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
DOI: 10.1128/JVI.01006-07

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Workflow for sample collection and sample preparation.

  • FIG. 2.
    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.
    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.
    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

  • 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
    wsv27717158380C, N14C 1501,131100No hitsThis study
    wsv36017158462C, M, N11N 105582100No hitsVP664 (14)
    wsv42117158523C, M, N4C 112301100SPVP28 (14)
    wsv23017158334C3C 151287100No hitsVP9 (33)
    wsv05117158155C2C 94152100No hitsThis study
    wsv20917158313C3C 87174100No hitsVP187 (32)
    wsv29417158396C, N2C 90144100No hitsThis study
    wsv32717158429C1C 103103100No hitsThis study
    wsv33217158434N2N 57111100No hitsVP75 (14)
    wsv25217158355C, N1N 9999100TMHThis study
    wsv18817158292C1C 8585100One TMHRR2 (17)
    wsv46517158566C, M, N2C 4885100No hitsVP136B (14)
    wsv34317158445C, N2N 5083100NTPaseThis study
    wsv07617158180C2C 5281100No hitsThis study
    wsv28917158391C1C 4444100No hitsVP190 (16)
    wsv28517158388N1N 6161100No hitsThis study
    wsv47717158578C2C 3858100No hitsWSV477 (21)
    wsv21617158320C, M, N1N 5757100One TMH, SPVP124 (35)
    wsv00217158106N1N 5353100SPVP24 (14)
    wsv30817158410N1N 5050100No hitsVP51C (14, 15)
    wsv01117158115C, N2C 2545100Three TMH, SPVP53A (14)
    wsv14317158247C, N1C 344299HMG domainThis study
    wsv17217158276C, N2C 224399One TMHRR1 (17)
    wsv25417158357N1N 434398No hitsVP36b (14, 15)
    wsv41517158517C, N1C 424298No hitsVP60B (14, 15)
    wsv19217158296C1C 334198RNP1, one TMH, recognition motif RNP-1This study
    wsv26017158363C, N1C 333796D/E/S-richThis study
    wsv39017158492C1C 363695No hitsVP38B (14)
    wsv22617158330C2C 313794No hitsThis study
    wsv28417158387C1C 292972One TMH, SPVP13 (14)
    wsv31117158413C1C 272759One TMH, SPVP26 (14)
    wsv00117158566C, N1C 272536No hitsVP180 (14, 15)
    wsv24217158346N1N 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

  • 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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