. cDNA encoding expressed Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody generated GST-VP466 Western blot showed mouse anti-GST-VP466 bound specifically 51-kDa WSSV. Immunogold labeling component viral envelope. Results investigation thus proved effectiveness proteomic approaches discovering new (WSSV), 1The abbreviations used are: white virus; ABC, ammonium bicarbonate; ESI-MS/MS, spectrometry; GST, S-transferase; p.i., post-infection; MALDI-TOF, ionization-time-of-flight; MS, ORF, frame; Q-TOF, quadrupole time-of-flight; RACE, rapid amplification ends; RT, reverse transcription; PBS, phosphate-buffered saline. considered be (1.Chen X.F. Chen P. Wu D.H. Study on bacilliform cultured shrimps.Sci. China Ser. C. 1997; 27: 415-420Google Scholar), penaeid shrimp. First appearing 1990s Taiwan, spread rapidly shrimp-farming areas around world causing large economic losses. broad host range, including other invertebrate aquatic organisms, such crab crayfish (2.Lo C.F. Ho C.H. Peng S.E. Hsu H.C. Chiu Y.L. Chang Liu K.F. Su M.S. Wang Kou G.H. baculovirus (WSBV) detected captured shrimp, crabs arthropods.Dis. Aquat. 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Vlak J.M. sequence.Virology. 286: 7-22Google Approximately 180 (ORFs) 50 amino acids or more sequences However, contrast insect baculovirus, best studied viruses, only few have reported (8.Zhang Hew C.-L. structure function gene basic peptide virus.Virus Res. 79: 137-144Google Scholar).With completion sequence, research now focused products. Essential end, approach using proven most effective technology identification (9.Naaby-Hansen Waterfield M.D. Cramer Proteomics-post-genomic cartography understand function.Trends Pharmacol. Sci. 22: 376-384Google Recently, (MALDI-TOF) (MS) (ESI-MS/MS) utilizing time-of-flight (Q-TOF) spectrometer tools characterization because their high sensitivity throughput (10.Mann Pandey A. Use spectrometry-derived data annotate nucleotide databases.Trends Biochem. 26: 54-61Google Scholar).In communication, SDS-PAGE analyzed MALDI-TOF MS ESI-MS/MS (Q-TOF), resulting spectrometric searched against theoretical ORF database One retrieved genes, vp466 gene, characterized.EXPERIMENTAL PROCEDURESProliferation Purification Shrimp WSSVProliferation Virion—The infected tissues (e.g. gill, stomach, midgut, etc.) homogenized TN buffer (20 mm Tris-HCl 400 NaCl, pH 7.4) 0.1 g/ml. After centrifugation 2000 × g 10 min, supernatant filtered (0.22-μm filter) injected (1:100 dilution 0.9% NaCl) intramuscularly into healthy Cambarus clarkii Hainan province, lateral area fourth abdominal segment. Four days later, hemolymph freshly extracted layered top 10–40% (w/v) continuous sodium bromide gradient centrifuged 110,000 SW41-Ti rotor Beckman ultracentrifuge (XL-90; Coulter) 2 h 4°C. Virus collected side puncture, diluted 1:10 TNE (50 Tris-HCl, 100 1 EDTA, 7.4), pelleted 119,000 pellets resuspended TNE. samples examined under transmission electron microscope (JEOL CXII) purity (11.Huang Xiao J.K. produced an alternate host: crayfish, clarkii.Virus 76: 115-125Google Scholar).Purification Nucleocapsid—Purified virion treated Triton X-100 15 min room temperature, 20–50% CsCl gradient, 24–48 SW 41-Ti. Viral capsid puncture then 1× (1:10), subsequently sedimented 120,000 45 min. pellet buffer, 7.4.Computer Analysis ORFs GenomeThe 305,107-bp Scholar) DNAMAN (Lynnon BioSoft, Vaudreuil, Canada) ORFs. total, 4443 starting ATG start codon lengths larger located both strands genome. From these ORFs, ∼181 ranging 61 6077 size likely encode These 181 designated putative assigned database. Homology searches performed BLAST BLAST2 programs. Protein motifs PROSITE release 16 (12.Hofmann K. Bucher Falquet Bairoch database, its status 1999.Nucleic Acids 215-219Google Scholar).Mass Spectrometric ProteinsIn-gel Digestion—The 12% stained Coomassie Blue