Article Figures & Data
Figures
- FIG. 1.
Workflow for sample collection and sample preparation.
- 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.
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.
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 ORF GI accession no. Fraction(s)b No. of peptides matched Best ion scorec Total ion score Total ion CI%d Predicted structure and functione Protein name and reference wsv277 17158380 C, N 14 C 150 1,131 100 No hits This study wsv360 17158462 C, M, N 11 N 105 582 100 No hits VP664 (14) wsv421 17158523 C, M, N 4 C 112 301 100 SP VP28 (14) wsv230 17158334 C 3 C 151 287 100 No hits VP9 (33) wsv051 17158155 C 2 C 94 152 100 No hits This study wsv209 17158313 C 3 C 87 174 100 No hits VP187 (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 N 2 N 57 111 100 No hits VP75 (14) wsv252 17158355 C, N 1 N 99 99 100 TMH This study wsv188 17158292 C 1 C 85 85 100 One TMH RR2 (17) wsv465 17158566 C, M, N 2 C 48 85 100 No hits VP136B (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 C 1 C 44 44 100 No hits VP190 (16) wsv285 17158388 N 1 N 61 61 100 No hits This study wsv477 17158578 C 2 C 38 58 100 No hits WSV477 (21) wsv216 17158320 C, M, N 1 N 57 57 100 One TMH, SP VP124 (35) wsv002 17158106 N 1 N 53 53 100 SP VP24 (14) wsv308 17158410 N 1 N 50 50 100 No hits VP51C (14, 15) wsv011 17158115 C, N 2 C 25 45 100 Three TMH, SP VP53A (14) wsv143 17158247 C, N 1 C 34 42 99 HMG domain This study wsv172 17158276 C, N 2 C 22 43 99 One TMH RR1 (17) wsv254 17158357 N 1 N 43 43 98 No hits VP36b (14, 15) wsv415 17158517 C, N 1 C 42 42 98 No hits VP60B (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 C 1 C 36 36 95 No hits VP38B (14) wsv226 17158330 C 2 C 31 37 94 No hits This study wsv284 17158387 C 1 C 29 29 72 One TMH, SP VP13 (14) wsv311 17158413 C 1 C 27 27 59 One TMH, SP VP26 (14) wsv001 17158566 C, N 1 C 27 25 36 No hits VP180 (14, 15) wsv242 17158346 N 1 N 23 23 0 No hits VP41B (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 name GenBank 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 2 37704007 —/—/—/1 —/—/—/54 —/—/—/98.5 —/—/—/0 Aldehyde dehydrogenase 66514094 —/—/—/1 —/—/—/62 —/—/—/99.8 —/—/—/0.3 Serum albumin 62113341 —/—/1/— —/—/90/— —/—/100/— —/—/0.4/— Heat shock protein 70 123585 —/—/—/1 —/—/—/50 —/—/—/95.3 —/—/—/0.4 FAMeT 85677401 —/1/—/— —/55/—/— —/98.5/—/— —/0.5/—/— CiTardbp 70571316 —/—/1/1 —/—/48/64 —/—/96.9/99.8 —/—/1.5/0.4 Beta-hemoglobin 29446 —/—/2/— —/—/105/— —/—/100/— —/—/0.5/— PACAP 18204192 —/—/—/1 —/—/—/49 —/—/—/94.7 —/—/—/0.6 Elongation factor 1 22128323 —/—/—/1 —/—/—/84 —/—/—/100 —/—/—/0.7 Clottable protein 6601498 2/3/1/3 109/254/69/195 100/100/100/100 0.8/1/1.8/0.6 Up-regulated proteins Arginine kinase 46401522 —/—/1/1 —/—/75/100 —/—/100/100 —/—/1.4/1.7 Hemocyanin 7414468 —/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) Score Expect value H/L ratio Tubulin group identified at 24 hpi (1) SIQFVDWCPTGFK + ICAT_heavy 1,762.88 1,762.88 56 0.0055 0.97 AYHEQLSVAEITNACFEPANQMVK + ICAT_heavy 2,928.43 2,928.42 83 1.3e−005 1.65 Tubulin group identified at 72 hpi (2) EIVHLQTGQCGNQIGTK + ICAT_light 2,052.04 2,052.04 132 2.8e−010 0.60 SIQFVDWCPTGFK + ICAT_light 1,753.83 1,753.85 73 0.00022 0.56 RSIQFVDWCPTGFK + ICAT_light 1,909.95 1,909.95 52 0.029 0.30 AVCMLSNTTAIAEAWAR + ICAT_light 2,034.00 2,034.00 66 0.0013 0.50 TIQFVDWCPTGFK + ICAT_light 1,767.86 1,767.87 68 0.00069 0.53 ATPase group identified at 24 hpi (3) IPIFSAAGLPHNEIAAQICR + ICAT_heavy 2,356.27 2,356.28 124 9.8e−010 1.41 KDHSDVSNQLYACYAIGK + ICAT_heavy 2,247.11 2,247.10 45 0.072 2.28 ATPase group identified at 72 hpi (4) IPIFSAAGLPHNEIAAQICR + ICAT_light 2,347.24 2,347.25 125 1.5e−009 0.64 KDHSDVSNQLYACYAIGK + ICAT_light 2,238.08 2,238.07 70 0.00045 0.38 ↵ a Details are shown in Table SVI in the supplemental material.
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
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Table SII (Details of WSSV protein identification by shotgun proteomics.)
Zipped PDF document, 980K. - Supplemental file 3
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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
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Table SV (The details of identification of differentially expressed cellular proteins by cICAT.)
Zipped PDF document, 363K. - Supplemental file 5
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Table SVI (The details of peptides identified in tubulin and ATPase groups by cICAT proteomics.)
Zipped PDF document, 377K. - Supplemental file 6
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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
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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.
- Supplemental file 1
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Table SI (Primer sets used for RT-PCR.)
