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Cloning of eIF5A from shrimp Penaeus monodon, a highly expressed protein involved in the survival of WSSV-infected shrimp
Aquaculture 265 (2007) 16 – 26 www.elsevier.com/locate/aqua-online
Cloning of eIF5A from shrimp Penaeus monodon, a highly expressed protein involved in the survival of WSSV-infected shrimp Amornrat Phongdara b,⁎, Yanisa Laoong-u-thai a , Warapond Wanna b b
a Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
Received 19 August 2006; received in revised form 22 December 2006; accepted 4 February 2007
Abstract Shrimp respond to viral infection with up- and down-regulation of many critical genes. In our previous work, we showed that white spot syndrome virus (WSSV) infection of Penaeus monodon (Pm) caused cellular syntenin levels to increase. In order to further explore the signal transduction pathway of syntenin, we constructed the cDNA library of WSSV-infected shrimp and performed a yeast two-hybrid screening of the library using syntenin as bait. Here we report that syntenin specifically binds eukaryotic translation initiation factor 5A (eIF5A), a 157 amino acid polypeptide that is implicated in cell proliferation and survival. GST-pull-down assays showed the presence of specific interaction between Pm-syntenin and eIF5A. Message analyses revealed that syntenin expression remained normal when shrimp were infected with WSSV but did not show any gross signs of disease. Syntenin expression, however, increased 1.4-fold when shrimp became dead. In contrast, eIF5A expression increased markedly during the stage of WSSV infection when there were no gross signs of disease, i.e., prior to the moribund stage. At the moribund stage, eIF5A expression was down-regulated. This data suggest that syntenin and eIF5A physically interact with each other in the setting of WSSV infection and that these two molecules exhibit dynamic changes in their expression patterns as infection progresses from grossly normal, infected shrimp to full-blown phase. © 2007 Elsevier B.V. All rights reserved. Keywords: eIF5A; Translation initiation; Syntenin; Protein–protein interaction; Yeast two hybrid
1. Introduction Penaeid shrimp production is a worldwide economic activity. The intensification of shrimp farming over the last few decades has been accompanied by development of infectious diseases of viral, bacterial, and, in some cases, fungal origin (Destoumieux-Grazon et al., 2001). Among the various viruses of the penaeid
⁎ Corresponding author. E-mail address: [email protected] (A. Phongdara). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.02.005
shrimp, the white spot syndrome virus (WSSV) is responsible for a major proportion of the diseases plaguing commercial shrimp ponds, and has resulted in high mortality and economic losses. (Lightner, 1996; Flegel, 1997, 2001). Shrimp exhibit a diverse response to viral infection, that is manifested in marked up- and down-regulation of a variety of genes (Gross et al., 2001; Supungul et al., 2002; Rojtinnakorn et al., 2002; Sotelo-Mundo et al., 2003; Hikima et al., 2003; He et al., 2005; De Lorgeril et al., 2005; Du et al., 2006). In our previous work, we identified the protein syntenin of the shrimp Penaeus
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monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly up-regulated in the acute phase of the infection (Bangrak et al., 2002). Reports from studies of other organisms indicate that syntenin is involved in the signal transduction pathway. Syntenin has no obviously catalytic domain and therefore is unlikely to have a signaling function by itself, but could serve as an adapter or scaffolding protein to attach other proteins and functions in the cell. As of now several synteninbinding proteins from other organisms have been identified and their functions have been described. These include syndecan (Grootjans et al., 1997), proTGFα (Femandez-Larrea et al., 1999), ephrin B (Torres et al., 1998; Lin et al., 1999), neurofascin (Grootjans et al., 2000), interleukin 5 receptor α subunit (IL5Rα), and EphA7 (Torres et al., 1998). In order to further explore the link between Pmsyntenin and viral infection, we performed a yeast twohybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. This experiment resulted in seven independent clones of syntenin-binding proteins being isolated. Among these syntenin-binding proteins, one (α2M) that plays an important role in immune mechanisms in shrimp has already been described (Tonganunt et al., 2005). Here we describe the identification and characterization of another syntenin-binding protein eIF5A and we follow its expression during the course of a WSSV infection.
1:1 × 107 dilution of a viral stock solution made in 0.85% NaCl and 100 to 150 μl of hemolymph was subsequently withdrawn at 6, 12, 24, 48 and 72 h postinjection (p.i.). The remaining shrimp were held up to the time when they became moribund (shrimp is immobile and unresponsive to touch but were still showing movement of gill rakers. At this time it was labeled as moribund, infected shrimp (MIS). When gill movement ceased, hemolymph was immediately withdrawn and labeled as dead shrimp (DIS). Hemolymph was stored at − 80 °C until used.