R 250. excised dehydrated several times acetonitrile. vacuum drying, gel incubated dithiothreitol bicarbonate (ABC buffer) 57°C 60 55 iodoacetamide (Sigma) ABC temperature Then gels washed dried. All digestions sequencing grade modified porcine trypsin (Promega, Madison, WI) 37°C h. Digests 6000 g. supernatants separated, pieces first 50% acetonitrile, 5% formic acid extracts combined original digesting supernatants, vacuum-dried, redissolved 0.5% trifluoroacetic acetonitrile (13.Shevchenko Wilm Vorm O. Mann Mass silver-stained polyacrylamide gels.Anal. Chem. 68: 850-858Google Scholar).MALDI-TOF MS—The matrix saturated solution α-cyano-4-hydroxycinnamic sample (1:1, v/v) loaded target plate. spectra peptides obtained Voyager-DE STR Biospectrometry work station (PerSeptive Biosystems, Inc., Framingham, MA). analyses positive ion reflector mode accelerating voltage 20 kV delayed extraction 150 ns. Typically scans averaged. autoproteolysis products internal calibrants. Data mining MS-Fit software deviation ppm usually allowed searches.Nano-ESI-MS/MS—The desalted C18 ZipTip (Millipore, Bedford, MA) dissolving μl acid, metallized glass capillary. capillary mounted nanoflow Z-spray source Q-TOF2 (Micromass, Manchester, United Kingdom). Flow rates varied 8 nL/min. Instrument operation, acquisition, MassLynx/BioLynx 3.5 (Micromass). collision energy optimized each sample. microchannel plate set 2200 V. Global Server (Micromass).Transcriptional Analyses GenesShrimp Infection WSSV—The tissue pathologically confirmed g, 1:100 NaCl filter). 0.2 ml filtrate At various post-infection (p.i.), four specimens selected random, hemolymphs collected. immediately frozen stored −70°C.RT-PCR—The total RNA WSSV-infected according manufacturer's instruction (NucleoSpin II; Macherey-Nagel GmbH & Co. KG, Germany). RT-PCR ORF-specific primers TITANIUM One-step kit (CLONTECH Laboratories, Inc.). cycles follows: 50°C h, 94°C 5 30 s, 65°C 68°C followed elongation min.Characterization GeneRapid Amplification Ends (5′ 3′ RACE)—The 5′ RACE. For gene-specific primer SP1 nested SP2 designed 5′-GCTCTCCATCCGCTTAGTCACATTGGC-3′ 5′- GCCGAAGCTGAAGGTTTTGGAGGTGC-3′, SP3 5′-GCAGTAGCAAATCTCACCGGACCTGTG-3′. RACE reaction instructions 5′/3′ (Roche Molecular Biochemicals).Expression Fusion coli—The amplified synthesized forward 5′-CACGGATCCATGTCTGCATCTTTAAT-3′ BamHI site (italic underlined) 5′-AGACCCGGGTTATGACACAAACCTAT-3′ SmaI underlined). plasmid vector pGEX-4T-2 + SmaI. ligation fragments, inserted downstream GST pGEX-4T-2-pLysS (Amersham Biosciences Corp.). resulted recombinant named pGEX466. competent cells E. BL-21 pLysS transformed pGEX466, colonies containing transformants screened colony PCR. identity pGEX466 restriction enzyme digestion (BamHI SmaI) sequencing.After overnight incubation 37°C, pGEX466-pLysS (BL-21 pGEX-4T-2, respectively) inoculated media ratio 1:100. When A600 reached 0.6, cultures induced IPTG continued grow 6 bacteria spun down (4000 g) suspended ice-cold saline (PBS) (containing 1% X-100, phenylmethanesulfonyl fluoride, 4 benzamidine, μg leupeptin, aprotinin) sonicated s ice. spinning 60,000 mixed PBS-buffered glutathione-agarose beads 4°C shaking device. PBS reducing reduced glutathione, 8.0) 1000 SDS-PAGE.Antibody Preparation—The immunize mice (Swiss Albino, 3–4 weeks) once every weeks intradermal injection over eight-week period. Titers antisera 1:20,000 determined enzyme-linked immunosorbent assay. A-SepharoseTM CL-4B isolate IgG manufacture's Biosciences). optimal IgG, after serial dilutions, 1:1,000 Horseradish peroxidase-conjugated goat anti-mouse Sigma. negative control, used.Western Blot—WSSV SDS-PAGE, transferring onto nitrocellulose membrane (Bio-Rad) electroblotting (Tris 25 mm, glycine 190 methanol 20%) 3 immersed blocking (3% bovine serum albumin, Tris, 0.1% Tween 20, 7.2) overnight, polyclonal anti-GST-P466 