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Cellular Response to Infection

White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag

Jinlu Wu, Qingsong Lin, Teck Kwang Lim, Tiefei Liu, Choy-Leong Hew
Jinlu Wu
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
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Qingsong Lin
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
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Teck Kwang Lim
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
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Tiefei Liu
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
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Choy-Leong Hew
Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
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  • For correspondence: dbshead@nus.edu.sg
DOI: 10.1128/JVI.01006-07
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  • FIG. 1.
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    FIG. 1.

    Workflow for sample collection and sample preparation.

  • FIG. 2.
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    FIG. 2.

    Relative abundance of viral proteins in different tissues of infected shrimp. (Left) Equal amounts (30 μg) of whole lysate proteins extracted from stomachs, gills, and subcuticular epithelia of WSSV-infected shrimp were resolved by SDS-PAGE and then probed with antibodies against β-actin, VP28 (WSSV structural protein), and VP9 (WSSV nonstructural protein), showing that the concentrations of both the structural and nonstructural proteins were relatively higher in the epithelial lysate. (Right) Equal amounts (10 μg) of cytosolic, membrane, and nuclear proteins from the epithelium were resolved by SDS-PAGE and probed with antibodies against histone 2A, GAPDH, VP28, and VP9, showing that subcellular fractionation can enrich viral proteins while reducing the nucleus-specific protein histone 2A in the cytosolic fraction.

  • FIG. 3.
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    FIG. 3.

    Temporal transcription of 11 novel viral protein genes. mRNAs were extracted from subcuticular epithelia sampled at 0, 2, 4, 6, 8, 12, 24, and 72 hpi. cDNAs were synthesized using random hexamers and normalized to equal concentrations by β-actin. As the time of infection elapsed, 10 of 14 genes showed increasing mRNA levels (group 1), 1 maintained a constant mRNA level (group 2), and 3 showed decreasing mRNA levels (group 3).

  • FIG. 4.
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    FIG. 4.

    Western blotting analysis of novel viral proteins. Three novel proteins were present in the total cell lysate and the cytosolic fraction of WSSV-infected epithelia but were not detected in the purified virus and the uninfected cell lysate by Western blotting using antibodies against wsv051, -076, -294, and -477 (a control for viral nonstructural proteins). Viral structural proteins, including the capsid protein VP664 and the envelope proteins (VP28 and VP26), were present in the infected cell lysate and the purified virus.

Tables

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  • TABLE 1.

