Differential display of grouper iridovirus-infected grouper cells by immunostaining

Biochemical and Biophysical Research Communications 372 (2008) 674–680

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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Differential display of grouper iridovirus-infected grouper cells by immunostaining Chiao-Hwa Yeh a,b, Yao-Sheng Chen b, Ming-Shan Wu b, Chien-Wen Chen b, Chung-Hsiang Yuan b, Kuo-Wen Pan b, Ya-Nan Chang b, Nin-Nin Chuang b,*, Chi-Yao Chang b a b

Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 13 May 2008 Available online 2 June 2008

Keywords: Differential display Iridovirus Immunostaining Grouper

a b s t r a c t Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a host’s cellular protein response to viral infection on a translational basis. Ó 2008 Elsevier Inc. All rights reserved.

Grouper iridovirus (GIV) [1–3] is a large icosahedral cytoplasmic DNA virus, of the genus Ranavirus and family Iridoviridae, which includes five genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus, and Megalocytivirus (NCBI Taxonomy browser: Iridoviridae). Members of the family can infect a diverse range of invertebrates and poikilothermic vertebrates hosts including insects, fish, amphibians, and reptiles, causing a wide range of different diseases [4]. Systemic iridovirus infections have been reported in a variety of freshwater and marine food fish species [5–8], and cause significant economic losses to aquaculture worldwide. The interplay between a virus and host cells is an intriguing topic. A dynamic series of events occurs in host cells upon viral invasion. How a virus hijacks a host cell’s apparatus to perform its own purposes, and how host cells defend against viral infection still need to be elucidated, especially for fish viruses. Recently, suppression subtractive hybridization (SSH) has been extensively used to investigate differentially expressed genes [9]. Using this technique, the transcriptional programs of fish host cells against different viral infections have been revealed, including mandarin fish (Siniperca chuatsi) spleen infected with infectious spleen and kidney necrosis virus (ISKNV) [10]; rainbow trout (Oncorhynchus mykiss) leukocytes infected with viral hemorrhagic septicemia virus (VHSV) [11]; the brain of sea bream (Sparus aurata) infected with * Corresponding author. Fax: +886 2 2785 8059. E-mail address: [email protected] (N.-N. Chuang). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.126

a nodavirus [12], and Atlantic cod (Gadus morhua) spleen responding to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC) [13]. Differentially expressed transcripts provide an overview of messenger (m)RNA expression profiles of infected cells; however, mRNA abundances do not truly reflect protein abundances; transcript levels only provide some indication of protein expression levels [14]. In addition, post-translational modification that determines whether or not a cellular protein functions properly cannot be predicted from its mRNA expression profile. To date, a small but increasing number of studies have been carried out to investigate cellular and viral proteomes using different proteomic approaches [15]. Although this proteomic strategy has become an important and promising technique and lots of data have been generated, some of these tools still have limitations. Besides the limitations of quality and characteristics of the protein samples themselves, the challenging techniques and relatively high capital costs for instruments are also drawbacks of this new approach [16]. In this study, we established phage display cDNA libraries of host cells and developed a immunostaining method to screen differentially expressed genes in response to GIV infection on a translational basis. To our knowledge, this is the first report using an immunostaining method to screen differentially expressed genes at the translational level upon viral infection. This new method provides an easier and more-economic way to reveal host cellular responses to viral infection on a translational basis.

C.-H. Yeh et al. / Biochemical and Biophysical Research Communications 372 (2008) 674–680