pre-immune serum, anti-GST Subsequently, horseradish secondary antibody, detection substrate 4-chloro-1-naphthol (Sigma).Localization Immunoelectron Microscopy—WSSV nucleocapsid Formvar-coated, carbon-stabilized nickel grids, respectively, grids blocked 2% AURION BSA-CTM (Electron Microscopy Sciences) primary (purified anti-VP466 1:1000 BSA-CTM) 2–3 washing conjugated nm colloid gold two briefly phosphotungstic (pH 7.0) CXII, Japan). control experiments, replaced following experimental steps.RESULTSIdentification Proteins MSWSSV proliferated clarkii. improved protocol (Fig. 1a). being ultracentrifugation, 1b).The More kDa visible staining 1c). them gel. Following alkylated MS. Peak lists tryptic masses search engine corresponding genes. Reliable matches covering 7 72% sequences, 24 gel-excised (Table I). Subsequently nano-ESI-MS/MS. derived MS/MS global server. 3–48% coverage respectively I).Table IWSSV productsBand No.GenePosition genomeSizeaSize (aa) predicted molecular (kDa).Estimated SDS-PAGEGenBank™ accession numberCharacteristics deduced proteinsGene transcription p.i.Mass spectrometrySequence coveragestart codonstop codonaamassMALDIESIkDah%1vp682281962279936877AF411464Not known18–36Q-TOF192vp9526722388951112AF402996Not known18MALDI724vp1212416372412751211317AF402997Not known6–24MALDI, Q-TOF28195vp1841731781737291842122AF402998Not known30MALDI76vp20811184952082324AF402999vp24 (Ref. 14)18–30MALDI, Q-TOF61337p221800361794252042225AF227911vp26 14)18–36MALDI, Q-TOF44488p2042442422448532042227.5AF308164vp28 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 445.445bSize 445.1684186180AY048547Collagen30MALDI22a Size (kDa).b 445. Open table tab 18 Q-TOF study, described previously, VP208, P22, P204, encoded vp208, p22, p204 (14.van Goldbach R.W. Three functionally diverged evolved duplication.J. Gen. 2000; 81: 2525-2529Google 15.Zhang envelope (p22) (WSSV).J. 2002; 83: 471-477Google time. P204 found 8, 9, 10, 12, P22 14, On hand, 13, 16, I).Structures Genes Homologies Known ProteinsA guanine residue beginning largest fragment point physical map positions numbers GenBankTM listed Table I. typical TATA box promoter regions indicating may essential vp184, vp300, vp674 polyadenylation signal (AATAAA) stop codons Among (ATGs) favorable context eukaryotic translation initiation (PuNNATGPu) (16.Kozak An 5′-noncoding 699 vertebrate messenger RNAs.Nucleic 1987; 15: 8125-8148Google Exceptions vp357, vp466, vp544, vp800 68 1684 I).Based homology BLAST2, could assigned. Two contained based 12 no any motifs. vp1684 (168 kDa) collagen family. This time intact virus. collagen-like repeat Gly-X-Y (X mostly proline, Y acid), but not clear. characteristics p22 immunoelectron microscopy our earlier studies (15.Zhang Scholar).Temporal Gene TranscriptionsRT-PCR detect transcripts RNAs adult stages (0, 6, 18, 24, 30, 36, 48 p.i) different results coding fidelity Based temporal analysis, vp121 p.i. until 17 suggesting course infection. However element, ATAAG, canonical baculoviruses, genes.Characterization GeneIdentification Spectrometry—The associated protein, (one minor bands) profile. Trypsin digests 2a). list product (termed protein). experimentally match within ppm, 21% sequence. measured calculated nano-ESI-MS spectrum shown Fig. 2b. corresponded searching database.Fig. 2Characterization spectrometry.a, 16. Peptides digestion. gene. Tryptic accuracy indicated dotted underline. b, pipetting through resin packed Millipore before loading Micromass system. solid underline.View Large Image Figure ViewerDownload (PPT)Location Gene—The 177,124 178,521 nucleotides, 1398-bp presumably 466-amino (hence termed gene; number AF395545; VP466), 51.2 isoelectric 6.51 (isoelectric computation, EMBL). same 6-kb vp26/p22 opposite orientation (TATAAAT) 37 nucleotides upstream site, 111 site. (poly(A)) 161 translational poly(A)