    WSSV proteins identified by shotgun proteomicsa

    Predicted ORFGI accession no.Fraction(s)bNo. of peptides matchedBest ion scorecTotal ion scoreTotal ion CI%dPredicted structure and functioneProtein name and reference
    wsv277 17158380 C, N 14 C 150 1,131 100 No hits This study
    wsv360 17158462 C, M, N11N 105582100No hitsVP664 (14)
    wsv421 17158523 C, M, N4C 112301100SPVP28 (14)
    wsv230 17158334 C3C 151287100No hitsVP9 (33)
    wsv051 17158155 C 2 C 94 152 100 No hits This study
    wsv209 17158313 C3C 87174100No hitsVP187 (32)
    wsv294 17158396 C, N 2 C 90 144 100 No hits This study
    wsv327 17158429 C 1 C 103 103 100 No hits This study
    wsv332 17158434 N2N 57111100No hitsVP75 (14)
    wsv252 17158355 C, N 1 N 99 99 100 TMH This study
    wsv188 17158292 C1C 8585100One TMHRR2 (17)
    wsv465 17158566 C, M, N2C 4885100No hitsVP136B (14)
    wsv343 17158445 C, N 2 N 50 83 100 NTPase This study
    wsv076 17158180 C 2 C 52 81 100 No hits This study
    wsv289 17158391 C1C 4444100No hitsVP190 (16)
    wsv285 17158388 N 1 N 61 61 100 No hits This study
    wsv477 17158578 C2C 3858100No hitsWSV477 (21)
    wsv216 17158320 C, M, N1N 5757100One TMH, SPVP124 (35)
    wsv002 17158106 N1N 5353100SPVP24 (14)
    wsv308 17158410 N1N 5050100No hitsVP51C (14, 15)
    wsv011 17158115 C, N2C 2545100Three TMH, SPVP53A (14)
    wsv143 17158247 C, N 1 C 34 42 99 HMG domain This study
    wsv172 17158276 C, N2C 224399One TMHRR1 (17)
    wsv254 17158357 N1N 434398No hitsVP36b (14, 15)
    wsv415 17158517 C, N1C 424298No hitsVP60B (14, 15)
    wsv192 17158296 C 1 C 33 41 98 RNP1, one TMH, recognition motif RNP-1 This study
    wsv260 17158363 C, N 1 C 33 37 96 D/E/S-rich This study
    wsv390 17158492 C1C 363695No hitsVP38B (14)
    wsv226 17158330 C 2 C 31 37 94 No hits This study
    wsv284 17158387 C1C 292972One TMH, SPVP13 (14)
    wsv311 17158413 C1C 272759One TMH, SPVP26 (14)
    wsv001 17158566 C, N1C 272536No hitsVP180 (14, 15)
    wsv242 17158346 N1N 23230No hitsVP41B (14, 15)
    • ↵ a Cytosolic, nuclear, and membrane fractions were isolated from WSSV-infected subcuticular epithelium for shotgun 2D-LC-MS/MS analysis. The last five proteins in the list (total ion score CI%, <95%) are not considered confident identifications. Proteins shown in bold were identified for the first time. Details of MS data are shown in Table SII in the supplemental material.

    • ↵ b C, M, and N, cytosolic, membrane, and nuclear fractions, respectively.

    • ↵ c Proteins that were best identified in the cytosol (C), membrane (M), or nucleus (N), with the highest ion score, total ion score, and total ion score CI%.

    • ↵ d The closer the CI% is to 100%, the more likely it is that the protein was correctly identified.

    • ↵ e SP, signal peptide, predicted using SignalP3.0 software; TMH, transmembrane helix, predicted using TMHMM-2.0 software; D/E/S, single-letter representations of the amino acids Asp/Glu/Ser.

  • TABLE 2.

    Summary of differentially expressed cellular proteins identified by time course cICAT analysisa

    Direction of regulation and protein nameGenBank accession no.No. of peptides (6/12/24/72 hpi)Total ion score (6/12/24/72 hpi)Total ion score CI% (6/12/24/72 hpi)Avg ICAT ratio (H/L) (6/12/24/72 hpi)
    Down-regulated proteins
        Elongation factor 237704007—/—/—/1—/—/—/54—/—/—/98.5—/—/—/0
        Aldehyde dehydrogenase66514094—/—/—/1—/—/—/62—/—/—/99.8—/—/—/0.3
        Serum albumin62113341—/—/1/——/—/90/——/—/100/——/—/0.4/—
        Heat shock protein 70123585—/—/—/1—/—/—/50—/—/—/95.3—/—/—/0.4
        FAMeT85677401—/1/—/——/55/—/——/98.5/—/——/0.5/—/—
        CiTardbp70571316—/—/1/1—/—/48/64—/—/96.9/99.8—/—/1.5/0.4
        Beta-hemoglobin29446—/—/2/——/—/105/——/—/100/——/—/0.5/—
        PACAP18204192—/—/—/1—/—/—/49—/—/—/94.7—/—/—/0.6
        Elongation factor 122128323—/—/—/1—/—/—/84—/—/—/100—/—/—/0.7
        Clottable protein66014982/3/1/3109/254/69/195100/100/100/1000.8/1/1.8/0.6
    Up-regulated proteins
        Arginine kinase46401522—/—/1/1—/—/75/100—/—/100/100—/—/1.4/1.7
        Hemocyanin7414468—/2/2/2—/126/146/226—/100/100/100—/1.7/1.6/2.1
    • ↵ a Pairs of epithelial lysates from uninfected and WSSV-infected Peneaus monodon shrimp at 6, 12, 24, and 72 hpi were prepared for cICAT analyses. Proteins with H/L ratios of <0.71 are regarded as down-regulated, while those with H/L ratios of >1.4 are regarded as up-regulated. Details are shown in Tables SV and SVII in the supplemental material. —, no peptide was detected; 0, only the peptide labeled with the light tag was detected.