Materials and methods Cell culture and viral infection. The grouper kidney GK-2 cell line was established from orange-spotted grouper (Epinephelus coioides) and maintained in Leibovitz’s L-15 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) at 28 °C. The GIV used in this study was isolated from spleen tissue of a diseased yellow grouper [1]. The propagation and purification of GIV in GK2 cells were as previously described [2]. Polyclonal antibody preparation. A total number of 1  107 GK-2 cells were inoculated with GIV filtrate at a multiplicity of infection (moi) of 10. One hundred and fifty micrograms of cell lysates was collected at 0, 6, and 12 h post-infection (hpi) and hypodermically injected into 6-month-old New Zealand white female rabbits. The first immunization used a mixture of cell lysates and an equal volume Freund’s complete adjuvant (Gibco). A similar boost was given after 2 weeks with incomplete adjuvant (Gibco) followed by three subsequent boosts. The rabbit was bled 5 days after the final injection. Immunoglobulin G (IgG) was then purified by protein A agarose columns (Pierce). Purified GIV viral particles were used to immunize mice to prepare the GIV antiserum. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and Western blot analysis. The cell lysate of GIV-infected GK-2 cells was collected at 0, 6, and 12 hpi and analyzed by SDS– PAGE using a 4% stacking gel (pH 6.8) and 10% separating gel (pH 8.8) in Tris–glycine buffer (pH 8.3). After electrophoresis, the separated proteins were silver-stained or transferred to Hybond-P membranes (Amersham Biosciences). After blocking, the membranes were hybridized with different antibodies. Following washing, the membranes were hybridized with alkaline phosphataseconjugated goat anti-rabbit or goat anti-mouse immunoglobulin and visualized with NBT/BCIP. Two-dimensional gel electrophoresis (2-DE) and Western blot analysis. Confluent GK-2 cells were collected, washed, and centrifuged. Pelleted cells were lysed with lysis buffer [7 M urea, 2 M thiourea, 4% CHAPS (w/v), 1% DTT (w/v), 0.5% IPG buffer (Amersham Biosciences) (w/v), and 10 mM spermin]. The isoelectric focusing (IEF) was performed at 25 °C on an Ettan IPGphor system (Amersham Biosciences). The 13-cm IPG strips (pH 4–7) were rehydrated for 16 h with 3.5 lg total protein in the lysis buffer. The strip was then equilibrated in an equilibration buffer [50 mM Tris–HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 0.002% bromophenol blue] containing 1% DTT for 20 min and incubated another 20 min in equilibration buffer containing 2.5% iodoacetamide. The equilibrated strips were resolved by 10% SDS–PAGE using a SE600 Ruby electrophoresis set. Gels were then silver-stained or transferred to Hybond-P membranes for Western blot analysis. Immunostaining screening. The cDNA libraries were prepared as previously described [17]. Briefly, mRNAs were prepared from 3  107 GK-2 or GB3 [18] cells using a FastTrack 2.0 mRNA extraction kit (Invitrogen). Five micrograms of mRNA was used to construct the complementary (c)DNA library with a cDNA Synthesis Kit ZAP-cDNA Gigapack III Gold Cloning kit (Stratagene). The UniZAP XR vector contained in the kit can be screened with antibody probes and allows in vivo excision of the pBlueScript phagemid to characterize the insert in a plasmid system. cDNA cloning was performed as described previously [17]. Briefly, the libraries were screened with the antiserum described above, followed by alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin. The signals were detected by BCIP/NBT. After three consecutive screenings, the inserts from the positive clones were excised using Exassist helper phage and the SOLR strain following the manufacturer’s instructions (Stratagene). To further confirm the antigenic changes, the membranes were cut into six equal pieces after plaque lifting,