2. Materials and methods
2.3. Yeast two-hybrid screening
2.1. Samples
Yeast two-hybrid screening was carried out with the MATCHMAKER Gal4 Two-hybrid System 3 (CLONTECH Laboratories, Inc.). Competent cells of the yeast strain AH109 were prepared and transformations were performed according to the manufacturer's protocols. Briefly, 1 μg of plasmid DNA was added to 100 μl of competent cells and mixed again with 36 μl of 1 M lithium acetate and 240 μl of 50% polyethylene glycol (PEG). The transformation mixture was then incubated at 30 °C for 30 min followed by a heat-shock at 42 °C for 15 min and subsequently spread on a drop-out-agar plate in the absence of leucine and tryptophan. The plates were incubated at 30 °C for 48 h to allow for yeast growth. PCR was used to confirm transformation with the target gene. A positive clone was inoculated into SD medium lacking leucine, tryptophan and histidine (SDLTH) and supplemented with 3-amino-1,2,4-triazole (SD-LTH + 5 mM 3-AT). The medium was shaken at 180 rpm at 30 °C for 96 h. Absorbance of the medium at 595 nm was measured in a spectrophotometer
All shrimp were obtained from a farm controlled by the Songkhla Coastal Fisheries Research and Development Center, Thailand. The shrimp were sourced from a pond stocked with postlavae that had tested negative for WSSV by nested PCR (Lo et al., 1996a,b) and that later, when the pond was harvested, showed no sign of WSSV infection. Prior to the beginning of the experiment, shrimp were kept individually in 60 l aquaria for 10 days, all samples were checked for WSSV infection by using PCR technology (Lo et al., 1996a,b). Hemolymph fluid (100–150 μl) was initially withdrawn and labeled as normal (NS) for individual shrimp whose PCR results proved negative for WSSV. Hemolymph from individual shrimp identified by PCR as WSSV positive, but showing no sign of the white spot syndrome, was labeled as originating from grossly normal, infected shrimp (GNIS). WSSV experimental infections were carried out by injection of 10 μl of a
2.2. Plasmid construction Proteins expressed using vector pGBKT7, were fused with amino acids 1–147 of the GAL4 DNA binding domain (BD). Proteins expressed using vector pGADT7 were fused with amino acids 768–881 of the GAL4 activation domain (AD). The control plasmids pGBKT7-53 and pGADT7-T were obtained from CLONTECH Laboratories, Inc. The Plasmid BDsyntenin containing a full-length Pm-syntenin gene was constructed by inserting the syntenin DNA inframe into an EcoRI and BamHI site. Plasmid construction was verified by sequencing cDNA from the hemocytes of WSSV-infected shrimp, ligated into the AD-prey vector that had been previously digested with the EcoRI and XhoI as reported from previous work (Tonganunt et al., 2005).
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method used here is reproducible and of equal or greater sensitivity in comparison with the β-galactosidase assay (Diaz-Camino et al., 2003). All of the yeast media used were prepared according to the standard protocols (Handbook PT3024-1, CLONTECH Laboratories, Inc.). 2.4. Sequence analysis
Fig. 1. Growth analysis results for the yeast two-hybrid assay. S. cerevisiae AH109 cell were cotransformed with BD-syntenin and ADsbp (marked as Syn + Sbp). Transformed cells were inoculated in SD medium. For the positive control yeast cells were transformed with pGBKT7-53 and pGADT7-T (positive) and the empty host cells were used as the negative control (negative).
(PerkinElmer instruments). This is an alternative method of screening colonies for transformants based on the growth curve analysis for yeast culture. The
Plasmid DNA was isolated from the clones constructed in Section 2.2 and transformed into Escherichia coli Top10F′ for plasmid recovery and sequencing. The sequence of the eIF5A gene of P. monodon was deposited at GenBank under accession number DQ851145. The searches for nucleotide and protein sequence similarities were conducted with BLAST algorithm at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST/). The eIF5A deduced amino acid sequence was analyzed with the NCBI Protein Sequence Analysis software. Multiple alignment of the eIF5A was performed with the ClustalX Multiple Alignment program (http://www.ncbi.nlm.nih.gov) and Multiple Alignment
Fig. 2. Nucleotide and deduced amino acid sequences of P. monodon eIF5A cDNA. Amino acids are indicated as single capital letters under each triplet codon of the nucleotide sequence. The 12-amino acid hypusine core region is shown in bold type while the hypusine modification site is shown in bold underlined type. An asterisk (⁎) indicates the stop codon. The polyadenylation signal (ATTAAA) is underlined.
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Fig. 3. Phylogenetic analysis of the eIF5A amino acid sequences of P. monodon and various other species: S. frugiperda, S. exigua, H. sapiens, B. mori, D. rerio, Rabbit, A. mellifera, C. familiaris, M. musculus, R. norvegicus, X. laevis, B. taurus, G. gallus, P. troglodytes, P. pygmaeus, X. tropicalis, and D. melanogaster obtained from GenBank. B. taurus was used as the out-group. Analysis was based on the completed amino acid sequences.
show by GENEDOC, version 2.6.001 (Nicholas and Nicholas, 1997). 2.5. Expression and purification of recombinant Pm-syntenin A BamHI-PstI fragment containing the entire coding region of Pm-syntenin was obtained from previous work (Bangrak et al., 2002). This recombinant plasmid as well as an insertless pQE40 expression vector were transformed into E. coli M15 and proteins were prepared according to Bangrak et al. (2002). 2.6. Expression and purification of GST-tagged eIF5A protein The AD-plasmid harboring the eIF5A gene was digested with EcoRI and XhoI, then ligated into the
pGEX-4T-1 expression vector (Amersham Biosciences) that had been digested with the same restriction enzymes. The resultant plasmid pGEX4T-1-eIF5A was transformed into E. coli BL21 (Amersham Biosciences). The cell was cultivated in medium containing 100 μg/ml of ampicillin and the expression of a recombinant protein was induced by the addition of 0.1 mM IPTG. Cells were harvested and the GST-eIF5A fusion protein was purified by using a Glutathione–Sepharose 4B resin column (Amersham Biosciences). The purified protein was detected as a single band on a 12% SDS-PAGE, and kept at − 80 °C until ready for use. 2.7. In vitro binding assays (in vitro GST-pull-down assays) GST-pull-down assay was performed using the Bulk GST purification Module (Amersham Biosciences). The purified GST-eIF5A fusion protein was adsorbed onto
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Fig. 4. In vitro binding assay. A glutathione–sepharose bead pull-down was performed on the combined proteins as shown in the table (nos. 1, 3, 4, 6). The eluted material was loaded on SDS-PAGE gels, transferred then detected using specific antibodies. A GST band and GST-eIF5A were detected with anti-GST antibody (lanes 4–6). The eluted material was also detected for syntenin with anti-His Tag antibody (lanes 1, 3). Lane 2 and lane 5 are purified 6xHis-syntenin and GST-eIF5A, used as a control for antibody detection.