  • TABLE 3.

    Peptides of tubulin and ATPase groups identified by cICAT analysisa

    Protein group and peptide sequence (section of Table SVI in the supplemental material)Mr (exptl)Mr (calculated)ScoreExpect valueH/L ratio
    Tubulin group identified at 24 hpi (1)
        SIQFVDWCPTGFK + ICAT_heavy1,762.881,762.88560.00550.97
        AYHEQLSVAEITNACFEPANQMVK + ICAT_heavy2,928.432,928.42831.3e−0051.65
    Tubulin group identified at 72 hpi (2)
        EIVHLQTGQCGNQIGTK + ICAT_light2,052.042,052.041322.8e−0100.60
        SIQFVDWCPTGFK + ICAT_light1,753.831,753.85730.000220.56
        RSIQFVDWCPTGFK + ICAT_light1,909.951,909.95520.0290.30
        AVCMLSNTTAIAEAWAR + ICAT_light2,034.002,034.00660.00130.50
        TIQFVDWCPTGFK + ICAT_light1,767.861,767.87680.000690.53
    ATPase group identified at 24 hpi (3)
        IPIFSAAGLPHNEIAAQICR + ICAT_heavy2,356.272,356.281249.8e−0101.41
        KDHSDVSNQLYACYAIGK + ICAT_heavy2,247.112,247.10450.0722.28
    ATPase group identified at 72 hpi (4)
        IPIFSAAGLPHNEIAAQICR + ICAT_light2,347.242,347.251251.5e−0090.64
        KDHSDVSNQLYACYAIGK + ICAT_light2,238.082,238.07700.000450.38
    • ↵ a Details are shown in Table SVI in the supplemental material.

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Table SI (Primer sets used for RT-PCR.)
      Zipped PDF document, 4K.
    • Supplemental file 2 - Table SII (Details of WSSV protein identification by shotgun proteomics.)
      Zipped PDF document, 980K.
    • Supplemental file 3 - Table SIII (List of peptides identified by shotgun proteomics.) Table SIV (List of shrimp epithelial cellular proteins identified by shotgun proteomics.)
      Zipped Excel document, 358K.
    • Supplemental file 4 - Table SV (The details of identification of differentially expressed cellular proteins by cICAT.)
      Zipped PDF document, 363K.
    • Supplemental file 5 - Table SVI (The details of peptides identified in tubulin and ATPase groups by cICAT proteomics.)
      Zipped PDF document, 377K.
    • Supplemental file 6 - Table SVII (The details of all proteins and peptides identified by cICAT proteomics, including the means and deviations of H/L ratios.)
      Zipped PDF document, 53K.
    • Supplemental file 7 - Fig. SI (Sequence alignment of ci-Tardbp with homologs.)
      Zipped PDF document, 44K.
    • Supplemental file 8 - Legends to Tables and Figure MS Word document, 29K.
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White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag
Jinlu Wu, Qingsong Lin, Teck Kwang Lim, Tiefei Liu, Choy-Leong Hew
Journal of Virology Oct 2007, 81 (21) 11681-11689; DOI: 10.1128/JVI.01006-07

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White Spot Syndrome Virus Proteins and Differentially Expressed Host Proteins Identified in Shrimp Epithelium by Shotgun Proteomics and Cleavable Isotope-Coded Affinity Tag
Jinlu Wu, Qingsong Lin, Teck Kwang Lim, Tiefei Liu, Choy-Leong Hew
Journal of Virology Oct 2007, 81 (21) 11681-11689; DOI: 10.1128/JVI.01006-07
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KEYWORDS

epithelium
Gene Expression Regulation, Viral
Penaeidae
proteomics
Viral Proteins
White spot syndrome virus 1

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