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

Shrimp, virus, and challenge.Virus inocula were prepared from stored hemolymph of WSSV-infected shrimp and intramuscularly injected into black tiger shrimp (Penaeus monodon; body weight, 10 to 15 g), while phosphate-buffered saline (PBS) vehicle was injected in parallel as a control (52). The challenge dose was optimized to ensure 100% mortality within 5 days of infection. The virus infection was confirmed by PCR using a pair of specific primers for WSSV.

Abundance of viral proteins in different tissues.The stomach, gill, and epithelium of the cephalothorax subcuticle were sampled from moribund individuals at 3 days postinfection for the extraction of whole-tissue lysates. Briefly, 0.5 g of each sample was homogenized with 1 ml precooled lysis buffer {7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris base, 2 mM tributylphosphine} and incubated on ice for 10 min. After centrifugation at 20,000 × g for 20 min at 4°C, the supernatant was treated using a 2-D cleanup kit (GE Healthcare), followed by dissolution in the denaturing buffer (0.1% sodium dodecyl sulfate [SDS], 50 mM Tris, pH 8.5) to keep the proteins under the same conditions as those used for shotgun and cICAT analyses. The protein concentration was determined by RC DC protein assay (Bio-Rad), using bovine γ-globulin as a standard. Equal amounts of proteins (30 μg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the relative abundance of viral proteins was analyzed by Western blotting.

Sample preparation for shotgun proteomic analysis.Cephalothorax subcuticular epithelium was sampled at 3 days postinfection. Cytosolic, membrane, and nuclear fractions were sequentially isolated using a Qproteome cell compartment kit (QIAGEN) with the following procedures. Tissue (0.5 g) was washed twice with ice-cold PBS and then homogenized with buffer CE1. After incubation on ice for 10 min, the lysate was centrifuged at 1,000 × g for 20 min at 4°C. The supernatant (cytosolic fraction) was transferred and stored on ice. The other two fractions were extracted using procedures described in the kit manual. Three fractions were then cleaned and redissolved in the denaturing buffer, followed by the determination of protein concentrations as described previously. Fractionation efficiency was examined by Western blotting before proceeding to trypsin digestion. Four hundred micrograms of proteins from each fraction was reduced with 2 mM triscarboxyethylphosphine and carbamidomethylated using 50 mM iodoacetamide. Porcine trypsin (Applied Biosystems) was added at an estimated enzyme-to-substrate ratio of 1:50 (wt/wt) and incubated overnight at 37°C. A strong-cation-exchange (SCX) column (Applied Biosystems) was used to remove SDS, trypsin, and other reagents from the peptide digestion. Sep-Pak cartridges (Waters) were used for peptide desalting.

Sample preparation for cICAT analysis.Cephalothorax subcuticular epithelia were collected at 6, 12, 24, and 72 h postinfection (hpi) from both WSSV- and PBS vehicle-injected individuals for time course cICAT analyses. To minimize deviations arising from individuals, the sample collected at each time point was a pool from five individuals for both the WSSV- and PBS-injected groups. The workflow for sampling and protein preparation is outlined in Fig. 1.

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FIG. 1.

Workflow for sample collection and sample preparation.

Whole-tissue proteins were extracted and quantitated using the same procedures as those described above for the abundance of viral proteins in different tissues. One hundred micrograms of proteins from the virus- and PBS-injected samples was reduced with 2 mM triscarboxyethylphosphine and labeled with heavy (H) and light (L) cICAT reagents (Applied Biosystems), respectively, for 2 h at 37°C in the dark. Labeled proteins were combined and digested with 25 μg trypsin at 37°C for 16 h, followed by cation-exchange chromatography and avidin affinity purification.