Glutathione–Sepharose beads (20 μl of a 50% bed slurry) for 1 h, then washed ten times and finally resuspended in phosphate-buffered saline, pH7. The beads carrying the GST-eIF5A fusion protein would serve as the bait protein in the subsequent steps. The prey protein, a purified 6xHistidine-tagged protein fused with syntenin (6xHis-syntenin) was combined with the bait protein and the mixture was gently shaken for 2 h at room temperature (RT). The proteins in solution were washed 6 times using 100 μl of phosphate-buffered saline and eluted by the addition of a buffer containing reduced glutathione. The eluted samples were analyzed by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The blots were incubated with an antiHis Tag antibody (His-Probe horseradish peroxidase conjugated, Pierce; diluted 1:20,000) and visualized using ECL detection reagent (Pierce). To confirm the presence of the GST-fusion protein, the blots were incubated with Anti-GST (Amersham Biosciences; 1:2000) and conjugated goat anti-mouse IgG-alkaline phosphatase (Pierce; diluted 1:20,000). Lumi-Phos was used as a substrate for detecting chemiluminescence. 2.8. Semi-quantitative RT-PCR Total cellular RNA of P. monodon hemocytes was isolated from NS (uninfected shrimp), GNIS and DIS using TRIZOL Reagent (Life Technologies). The total RNA (800 ng) from each sample was reverse transcribed to produce cDNA using SuperScript™ III Reverse Transcriptase (Invitrogen). The resulting cDNA
(500 ng) was amplified by Taq DNA polymerase (Promega) and 0.2 μM of primers specific for each gene. The following thermal profile was used for PCR amplification of the cDNA on a GeneAmp® PCR system 9700 (Applied Biosystems): 1 cycle of 94 °C for 5 min followed by 25 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR products were electrophoretically separated on a 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light by Gel Doc 1000 genetic Analyzer (BIO RAD). Semiquantitation by RT-PCR was carried out in triplicate. 2.9. SYBR Green RT-PCR cDNA was synthesized in a 10 μl reaction volume containing 800 ng of DNase I treated total RNA and SuperScript™ III Reverse Transcriptase (Invitrogen). For comparing the detection limits of the amplicon in SYBR Green PCR, 300 ng cDNA was used per reaction. The reaction mixture contained 12.5 μl of iQ™ SYBR® Green Super Mix (BIO RAD) and 0.2 μM of each forward and reverse primer. The thermal profile for the PCR amplification was 95 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min. A post-PCR curve was run according to the MX3000P™ (STRATAGENE®): a 1 min hold at 95 °C, a 30 s hold at 50 °C, and a 30 min slow ramp from 50 to 95 °C. Each sample was comprised of 3 replicates and all reactions were independently repeated twice to ensure the reproducibility of the results. For real-time
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Fig. 5. (A) RT-PCR assay of eIF5A, syntenin and α2M gene expression in hemocytes of 4 individuals of uninfected P. monodon (NS), grossly normal, infected shrimp (GNIS) and dead shrimp (DIS). The expression is compared to elongation factor (EF) as an internal control. (B) Normalized eIF5A, syntenin, α2M and eIF4A expression were calculated from the images using Scion Images software. The bars represent means with standard deviation (SD) for results obtained from 4 individuals in each sample set.
RT-PCR quantification of eIF5A and syntenin expression, the shrimp gene elongation factor-1α (EF) (Dhar et al., 2002) was used as an internal control. Anova comparison tests were used for statistical analysis by SPSS software (version 14.0). Values were considered to be significant at P b 0.05. 3. Results 3.1. Identification of Pm-syntenin-binding protein Using the yeast two-hybrid method with Pmsyntenin as the bait and a WSSV-infected shrimp hemocyte library as the prey, we identified 7 yeast clones that contained possible syntenin-binding proteins (Sbps) as a result of their growth in high stringency medium (SD-LTH + 5.0 mM 3-AT). An example of the growth analysis is shown in Fig. 1 and includes the clone showing the best growth (Syn + Sbp). By contrast, empty host control cells gave no growth (Fig. 1). The prey vector from this clone was extracted and transformed into E. coli for plasmid recovery and sequencing.
3.2. Characterization and comparison of synteninbinding protein (Sbp) sequences Sequencing of the cDNA insert in the plasmid vector of the fastest growing yeast clone revealed a deduced protein sequence of 157 amino acids with a predicted molecular mass of 17.27 kDa (Fig. 2). The deduced sequence showed 81% identity to the protein eIF5A of the moth Spodoptera frugiperda (GenBank accession number AAF13316.1). Alignment of the P. monodon protein sequence with other eIF5A sequences at GenBank revealed that eIF5A is a highly conserved protein, particularly at the N-terminal portion. In addition to the complete conservation of 12 amino acids constituting the hypusine core region (bold letters in Fig. 2), several other amino acids were also conserved. These included 5 proline residues, indicating conservation of the secondary structure of the protein. Phylogenetic analysis of eIF5A amino acid sequences of P. monodon and eIF5As from various other species showed that the eIF5A of P. monodon fell within a group of arthropod (insect) proteins (Fig. 3).
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Fig. 6. Real-time PCR machine output and standard curves for eIF5A, syntenin and α2M.
3.3. Interaction of syntenin with eIF5A in vitro
3.4. Gene expression by semi-quantitative RT-PCR
Pull-down assays using soluble His-labeled Pmsyntenin with GST-labeled eIF5A bound to sepharose beads confirmed binding between the two proteins (Fig. 4). Specifically, western blot detection of His and GST labels revealed that both were present in the eluant (Fig. 4, lanes 3 and 6) from glutathione treated beads after exposure to His-labeled syntenin. However, no Hislabeled syntenin was found when GST alone was bound to the beads (Fig. 4, lanes 1 and 4). This test confirmed that syntenin binds specifically to eIF5A and not to GST.