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and three pieces were hybridized at three different time points (0, 6, and 12 hpi) with polyclonal antibodies as described above. Sequencing and BLAST homology search. Plasmid DNA was extracted with a MiniPlus Plasmid DNA Extraction System (Viogene), and DNA sequencing was performed using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit with FS AmpliTaq DNA polymerase (Perkin-Elmer) and was analyzed on an ABI 3730 DNA analyzer. DNA sequences thus obtained were edited by removing vector sequences and ambiguous regions, and then they were transformed to amino acid sequences using the MegAlign subprogram of the DNASTAR software. The resulting amino acid sequences were used as a query sequence to search the translated nucleotide database at the National Center for Biotechnology Information (NCBI) with the BLAST program, tblastn. Microarray and image analysis. Total RNA was extracted from GIV-infected GK-2 cells at 0, 1, 3, 6, 9, and 12 hpi using TRIZOL (Invitrogen) and was subjected to DNase I treatment (Qiagen) following the manufacturer’s instructions. The cDNA was then synthesized from 10 lg purified total RNA with an anchored oligo (dT)20 primer (Invitrogen) and was indirectly coupled with Alex Fluor 555 and 647 dyes (Invitrogen). Meanwhile, plasmid DNAs with previously obtained confirmed sequences were spotted in duplicate onto glass slides. Labeled cDNAs were hybridized to microarrays at 42 °C overnight. After washing, microarrays were dried and scanned using a GenePix 4000B array scanner. Obtained images were analyzed by GeneSpring software (Agilent Technologies) and clustered using GeneTree (Agilent Technologies). Results and discussion Cytopathic effect and antigenic analysis of GK-2 cells infected with GIV GK-2 cells were infected with GIV filtrate at an moi of 10, and morphological changes in cells were recorded throughout infection. As shown in Fig. 1A, the specific cytopathic effect initially developed from 6 hpi, with rounded, granular, and refractile cells which spread more significantly through the cell sheet until 18 hpi when all cells had degenerated and detached. The GIV susceptibility of GK-2 cells implies massive interactions between the virus and host cells, including disruption during infection of the cytoskeleton and extracellular matrix, which help maintain a cell’s integrity. SDS–PAGE and Western blotting were further performed to understand the protein content changes after infection. There was no significant change in the silver-stained protein level of GK-2 cells after GIV infection for up to 12 hpi. However, increasing and decreasing antigenic proteins that were not viral proteins were observed by the more-sensitive immunostaining method throughout GIV infection (Fig. 1B). Two-dimensional Western blot profile of GK-2 cells To obtain a further detailed cellular protein profile of viral infection and exclude possible confusion arising from viral proteins, cell lysates of GK-2 cells were extracted for 2-DE and immunostained. As rabbit immunoglobulin can retain a memory of antigenicity, it can be used as a tracing tool to determine qualitative and quantitative changes in protein levels of GK-2 cells after GIV infection. On the basis of the intensity of the protein spots, 14 groups of protein spots were found to have dynamically changed at different time points of infection. Two groups of protein spots were significantly downregulated at 6 hpi, while, 10 groups were significantly upregulated at 6 hpi, and six groups were upregulated through 12 hpi (Fig. 2). It appears that GK-2 cells had different antigenic responses at differ-

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and GB3 cell cDNA libraries. To further investigate the differential antigenic patterns of these identified clones in response to different time points of infection, about 100 plaque-forming units (pfu) of each phage clone was transferred to membranes to reveal their antigenic display. Four groups of antigenic change patterns were categorized (Fig. 3). Not surprisingly, the number of genes with antigenic changes agreed with the results of 2-DE immunostaining (Table 1).

A 0h

1h

3h

6h

9h

12 h

15 h

18 h

Blast and annotation

B

Anti-serum

Silver stain 0

6 12

All isolated gene sequences were edited and used to search for homologous proteins in the database using the NCBI tblastn program that showed significance with known genes with identities varying from 46% to 100%. Among these sequences, 63 had already been found in teleosts, and three were first found in fish. Based on the function of the identified cellular proteins, all of these sequences could be grouped into 10 categories including replication (1.5%), transcription (9.1%), translation (28.8%), immunity (6.0%), protein modification (7.6%), structure (21.2%), metabolism (9.1%), energy (6.1%), apoptosis (4.5%), and homeostasis (6.0%) (Table 1).

0h 0

6

6h 12

0

6

12 h 12

0

6

GIV 12

0

6

12 h

kDa 170 130 100 72

Transcriptional microarray analysis

55 40

To compare the gene expression profiles from the transcriptional level point of view, plasmid DNA of 66 isolated clones was spotted onto glass slides and hybridized with probes prepared from the mRNA of GIV-infected GK-2 cells. As shown in Fig. 4, at 0 hpi, about 31.8% clones were expressed at a higher than intermediate level. The number of expressed clones significantly increased to 78.8% at 1 hpi, and reached the highest at 96% at 6 hpi. However, the number of expressed clones decreased to 63% at 12 hpi. The overall expression pattern of the 66 clones basically agreed with the expression results obtained from the 2-DE analysis and immunostaining screening, which all showed a tendency for increased signals at 6 hpi and decreased signals at 12 hpi. Since various clones had their own expression kinetics, we then compared the results of the microarray and immunostaining screening of each clone and found 53 of 66 (80%) clones had the same expression kinetics, representing accordance between the transcriptional and translational levels.