2D-LC separation of peptide mixtures.Peptide mixtures for both shotgun and cICAT proteomics were separated using an Ultimate dual-gradient LC system (Dionex-LC Packings) equipped with a Probot matrix-assisted laser desorption ionization (MALDI) spotting device, as follows. The peptide mixture was dissolved in 98% H₂O-2% acetonitrile (ACN) with 0.05% trifluoroacetic acid (TFA) and injected into a 0.3- by 150-mm strong-cation-exchange (SCX) chromatography column (FUS-15-CP; Poros 10S [Dionex-LC Packings]) for first-dimension separation. Mobile phases A and B were 5 mM KH₂PO₄ buffer, pH 3, plus 5% ACN and 5 mM KH₂PO₄ buffer, pH 3, plus 5% ACN plus 500 mM KCl, respectively. The flow rate for the SCX column was 6 μl/min. Nine fractions were separated by step gradients of mobile phase B (unbound, 0 to 5%, 5 to 10%, 10 to 15%, 15 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, and 50 to 100%). The eluted fractions were captured alternately on two 0.3- by 1-mm trap columns (3-μm C₁₈ PepMapTM; 100 Å [Dionex-LC Packings]) and washed with 0.05% TFA, followed by gradient elution to a 0.2- by 50-mm reverse-phase column (Monolithic PS-DVB; Dionex-LC Packings). Mobile phases A and B used for the second-dimension separation were 98% H₂O-2% ACN with 0.05% TFA and 80% H₂O-20% ACN with 0.04% TFA, respectively. The gradient elution step was 0 to 60% mobile phase B over 15 min at a flow rate of 2.7 μl/min. The LC fractions were mixed with MALDI matrix solution (7 mg/ml α-cyano-4-hydroxycinnamic acid and 130 μg/ml ammonium citrate in 75% ACN) at a flow rate of 5.4 μl/min through a 25-nl mixing tee (Upchurch Scientific) before being placed as spots onto 192-well stainless steel MALDI target plates (Applied Biosystems, Foster City, CA), using a Probot Micro fraction collector (Dionex-LC Packings) with a speed of 5 s per well.

MS.The samples were analyzed by MALDI-tandem time of flight-MS/MS (MALDI-TOF/TOF-MS/MS) (ABI 4700 proteomic analyzer; Applied Biosystems) as previously reported (5). For cICAT samples, ICAT pairs with normalized ratio changes (normalized against the median ratio of all ICAT pairs detected) of ≥40% and the more intense peaks with signal-to-noise ratios of ≥30 were selected as precursor ions. Singletons with signal-to-noise ratios of ≥50 were also selected for MS/MS analysis. Raw MS data are accessible via hyperlink at ftp://dbswjl:dbswjl@137.132.35.227/ .

Proteomic data analysis.The MS together with MS/MS spectra were searched using GPS Explorer software, version 3.0, and the MASCOT 2.0 search engine (Matrix Science), allowing one missed cleavage (trypsin). Precursor error tolerance and MS/MS fragment error tolerance were set to 150 ppm and 0.4 Da, respectively. Only fully tryptic peptides with seven amino acids or more and a MS/MS score above 20 were accepted for positive peptide identification.

For protein identification by shotgun proteomics, carbamidomethyl cysteine N-terminal acetylation, pyroglutamation (E or Q), and methionine oxidation were selected as variable modifications. A local database containing all predicted WSSV ORFs (2,013 entries from three complete WSSV genomes [GenBank accession no. AF440570, AF332093, and AF369029]) and the IPI Human Database v. 3.07 (62,322 entries) (http://www.ebi.ac.uk/IPI/IPIhelp.html ) were used for identification of WSSV proteins. Proteins with a total ion score confidence interval percentage (CI%) above 95% were considered positive identifications. Only a few proteins with total ion score CI%s of <95% were checked manually and are presented in Results for reference only. Identified novel viral proteins were further confirmed by Western blotting and/or reverse transcription-PCR (RT-PCR).

An NCBInr database (4,111,659 entries) was used for host protein identification, but only those peptides with ion scores above 46 (corresponding to total ion score CI%s of >50%) were accepted. All identified cellular proteins were further analyzed using in-house software to group them into either “distinct” proteins [not sharing a peptide(s) with other proteins] or “indistinct” proteins [sharing a peptide(s) with multiple proteins] to clarify possible ambiguous identifications. Unique and shared peptides were numbered and tabulated. If peptides matched multiple members of a protein family, default-setting criteria of MASCOT 2.0 software were used to select the one to be reported. In addition, we randomized the same NCBInr database by using a pearl script downloaded from the Matrix Science website (http://www.matrixscience.com/help/decoy_help.html ). The same MS and MS/MS data were searched against the randomized database (i.e., decoy database), using the same criteria and software. The numbers of matches, including peptide and protein matches, were counted for the calculation of false-positive rates.