Semi-quantitative RT-PCR during the course of a WSSV infection revealed that the expression of eIF5A was high in grossly normal WSSV-infected shrimp (GNIS) when compared to normal shrimp (NS) and dead WSSV-infected shrimp (DIS) (Fig. 5). Since α2M is also known as a syntenin-binding protein, we also measured its expression during the course of WSSV infection and found that it was also high in GNIS when compared to NS and DIS. In addition, we measured the expression of eIF4A, a member of the initiation factor
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Fig. 7. eIF5A, syntenin and α2M gene expression by quantitative real-time RT-PCR. (A) Fold-change (x-change, mean ± S.D.) of eIF5A and syntenin mRNA expression in hemocytes of 3 individuals of uninfected P. monodon (normal, NS), grossly normal, infected shrimp (GNIS) and dead shrimp (DIS). The expression level was compared to elongation factor as an internal control. The standard curve method was used for the quantification of mRNA, NC = normal control. (B) Fold-change (x-change, mean ± S.D.) of eIF5A and syntenin mRNA expression in hemocytes of experimentally infected shrimp at various times post challenge (6, 12, 24, 48, 72 h postinjection; MIS = moribund infected shrimp; DIS = dead shrimp).
complex that controls protein translation (Palacios et al., 2004), and we found that its expression differed markedly from that of eIF5A (Fig. 5). Expression was relatively low for all samples. 3.5. Syntenin and eIF5A gene expression by quantitative RT-PCR assay Real-time PCR amplification curves for the three genes generated by the MX3000P™ (STRATAGENE®) were very reproducible and indicated that the primers were selective and effective in producing the specific PCR products (Fig. 6A,C,E). The accuracy of mRNA quantification, and sensitivity and linearity of SYBR Green-based Q-RT-PCR were examined using a 10-fold serial dilution of each DNA standard. The relationship between threshold cycle (CT) and the log copy number of the DNA standard was linear with r2 N 0.97 for the three genes, indicating that the CT values changed
proportionally with serial dilution of the samples. The reproducibility of the techniques within and between assays was tested, using serial dilutions of eIF5A, syntenin and α2M cDNA standards. By reference to the standard curves, expression of eIF5A and α2M was high in GNIS but low in DIS when compared to NS (Fig. 7A). The increased expression in GNIS when compared to NS was 2.7-fold for eIF5A and 4.6-fold for α2M. However, both then decreased by 3.2fold and 4.7-fold, respectively in DIS. By contrast, syntenin expression appeared to increase gradually as the disease progressed, reaching 1.4-fold that of NS in DIS. A more detailed time-course examination of eIF5A and syntenin expression during the course of WSSV infection (Fig. 7B), revealed general trends for eIF5A expression. An initial rise was followed by a decline as the shrimp approached death, while syntenin expression fell initially and finally rose sharply as the shrimp approached death.
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4. Discussion The yeast two-hybrid assay is a powerful method for identifying protein–protein interactions that we have previously used to successfully identify several proteins binding to known bait proteins (Tonganunt et al., 2005; Senapin and Phongdara, 2006). After protein identification, we can further investigate and characterize the function of these binding proteins. Here, we have shown that eIF5A derived from a cDNA library of hemocytes from WSSV-infected shrimp can specifically bind with syntenin, a suggested adapter protein previously identified in shrimp hemocytes. Although eIF5A binding with syntenin has been previously reported by Li et al. (2004), ours is the first report of the phenomenon from the cDNA library of a marine animal. We earlier reported that syntenin was upregulated at the acute stage of WSSV infections in shrimp (Bangrak et al., 2002) and similar results were obtained again here using both semi-quantitative and quantitative RT-PCR analysis. The initial increase in eIF5A expression during the period of infection, when no gross signs of disease are visible, indicates that eIF5A may play some role in slowing disease progression. This is supported by the fact that its expression falls as the shrimp approach death. It has been suggested (Li et al., 2004) that binding of syntenin and eIF5A may act to prevent apoptosis and apoptosis has been proposed as a cause of death in WSSV-infected shrimp (Flegel, 2006). Thus, we may hypothesize that eIF5A binding to syntenin prevents apoptosis during the early stages of WSSV infection but fails to do so as the expression of eIF5A declines with disease progression. Although the function of α2M has been discussed in our earlier work (Chotigeat et al., 2006), the function of eIF5A remains an open question. The results from our work and elsewhere indicates that it is responsible for supporting viral replication while keeping the cell viable. Recent research has revealed that eIF5A is not an initiator of protein translation (Kang and Hershey, 1994; Zuk and Jacobson, 1998) although it could be involved in the translation of a specific subset of mRNAs (Park et al. 1997). It has also been correlated with cell proliferation (Kang and Hershey, 1994). Li et al. (2004) demonstrated the binding of eIF5A and syntenin and proposed a new biological activity for eIF5A as a regulator of p53. Their data showed that the eIF5A interaction with syntenin prevented it from inducing increased expression of p53 protein. In addition, eIF5A expression itself is significantly increased during dendritic cell maturation, and hypusine formation appears to be essential for this process (Kruse et al.,
2000). Ling et al. reported that eIF5A was involved in the apoptosis of tumor cells induced by inhibition of ubiquitin proteasomes. These reports indicate that eIF5A may be involved in cell growth and apoptosis. The finding that eIF5A is a cellular cofactor of HIV-1 Rev and HTLV-I Rex transactivator proteins indicates that eIF5A functions as part of or provides access to a cellular nuclear-cytoplasmic RNA transport system and therefore supports viral replication (Bevec et al., 1996; Junker et al., 1996; Ruhl et al., 1993; Katahira et al., 1995; Schatz et al., 1998; Elfgang et al., 1999). Van Oers et al. (1999) reported that the S. frugiperda genome had a single copy of an eIF5A gene but that it was transcribed into 4 different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decrease in the number of all four transcripts as soon as 12 h after infection. They suggested that eIF5A was an essential protein in insect species and that depletion of this factor towards the end of a baculovirus infection was likely to reduce cell viability. A perplexing aspect of the eIF5A protein is that high expression occurs only in grossly normal, infected shrimp (GNIS) but not in uninfected normal shrimp (NS) or in moribund/dead shrimp. Perhaps this is a result of the cell's attempt to survive viral infection. It is expected that future research will result in the identification of more components of both the viral and host genomes and that this will allow us to better understand how host cells survive viral infection. Acknowledgements This work was supported by National Research Council of Thailand, The Royal Golden Jubilee Graduate Program from the Thailand Research Fund to Ms. Yanisa Laoong-u-thai (PHD/0250/2546). cDNA library was constructed from the project: Genomic Researches for Increasing Culture Efficiency of the Black Tiger Shrimp (Penaeus monodon) Phase I and Phase II: (BT-B-06-SG-09-4603) supported by NSTDA, Thailand. We thank Prof. Dr. Kenichi Fujise, University of Texas Health Science Center at Houston, and Prof. Dr. Brian Hodgson, Prince of Songkla University for reading the manuscript and making valuable comments. References Bangrak, P., Graidist, P., Chotigeat, W., Supamattaya, K., Phongdara, A., 2002. A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection. Dis. Aquat. Org. 49, 19–25.