33

Fig. 1. Morphological and antigenic changes in grouper kidney (GK-2) cells after grouper iridovirus (GIV) infection. (A) Cytopathic effect of GK-2 cells infected with GIV for various times at a multiplicity of infection (moi) of 10. (B) Silver-staining and Western blot analysis of GIV-infected GK-2 cells at 0, 6, and 12 h post-infection (hpi). Antiserums were prepared by immunized rabbits or mice with GIV-infected GK-2 cell lysates (at 0, 6, and 12 hpi) or GIV viral particles, respectively. Arrows indicate antigenic-decreasing proteins. Arrowheads indicate antigenic-increasing proteins. Stars indicate GIV viral proteins.

ent time points to viral infection, and the antigenicity significantly increased at 6 hpi. Immunostaining screening To search the corresponding cellular genes for target proteins that respond to viral infection, GK-2 and GB3 phage display cDNA libraries were screened by immunostaining. In total, 158 clones were isolated from the GK-2 library and 58 clones were isolated from the GB3 library. After removing clones with repetitive sequences and those for which a homolog was not found in the database, 35 and 31 clones were, respectively, obtained from the GK-2

Sliver stain pH4

Discovery and discussion of identified genes Among the differentially displayed genes, the most abundant group (28.8%) fell in the category of translation, which consisted

0h pH7

pH4

6h pH7

pH4

12 h pH7

pH4

pH7

130 100 72 55 40 33 24

Fig. 2. Two-dimensional profiles of grouper kidney (GK-2) cells against different antiserums. Broken-line circles indicate antigenic-decreasing proteins. Solid-line circles indicate antigenic-increasing proteins.

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A

B +

++

++

0

6

12

+++

++++

++

0

6

12

h

C

+

+++

+

0

6

12

++++

++

+

0

6

12

h

D

h

h

Fig. 3. Characteristic patterns of immunostaining screening. Three equal cut pieces of membranes containing transferred phages were hybridized with different antiserums. +, ++, +++, and ++++ represent the intensity of the signal. (A) Ictacalcin; (B) zinc finger-containing protein; (C) non-muscle myosin heavy polypeptide 9, and (D) AHNAK nucleoprotein as representative examples.

of 13 ribosomal proteins, two transfer (t)RNA synthetases, two translation elongation factors, one translation initiation factor, and a U3 small nucleolar RNP. Different translational protein expression patterns may imply that GIV conditionally uses the host translation apparatus for infection. The second most abundant group of identified genes consisted of structural proteins (21.2%). A recent study revealed that various viruses can interact with the host cell cytoskeleton and utilize it as a track to promote viral infection [19]. Therefore, many actin and tubulin proteins were upregulated [20]. Vimentin, a class III intermediate filament protein, has also been found to be associated with mature virus particle capsid protein and to contribute to virus egress [21]. AHNAK is a member of the scaffold protein family, highly expressed by CD4+ T cells, and is a critical component of calcium signaling [22]. In this study, AHNAK showed the most surprising difference between the transcription and translation levels. AHNAK was the most abundant clone (45 repetitive) in the screening. The immunostaining results of AHNAK showed the strongest intensity at 0 hpi, but decreased intensity at 6 hpi, and it had almost disappeared by 12 hpi (Fig. 3D). However, the microarray data showed that the transcriptional level of AHNAK was below intermediate at 0 hpi, increased at 6 hpi and decreased at 12 hpi. The significantly different antigenicities between the transcriptional and translational levels imply that AHNAK undergoes interesting qualitative and quantitative changes during viral infection. Another interesting category is apoptosis-related genes, among which we identified three genes: ubiquitin-like 7, lysophosphatidic acid G-protein-coupled receptor 4, and the cathepsin B precursor. The ubiquitin-conjugating enzyme 7-interacting protein was also described as being upregulated in the nodavirus-infected brain of sea bream [12]. Some other ubiquitin–proteasome pathway-related genes like polyubiquitin, ubiquitin-activating enzyme E1, and the proteasome activation subunit 2 genes were also upregulated in ISKNV-infected mandarin fish spleen cells [10]. Since viruses are known to utilize protein ubiquitination pathways [23,24], and ubiquitination is involved in regulating many critical