For the analysis of differential expression of cellular proteins by cICAT, an NCBInr database (4,111,659 entries) was used for the search. H- and L-cICAT-labeled cysteine, N-terminal acetylation, pyroglutamation (E or Q), and methionine oxidation were selected as variable modifications. The maximum peptide rank was set to 1, and the minimum ion score (peptide) was set to 40. cICAT quantification was performed using GPS Explorer v. 3.0 software, and data were normalized against the median ratio obtained from all the ICAT peptide pairs detected in one sample. Cutoff values for protein up-regulation and down-regulation were set at H/L ratios of ≥1.4 and ≤0.71, respectively.

Time course analysis of 11 novel WSSV protein genes by RT-PCR.Epithelium was collected in the same way as that described for the cICAT analysis, but with three additional time points, 2, 4, and 8 hpi. Total RNAs were isolated from specimens by using an RNeasy mini kit (QIAGEN). Isolated RNAs (5 μg) were reverse transcribed with SuperScript III reverse transcriptase and random hexamers (Invitrogen). The first-strand cDNA products were subjected to PCRs with the primer sets shown in Table SI in the supplemental material. The β-actin transcript was amplified and used as an internal control for RNA quality, efficiency of first-strand cDNA synthesis, and loading amounts for PCRs. One known early gene (wsv477) (14) and two structural protein genes (wsv209 [encoding VP187] and wsv465 [encoding VP124]) were included for comparison.

Antibody preparation and Western blotting.Full-length wsv051, -076, -294, and -477 genes were cloned into pET-32a (+) vector (Novagen Inc.) and expressed in Escherichia coli BL21, and protein expression was confirmed by both DNA sequencing and MALDI-TOF-MS. Purified fusion proteins were subjected to SDS-PAGE analysis prior to immunizing rabbits to generate polyclonal antibodies. Polyclonal antibodies against WSSV VP9, VP26, and VP28 were generated previously (28, 40). Anti-VP664 was kindly provided by C.-F. Lo, National Taiwan University. The following antibodies were purchased: anti-actin (A2066; Sigma), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (FL-335; Santa Cruz Biotechnology), and anti-histone 2A (H-124; Santa Cruz Biotechnology). Western blotting was carried out as described elsewhere (28).

Computational annotation.Topology predictions were performed using the TMHMM predictor (www.cbs.dtu.dk/services/TMHMM/ ), and signal peptide predictions were performed using SignalP3.0 (www.cbs.dtu.dk/services/SignalP/ ). Protein families, domains, and functional sites were annotated by searching the InterPro database (http://www.ebi.ac.uk/InterProScan/ ).

Identification of WSSV proteins by shotgun proteomics.Initial experiments were carried out to determine which of the three tissues contained higher concentrations of viral proteins. Western blotting analysis showed that both the structural protein (VP28) and the nonstructural protein (VP9) were more abundant in the subcuticular epithelium and the gill than in the stomach of WSSV-infected shrimp (Fig. 2, left panel). The epithelium was hence chosen for subsequent proteomic analysis.

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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.

To improve the identification of viral proteins, cytosolic, nuclear, and membrane fractions were isolated from the epithelium. The efficiency of isolation was examined by Western blotting analysis. The results showed that VP28 and VP9 had the highest concentrations in the cytosolic fraction and the lowest concentrations in the membrane fraction (Fig. 2, right panel). The cellular protein GAPDH, specific to the cytosolic fraction, was the most abundant in the cytosol but could also be detected in the other two fractions. On the other hand, histone 2A, specific to the nuclear fraction, was the most abundant in the nuclear fraction. These results showed that subcellular fractionation could enrich cytosolic and nuclear proteins into their respective fractions.

Twenty-seven, 20, and 4 viral proteins were identified from the cytosolic, nuclear, and membrane fractions, respectively. In total, 33 unique proteins were identified from the three fractions. Twenty-eight of these proteins showed high scores (total ion score CI%, >95%), while the remaining five proteins with low scores (total ion score CI%, ≤95%) are also listed (Table 1; see Table SII in the supplemental material). Among the 28 unique proteins, 11 proteins were identified for the first time in this study (shown in bold in Table 1). Of the remaining proteins, there were 13 structural and 4 nonstructural proteins reported previously. Of five proteins with low scores, wsv226 is reported here for the first time, while the remaining four are previously reported structural proteins. All identified proteins, with either high or low ion scores, have distinct peaks of fragment ions in MS/MS spectra, as found by manual examination. A total of eight proteins were predicted to have transmembrane helixes. Four proteins have signal peptides.