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Hikima, S., Hikima, J.I., Rojtinnakorn, J., Hirono, I., Aoki, T., 2003. Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species. Gene 316, 187–195. Junker, U., Bevec, D., Barske, C., Kalfoglou, C., Escaich, S., Dobrovnik, M., Hauber, J., Böhnlein, E., 1996. Intracellular expression of cellular eIF5A mutants inhibits HIV-1 replication in human T cells: a feasibility study. Hum. Gene Ther. 7, 1861–1869. Kang, H.A., Hershey, J.W., 1994. Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J. Biol. Chem. 269, 3934–3940. Katahira, J., Ishizaki, T., Sakai, H., Adachi, A., Yamamoto, K., Shida, H., 1995. Effects of translation initiation factor eIF-5A on the functioning of human T-cell leukemia virus type I Rex and human immunodeficiency virus Rev inhibited trans dominantly by a Rex mutant deficient in RNA binding. J. Virol. 69, 3125–3133. Kruse, M., Rosorius, O., Kratzer, F., Bevec, D., Kuhnt, C., Steinkasserer, A., Schuler, G., Hauber, J., 2000. Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with Nuclear Export of CD83 mRNA. J. Exp. Med. 191, 1581–1590. Li, A.L., Li, H.Y., Jin, B.F., Ye, Q.N., Zhou, T., Yu, X.D., Pan, X., Man, G.H., He, K., Yu, M., Hu, M.R., Wang, J., Yang, S.C., Shen, B.F., Zhang, X.M., 2004. A Novel eIF5A complex functions as a regulator of p53 and p53-dependent apoptosis. J. Biol. Chem. 279, 49251–49258. Lightner, D.V., 1996. A Handbook of Pathology and Diagnostic Procedures for Diseases of Penaeid Shrimp. World Aquaculture Society, Boton Rouge, LA, USA, p. 305. Lin, D., Gish, G.D., Songyang, Z., Pawson, T., 1999. The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif. J. Biol. Chem. 274, 3726–3733. Lo, C.F., Ho, C.H., Peng, S.E., Chen, C.H., Hsu, H.C., Chiu, Y.L., Chang, C.F., Liu, K.F., Su, M.S., Wang, C.H., Kou, G.H., 1996a. White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods. Dis. Aquat. Org. 27, 215–225. Lo, C.F., Leu, J.H., Ho, C.H., Chen, C.H., Peng, S.E., Chen, Y.T., Chou, C.M., Yeh, P.Y., Huang, C.J., Chou, H.Y., Wang, C.H., Kou, G.H., 1996b. Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis. Aquat. Org. 25, 133–141. Nicholas, K.B., Nicholas Jr., H.B., 1997. GENEDOC: A Tool for Editing and Annotating Multiple Sequence Alignments. http:// www.cris.com/ketchup/genedoc.html. Palacios, I.M., Gatfield, D., St Johnston, D., Izaurralde, E., 2004. An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 427, 753–757. Park, M.H., Lee, Y.B., Joe, Y.A., 1997. Hypusine is essential for eukaryotic cell proliferation. Biol. Signals 6, 115–123. Rojtinnakorn, J., Hirano, I., Itami, T., Takahashi, Y., Aoki, T., 2002. Gene expression in hemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach. Fish Shellfish Immunol. 13, 69–83. Ruhl, M., Himmelspach, M., Bahr, G.M., Hammerschmid, F., Jaksche, H., Wolff, B., Aschauer, H., Farrington, G.K., Probst, H., Bevec, D., Hauber, J., 1993. Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation. J. Cell Biol. 123, 1309–1320. Schatz, O., Oft, M., Dascher, C., Schebesta, M., Rosorius, O., Jaksche, H., Dobrovnik, M., Bevec, D., Hauber, J., 1998. Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5. Cell Biol. 95, 1607–1612. Senapin, S., Phongdara, A., 2006. Detection of binding interactions of shrimp cellular proteins to Taura syndrome viral capsid proteins; VP1, VP2 and VP3. Virus Res. 122, 69–77.