cellular physiological functions including controlling the lifetime and activities of proteins [25], how these genes are involved in GIV-infected cells and if they are involved in apoptosis need to be investigated. The lysophosphatidic acid (LPA) G-protein-coupled receptor (GPCR) was first found in fish. Previous studies suggested that the LPA GPCR might play a role in the development of several human cancers including ovarian, prostate, and breast cancers [26]. However, its role in virus-host interactions still remains unknown. Cathepsin is a member of the lysosomal protease family and shows the highest activity at pH 5–6 [27]. This property and its location in the lysosomal compartment suggests that cathspsin B plays a role as a host cell protease and is involved in the membrane fusion of Molony MLV-infected mouse NIH 3T3 cells [28]. However, cathepsin is also involved in many important physiological and pathological processes including apoptosis, inflammation, parasite infection, and cancer in humans. Its role in the interaction of GIV and GK-2 cells would be an interesting topic in the future. Based on the immunostaining strategy we used, only virus-infected cellular proteins expressed in sufficient quantity or with enough antigenicity could induce an immune response in the rabbit, and then the responding genes could be recognized in the subsequent cDNA library screening. Compared to other strategies used in differential expression studies, the advantages of this method can be summarized as follows: (1) it is able to screen genes with full length and also novel genes; (2) it is able to effectively express selected genes in an Escherichia coli expression system; (3) the genes identified by this method have either a high expression level or high antigenicity upon viral infection; and (4) genes with antigenic changes due to the protein structure (e.g., post-translational modification) induced by viral infection are easily identified by this method. To summarize, we identified 66 genes related to GIV-infected grouper cells by an immunostaining screening method at the translational level, which were confirmed by the DNA microarray at the transcriptional level. These genes are related to replication, transcription, translation, immunity, apoptosis, structure, metabolism, energy, protein modification, and homeostasis. The revealed

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Table 1 Annotation of immunocloning genes from grouper iridovirus-infected grouper cells Category

BLAST annotation

Accession No.

Intensity of signal (hpi) 0

6

12

Replication EU714161

Helentron 6 helitron-like transposon replicase

++

++

+

Transcription EU714155 EU714159 EU714160 EU042108 EU714177 EU714181

Chromatin modifying protein 1B Glyceraldehyde-3-phosphate dehydrogenase Zinc finger, CCHC domain containing 9 Heterogeneous nuclear ribonucleoprotein A0 Heterogeneous nuclear ribonucleoprotein G Splicing factor, arginine/serine-rich 7

++ + + + + +

+++ ++ +++ ++ ++ ++

++ + + ++ ++ +

Translation EU042099 EU042114 EU042113 EU714141 EU714142 EU714143 EU714144 EU714145 EU042118 EU042117 EU714146 EU042116 EU714147 EU714172 EU042120 EU714175 EU042122 EU714176 EU714178

Ribosomal protein L27 Ribosomal protein L23 Ribosomal protein L14 Ribosomal protein L7 Ribosomal protein LP1 Ribosomal protein LP0 Ribosomal protein LP0-like protein Ribosomal protein S26 Ribosomal protein S18 Ribosomal protein S13 Ribosomal protein S11 Ribosomal protein S10 Ribosomal protein S7 Cysteinyl-tRNA synthetase Seryl-tRNA synthetase Elongation factor 1-alpha Translation elongation factor 1-delta Translation initiation factor 3, subunit 10 IMP4, U3 small nucleolar RNP

+ + + ++ + + + + + + + + + + + ++ + + +

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++

+ + + + + + + + + + + + + + + + + ++ +

Immunity EU714152 EU042111 EU714169 EU714171

Mitogen-activated protein kinase 6 Natural killer cell enhancement factor Cytokine induced protein Growth arrest-specific 6

+ + + +

++ ++ ++ ++

++ + + +

Apoptosis EU714174 EU042110 EU714151

Ubiquitin-like 7 Lysophophatidic acid G-protein-coupled receptor 4 Cathepsin B precursor

+ + +

++ ++ ++

++ ++ +

Structure EU714139 EU714184 EU714157 EU714168 EU042119 EU714140 EU714148 EU714149 EU714150 EU714167 EU042121 EU714182 EU714183 EU714138

Non-muscle myosin heavy polypeptide 9 Keratin 18 Keratin 8 Vimentin S100-like calcium binding protein Ictacalcin Gelsolin a Gelsolin (amyloidosis, Finnish type) Alpha actinin 4 Collagen type IX, alpha 1 Smooth muscle cell-specific protein SM22 alpha-b Actin related protein 2/3 complex subunit 1B Beta actin AHNAK nucleoprotein isoform 1

+++ + + + ++ + ++ + + + + +++ + ++++

++++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++

++ + + + + ++ ++ + + ++ + + + +

Metabolism EU714153 EU714154 EU714158 EU042104 EU042103 EU714180

Pyrroline-5-carboxylate reductase 2 Nucleoside diphosphate kinase Quinoid dihydropteridine reductase a Endozepine Brain-type fatty acid binding protein Fatty acid binding protein H6-isoform