Data set of host proteins identified by shotgun proteomics.To investigate the protein composition of shrimp epithelium, MS and MS/MS spectra from shotgun proteomics of epithelial cytosolic, nuclear, and membrane fractions were combined and searched against an NCBInr database. A total of 1,999 peptides with ion scores of ≥20 were identified, of which 1,669 peptides were identified as having ion scores of ≥46 (see Table SIII in the supplemental material). We identified 429 proteins with total ion score CI%s of >95%, of which 144 were grouped into “distinct” proteins (see Table SIVa in the supplemental material), while the remaining 285 proteins were grouped into “indistinct” proteins (see Table SIVb in the supplemental material). Using the randomized database, we identified 95 false-positive matches at the peptide level (ion score, ≥46) and 9 false-positive matches at the protein level, with total ion score CI%s of >95%, which correspond to false-positive rates of 5.7% (i.e., 95 of 1,669 peptides) and 2.1% (i.e., 9 of 429 proteins) for peptide and protein identifications, respectively.

Data set of differentially expressed host proteins identified by cICAT.To understand the shrimp response to WSSV infection, subcuticular epithelia of WSSV- and PBS (uninfected control)-injected shrimps were sampled in parallel at 6, 12, 24, and 72 hpi, and whole-tissue lysates were then extracted and prepared for cICAT analysis. A total of 12 proteins showing differential expression were identified at the 95% confidence level (Table 2; see Tables SV and SVII in the supplemental material). As previously noted, peptides from the virus-infected group had an H tag, and peptides from the uninfected group had an L tag. Of the 12 proteins, 10 proteins (83.3% of differentially expressed proteins) were down-regulated (H/L ratio, ≤0.71) and 2 proteins were up-regulated (H/L ratio, ≥1.4). The means and standard deviations of H/L ratios are shown in Table SVII in the supplemental material. The expression of the clottable protein was identified to be unchanged at 6 and 12 hpi, up-regulated at 24 hpi, and down-regulated at 72 hpi. Of the 10 down-regulated proteins, 1, 2, and 7 of them were identified at 12, 24, and 72 hpi, respectively, showing an increasing number of down-regulated proteins with increasing times of infection. For the two up-regulated proteins, up-regulation of hemocyanin was first detected at 12 hpi and lasted to 72 hpi, while up-regulation of arginine kinase was detected only at 72 hpi. Neither down-regulated nor up-regulated proteins were detected at 6 hpi.

Two peptides of the tubulin group were detected at 24 hpi (Table 3; see Tables SVI and SVII in the supplemental material); one peptide was up-regulated (H/L ratio = 1.65), and the other did not change significantly (H/L ratio = 0.97). All peptides of the tubulin group identified at 72 hpi showed down-regulation (H/L ratio, <0.7). The same two peptides of the ATPase group were detected at both 24 and 72 hpi (Table 3; see Tables SV and SVII in the supplemental material); one was up-regulated at 24 hpi (H/L ratio = 2.28) and then down-regulated at 72 hpi (H/L ratio = 0.38), and the other did not change significantly at these two time points.

Time course analysis of 11 novel WSSV protein genes by RT-PCR.To investigate temporal gene transcription of 11 novel viral proteins and to provide further evidence of their presence at the mRNA level, a time course semiquantitative RT-PCR was performed, with β-actin as an internal control and three other genes (wsv209, -465, and -477) as positive controls. Among 14 genes, 10 showed increasing mRNA levels with the time course of infection (group 1), 1 showed a constant mRNA level at all time points (group 2), and 3 showed decreasing mRNA levels with the time course of infection (group 3) (Fig. 3). On the other hand, six genes (wsv076, -192, -143, -277,-343, and -477) commenced their expression at a very early stage of the infection (2 hpi), while two genes (wsv285 and -294) commenced expression at a later stage of the infection (24 hpi).

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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).

Western blotting.Western blotting analysis was performed to verify the presence of proteins in the virus-infected tissue and in the purified virus. Four proteins (wsv051, −076, and −294 and a known nonstructural viral protein, wsv477, as a positive control) were detected in the virus-infected whole-tissue lysate and the cytosolic fraction but were not detected in the purified virus and the uninfected cell lysate (Fig. 4). Positive controls using known viral structural proteins, including envelope proteins VP28 and VP26 as well as the nucleocapsid protein (VP664), showed that these known structural proteins were present in both tissue lysates and purified virus. The results indicated that the three novel proteins, i.e., wsv051, -076, and -294, might not be viral structural proteins.

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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.