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Cloning of eIF5A from shrimp Penaeus monodon, a highly expressed protein involved in the survival of WSSV-infected shrimp Amornrat Phongdara b,⁎, Yanisa Laoong-u-thai a , Warapond Wanna b b
a Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
Received 19 August 2006; received in revised form 22 December 2006; accepted 4 February 2007
Abstract Shrimp respond to viral infection with up- and down-regulation of many critical genes. In our previous work, we showed that white spot syndrome virus (WSSV) infection of Penaeus monodon (Pm) caused cellular syntenin levels to increase. In order to further explore the signal transduction pathway of syntenin, we constructed the cDNA library of WSSV-infected shrimp and performed a yeast two-hybrid screening of the library using syntenin as bait. Here we report that syntenin specifically binds eukaryotic translation initiation factor 5A (eIF5A), a 157 amino acid polypeptide that is implicated in cell proliferation and survival. GST-pull-down assays showed the presence of specific interaction between Pm-syntenin and eIF5A. Message analyses revealed that syntenin expression remained normal when shrimp were infected with WSSV but did not show any gross signs of disease. Syntenin expression, however, increased 1.4-fold when shrimp became dead. In contrast, eIF5A expression increased markedly during the stage of WSSV infection when there were no gross signs of disease, i.e., prior to the moribund stage. At the moribund stage, eIF5A expression was down-regulated. This data suggest that syntenin and eIF5A physically interact with each other in the setting of WSSV infection and that these two molecules exhibit dynamic changes in their expression patterns as infection progresses from grossly normal, infected shrimp to full-blown phase. © 2007 Elsevier B.V. All rights reserved. Keywords: eIF5A; Translation initiation; Syntenin; Protein–protein interaction; Yeast two hybrid
1. Introduction Penaeid shrimp production is a worldwide economic activity. The intensification of shrimp farming over the last few decades has been accompanied by development of infectious diseases of viral, bacterial, and, in some cases, fungal origin (Destoumieux-Grazon et al., 2001). Among the various viruses of the penaeid
⁎ Corresponding author. E-mail address: [email protected] (A. Phongdara). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.02.005
shrimp, the white spot syndrome virus (WSSV) is responsible for a major proportion of the diseases plaguing commercial shrimp ponds, and has resulted in high mortality and economic losses. (Lightner, 1996; Flegel, 1997, 2001). Shrimp exhibit a diverse response to viral infection, that is manifested in marked up- and down-regulation of a variety of genes (Gross et al., 2001; Supungul et al., 2002; Rojtinnakorn et al., 2002; Sotelo-Mundo et al., 2003; Hikima et al., 2003; He et al., 2005; De Lorgeril et al., 2005; Du et al., 2006). In our previous work, we identified the protein syntenin of the shrimp Penaeus
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monodon (Pm) as a dynamic responder to white spot syndrome virus (WSSV) infection, its message being greatly up-regulated in the acute phase of the infection (Bangrak et al., 2002). Reports from studies of other organisms indicate that syntenin is involved in the signal transduction pathway. Syntenin has no obviously catalytic domain and therefore is unlikely to have a signaling function by itself, but could serve as an adapter or scaffolding protein to attach other proteins and functions in the cell. As of now several synteninbinding proteins from other organisms have been identified and their functions have been described. These include syndecan (Grootjans et al., 1997), proTGFα (Femandez-Larrea et al., 1999), ephrin B (Torres et al., 1998; Lin et al., 1999), neurofascin (Grootjans et al., 2000), interleukin 5 receptor α subunit (IL5Rα), and EphA7 (Torres et al., 1998). In order to further explore the link between Pmsyntenin and viral infection, we performed a yeast twohybrid screening of a P. monodon cDNA library, using Pm-syntenin as bait. This experiment resulted in seven independent clones of syntenin-binding proteins being isolated. Among these syntenin-binding proteins, one (α2M) that plays an important role in immune mechanisms in shrimp has already been described (Tonganunt et al., 2005). Here we describe the identification and characterization of another syntenin-binding protein eIF5A and we follow its expression during the course of a WSSV infection.
1:1 × 107 dilution of a viral stock solution made in 0.85% NaCl and 100 to 150 μl of hemolymph was subsequently withdrawn at 6, 12, 24, 48 and 72 h postinjection (p.i.). The remaining shrimp were held up to the time when they became moribund (shrimp is immobile and unresponsive to touch but were still showing movement of gill rakers. At this time it was labeled as moribund, infected shrimp (MIS). When gill movement ceased, hemolymph was immediately withdrawn and labeled as dead shrimp (DIS). Hemolymph was stored at − 80 °C until used.
2. Materials and methods
2.3. Yeast two-hybrid screening
2.1. Samples
Yeast two-hybrid screening was carried out with the MATCHMAKER Gal4 Two-hybrid System 3 (CLONTECH Laboratories, Inc.). Competent cells of the yeast strain AH109 were prepared and transformations were performed according to the manufacturer's protocols. Briefly, 1 μg of plasmid DNA was added to 100 μl of competent cells and mixed again with 36 μl of 1 M lithium acetate and 240 μl of 50% polyethylene glycol (PEG). The transformation mixture was then incubated at 30 °C for 30 min followed by a heat-shock at 42 °C for 15 min and subsequently spread on a drop-out-agar plate in the absence of leucine and tryptophan. The plates were incubated at 30 °C for 48 h to allow for yeast growth. PCR was used to confirm transformation with the target gene. A positive clone was inoculated into SD medium lacking leucine, tryptophan and histidine (SDLTH) and supplemented with 3-amino-1,2,4-triazole (SD-LTH + 5 mM 3-AT). The medium was shaken at 180 rpm at 30 °C for 96 h. Absorbance of the medium at 595 nm was measured in a spectrophotometer
All shrimp were obtained from a farm controlled by the Songkhla Coastal Fisheries Research and Development Center, Thailand. The shrimp were sourced from a pond stocked with postlavae that had tested negative for WSSV by nested PCR (Lo et al., 1996a,b) and that later, when the pond was harvested, showed no sign of WSSV infection. Prior to the beginning of the experiment, shrimp were kept individually in 60 l aquaria for 10 days, all samples were checked for WSSV infection by using PCR technology (Lo et al., 1996a,b). Hemolymph fluid (100–150 μl) was initially withdrawn and labeled as normal (NS) for individual shrimp whose PCR results proved negative for WSSV. Hemolymph from individual shrimp identified by PCR as WSSV positive, but showing no sign of the white spot syndrome, was labeled as originating from grossly normal, infected shrimp (GNIS). WSSV experimental infections were carried out by injection of 10 μl of a
2.2. Plasmid construction Proteins expressed using vector pGBKT7, were fused with amino acids 1–147 of the GAL4 DNA binding domain (BD). Proteins expressed using vector pGADT7 were fused with amino acids 768–881 of the GAL4 activation domain (AD). The control plasmids pGBKT7-53 and pGADT7-T were obtained from CLONTECH Laboratories, Inc. The Plasmid BDsyntenin containing a full-length Pm-syntenin gene was constructed by inserting the syntenin DNA inframe into an EcoRI and BamHI site. Plasmid construction was verified by sequencing cDNA from the hemocytes of WSSV-infected shrimp, ligated into the AD-prey vector that had been previously digested with the EcoRI and XhoI as reported from previous work (Tonganunt et al., 2005).