+ + + + + +

++ ++ ++ ++ ++ ++

+ + + + ++ ++

Energy EU714164 EU714165 EU042102 EU042100

Mitochondrial cytochrome C oxidase subunit Vb precursor ATP synthase, H+ transporting, mitochondrial F0 complex, subunit g ATP synthase, H+ transporting, mitochondria F0 complex, subunit c ADP-ATP translocase

++ + + +

+++ ++ ++ ++

+ + + +

Protein modification EU714162 EU714163 EU714173 EU042105

Chaperonin containing TCP-1 subunit 6A Chaperonin containing TCP-1 delta Protein disulfide isomerase associated 4 GABA(A) receptor-associated protein

+ + + +

++ ++ ++ ++

+ + + ++

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C.-H. Yeh et al. / Biochemical and Biophysical Research Communications 372 (2008) 674–680 Table 1 (continued) Category

BLAST annotation

Intensity of signal (hpi)

Accession No.

0

6

12

EU042101

ADP-ribosylation factor binding protein

+

++

+

Homeostasis EU714156 EU714179 EU714166 EU714170

Metallothionein 1 Claudin 3a Ferritin, heavy subunit Taurine transporter

+ + + ++

++ ++ ++ ++

++ + ++ +

hpi, hours post-infection; +, ++, +++, and ++++ represent the intensity of immunostaining.

0

1

3

6

9

12

hpi Ribosomal protein L27 Collage type IX, alpha I Smooth muscle cell-specific protein SM22 alpha Ribosomal protein S18 Ribosomal protein L23 Ribosomal protein L14 Translation initiation factor 3, subunit 10 Heterogeneous nuclear ribonucleoprotein G Pyrroline-5-carboxylate reductase 2 Ribosomal protein S13 Translation elongation factor 1-delta Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) Glyceraldehyde-3-phosphate Ribosomal protein S10 Vimentin (Vim) Cytokine induced protein ADP-ATP translocase Seryl-tRNA synthetase Fatty acid binding protein H6 Lysophosphatidic acid G-protein-coupled receptor 4 GABA(A) receptor associated protein Splicing factor, arginine/serine-rich 7 Hnrpa01 protein Fatty acid binding protein ADP-ribosylation factor binding protein Quinoid dihydropteridine reductase (qdpra) Protein disulfide isomerase associated 4 Ubiquitin-like 7 Actin-related protein 2/3 complex subunit 1B Ribosomal protein S7 Cathepsin B (thiol protease) Ictacalcin Endozepine Nucleoside diphosphate kinase a ATP synthase, transporting, mitochondrial F0 complex complex, subunit g synthase H+ transporting IMP4, U3 small nucleolar ribonucleoprotein homologue Natural killer cell enhancement factor (NKEF) a Ribosomal protein LP0 Ribosomal protein S11 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c Mitogen-activated protein kinase Ferritin, heavy polypeptide 1 (fth 1) Growth arrest specific 6 Metallothionein Keratin 8 Zinc finger, CCHC domain containing Claudin 3a Chromatin modifying protein 1B AHNAK Beta actin Taurine transporter Ribosomal protein S26 Elongation factor 1-alpha Ribosomal protein LP0-like protein Keratin 18 Actinin alpha4 Helentron 6 helitron-like transposon replicase Gelsolin a Gelsolin (amyloidosis, Finnish type) Ribosomal protein LP1 Myosin heavy chain 9 Chaperonin containing TCP 1 subunit 4 (Tcp-1-delta) Cysteinyl-tRNA synthetase isoform 5 S 100 calcium binding protein) Mitochondrial cytochrome C oxidase subunit Vb Chaperonin containing TCP 1 subunit 6A (Tcp-1-zeta) Ribosomal protein L7

Fig. 4. GeneTree analysis of the immunocloned gene transcriptional patterns throughout infection. Probes were prepared from grouper iridovirus (GIV)-infected grouper kidney (GK-2) cells for various times at a multiplicity of infection (moi) of 10. Black, green, and red colors indicate intermediate, low, and high expression levels, respectively.

expression profile provided important insights into the cellular proteomes induced by viral infection and the apparatus that viruses hijack to fulfill their own goals. This is the first immunostaining screening method designed to reveal virus-infected cellular responses at the protein level, and also the first study on iridovirus-infected host cells and on grouper in particular. Acknowledgment This study was supported by Grant No. NSC95-2311-B-001069-MY3 from the National Science Council, Taiwan.

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