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method used here is reproducible and of equal or greater sensitivity in comparison with the β-galactosidase assay (Diaz-Camino et al., 2003). All of the yeast media used were prepared according to the standard protocols (Handbook PT3024-1, CLONTECH Laboratories, Inc.). 2.4. Sequence analysis
Fig. 1. Growth analysis results for the yeast two-hybrid assay. S. cerevisiae AH109 cell were cotransformed with BD-syntenin and ADsbp (marked as Syn + Sbp). Transformed cells were inoculated in SD medium. For the positive control yeast cells were transformed with pGBKT7-53 and pGADT7-T (positive) and the empty host cells were used as the negative control (negative).
(PerkinElmer instruments). This is an alternative method of screening colonies for transformants based on the growth curve analysis for yeast culture. The
Plasmid DNA was isolated from the clones constructed in Section 2.2 and transformed into Escherichia coli Top10F′ for plasmid recovery and sequencing. The sequence of the eIF5A gene of P. monodon was deposited at GenBank under accession number DQ851145. The searches for nucleotide and protein sequence similarities were conducted with BLAST algorithm at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST/). The eIF5A deduced amino acid sequence was analyzed with the NCBI Protein Sequence Analysis software. Multiple alignment of the eIF5A was performed with the ClustalX Multiple Alignment program (http://www.ncbi.nlm.nih.gov) and Multiple Alignment
Fig. 2. Nucleotide and deduced amino acid sequences of P. monodon eIF5A cDNA. Amino acids are indicated as single capital letters under each triplet codon of the nucleotide sequence. The 12-amino acid hypusine core region is shown in bold type while the hypusine modification site is shown in bold underlined type. An asterisk (⁎) indicates the stop codon. The polyadenylation signal (ATTAAA) is underlined.
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Fig. 3. Phylogenetic analysis of the eIF5A amino acid sequences of P. monodon and various other species: S. frugiperda, S. exigua, H. sapiens, B. mori, D. rerio, Rabbit, A. mellifera, C. familiaris, M. musculus, R. norvegicus, X. laevis, B. taurus, G. gallus, P. troglodytes, P. pygmaeus, X. tropicalis, and D. melanogaster obtained from GenBank. B. taurus was used as the out-group. Analysis was based on the completed amino acid sequences.
show by GENEDOC, version 2.6.001 (Nicholas and Nicholas, 1997). 2.5. Expression and purification of recombinant Pm-syntenin A BamHI-PstI fragment containing the entire coding region of Pm-syntenin was obtained from previous work (Bangrak et al., 2002). This recombinant plasmid as well as an insertless pQE40 expression vector were transformed into E. coli M15 and proteins were prepared according to Bangrak et al. (2002). 2.6. Expression and purification of GST-tagged eIF5A protein The AD-plasmid harboring the eIF5A gene was digested with EcoRI and XhoI, then ligated into the
pGEX-4T-1 expression vector (Amersham Biosciences) that had been digested with the same restriction enzymes. The resultant plasmid pGEX4T-1-eIF5A was transformed into E. coli BL21 (Amersham Biosciences). The cell was cultivated in medium containing 100 μg/ml of ampicillin and the expression of a recombinant protein was induced by the addition of 0.1 mM IPTG. Cells were harvested and the GST-eIF5A fusion protein was purified by using a Glutathione–Sepharose 4B resin column (Amersham Biosciences). The purified protein was detected as a single band on a 12% SDS-PAGE, and kept at − 80 °C until ready for use. 2.7. In vitro binding assays (in vitro GST-pull-down assays) GST-pull-down assay was performed using the Bulk GST purification Module (Amersham Biosciences). The purified GST-eIF5A fusion protein was adsorbed onto
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Fig. 4. In vitro binding assay. A glutathione–sepharose bead pull-down was performed on the combined proteins as shown in the table (nos. 1, 3, 4, 6). The eluted material was loaded on SDS-PAGE gels, transferred then detected using specific antibodies. A GST band and GST-eIF5A were detected with anti-GST antibody (lanes 4–6). The eluted material was also detected for syntenin with anti-His Tag antibody (lanes 1, 3). Lane 2 and lane 5 are purified 6xHis-syntenin and GST-eIF5A, used as a control for antibody detection.
Glutathione–Sepharose beads (20 μl of a 50% bed slurry) for 1 h, then washed ten times and finally resuspended in phosphate-buffered saline, pH7. The beads carrying the GST-eIF5A fusion protein would serve as the bait protein in the subsequent steps. The prey protein, a purified 6xHistidine-tagged protein fused with syntenin (6xHis-syntenin) was combined with the bait protein and the mixture was gently shaken for 2 h at room temperature (RT). The proteins in solution were washed 6 times using 100 μl of phosphate-buffered saline and eluted by the addition of a buffer containing reduced glutathione. The eluted samples were analyzed by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. The blots were incubated with an antiHis Tag antibody (His-Probe horseradish peroxidase conjugated, Pierce; diluted 1:20,000) and visualized using ECL detection reagent (Pierce). To confirm the presence of the GST-fusion protein, the blots were incubated with Anti-GST (Amersham Biosciences; 1:2000) and conjugated goat anti-mouse IgG-alkaline phosphatase (Pierce; diluted 1:20,000). Lumi-Phos was used as a substrate for detecting chemiluminescence. 2.8. Semi-quantitative RT-PCR Total cellular RNA of P. monodon hemocytes was isolated from NS (uninfected shrimp), GNIS and DIS using TRIZOL Reagent (Life Technologies). The total RNA (800 ng) from each sample was reverse transcribed to produce cDNA using SuperScript™ III Reverse Transcriptase (Invitrogen). The resulting cDNA
(500 ng) was amplified by Taq DNA polymerase (Promega) and 0.2 μM of primers specific for each gene. The following thermal profile was used for PCR amplification of the cDNA on a GeneAmp® PCR system 9700 (Applied Biosystems): 1 cycle of 94 °C for 5 min followed by 25 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR products were electrophoretically separated on a 1.5% agarose gel, stained with ethidium bromide and visualized under ultraviolet light by Gel Doc 1000 genetic Analyzer (BIO RAD). Semiquantitation by RT-PCR was carried out in triplicate. 2.9. SYBR Green RT-PCR cDNA was synthesized in a 10 μl reaction volume containing 800 ng of DNase I treated total RNA and SuperScript™ III Reverse Transcriptase (Invitrogen). For comparing the detection limits of the amplicon in SYBR Green PCR, 300 ng cDNA was used per reaction. The reaction mixture contained 12.5 μl of iQ™ SYBR® Green Super Mix (BIO RAD) and 0.2 μM of each forward and reverse primer. The thermal profile for the PCR amplification was 95 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min and 72 °C for 1 min. A post-PCR curve was run according to the MX3000P™ (STRATAGENE®): a 1 min hold at 95 °C, a 30 s hold at 50 °C, and a 30 min slow ramp from 50 to 95 °C. Each sample was comprised of 3 replicates and all reactions were independently repeated twice to ensure the reproducibility of the results. For real-time
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Fig. 5. (A) RT-PCR assay of eIF5A, syntenin and α2M gene expression in hemocytes of 4 individuals of uninfected P. monodon (NS), grossly normal, infected shrimp (GNIS) and dead shrimp (DIS). The expression is compared to elongation factor (EF) as an internal control. (B) Normalized eIF5A, syntenin, α2M and eIF4A expression were calculated from the images using Scion Images software. The bars represent means with standard deviation (SD) for results obtained from 4 individuals in each sample set.
RT-PCR quantification of eIF5A and syntenin expression, the shrimp gene elongation factor-1α (EF) (Dhar et al., 2002) was used as an internal control. Anova comparison tests were used for statistical analysis by SPSS software (version 14.0). Values were considered to be significant at P b 0.05. 3. Results 3.1. Identification of Pm-syntenin-binding protein Using the yeast two-hybrid method with Pmsyntenin as the bait and a WSSV-infected shrimp hemocyte library as the prey, we identified 7 yeast clones that contained possible syntenin-binding proteins (Sbps) as a result of their growth in high stringency medium (SD-LTH + 5.0 mM 3-AT). An example of the growth analysis is shown in Fig. 1 and includes the clone showing the best growth (Syn + Sbp). By contrast, empty host control cells gave no growth (Fig. 1). The prey vector from this clone was extracted and transformed into E. coli for plasmid recovery and sequencing.
3.2. Characterization and comparison of synteninbinding protein (Sbp) sequences Sequencing of the cDNA insert in the plasmid vector of the fastest growing yeast clone revealed a deduced protein sequence of 157 amino acids with a predicted molecular mass of 17.27 kDa (Fig. 2). The deduced sequence showed 81% identity to the protein eIF5A of the moth Spodoptera frugiperda (GenBank accession number AAF13316.1). Alignment of the P. monodon protein sequence with other eIF5A sequences at GenBank revealed that eIF5A is a highly conserved protein, particularly at the N-terminal portion. In addition to the complete conservation of 12 amino acids constituting the hypusine core region (bold letters in Fig. 2), several other amino acids were also conserved. These included 5 proline residues, indicating conservation of the secondary structure of the protein. Phylogenetic analysis of eIF5A amino acid sequences of P. monodon and eIF5As from various other species showed that the eIF5A of P. monodon fell within a group of arthropod (insect) proteins (Fig. 3).
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Fig. 6. Real-time PCR machine output and standard curves for eIF5A, syntenin and α2M.
3.3. Interaction of syntenin with eIF5A in vitro
3.4. Gene expression by semi-quantitative RT-PCR
Pull-down assays using soluble His-labeled Pmsyntenin with GST-labeled eIF5A bound to sepharose beads confirmed binding between the two proteins (Fig. 4). Specifically, western blot detection of His and GST labels revealed that both were present in the eluant (Fig. 4, lanes 3 and 6) from glutathione treated beads after exposure to His-labeled syntenin. However, no Hislabeled syntenin was found when GST alone was bound to the beads (Fig. 4, lanes 1 and 4). This test confirmed that syntenin binds specifically to eIF5A and not to GST.
Semi-quantitative RT-PCR during the course of a WSSV infection revealed that the expression of eIF5A was high in grossly normal WSSV-infected shrimp (GNIS) when compared to normal shrimp (NS) and dead WSSV-infected shrimp (DIS) (Fig. 5). Since α2M is also known as a syntenin-binding protein, we also measured its expression during the course of WSSV infection and found that it was also high in GNIS when compared to NS and DIS. In addition, we measured the expression of eIF4A, a member of the initiation factor
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Fig. 7. eIF5A, syntenin and α2M gene expression by quantitative real-time RT-PCR. (A) Fold-change (x-change, mean ± S.D.) of eIF5A and syntenin mRNA expression in hemocytes of 3 individuals of uninfected P. monodon (normal, NS), grossly normal, infected shrimp (GNIS) and dead shrimp (DIS). The expression level was compared to elongation factor as an internal control. The standard curve method was used for the quantification of mRNA, NC = normal control. (B) Fold-change (x-change, mean ± S.D.) of eIF5A and syntenin mRNA expression in hemocytes of experimentally infected shrimp at various times post challenge (6, 12, 24, 48, 72 h postinjection; MIS = moribund infected shrimp; DIS = dead shrimp).
complex that controls protein translation (Palacios et al., 2004), and we found that its expression differed markedly from that of eIF5A (Fig. 5). Expression was relatively low for all samples. 3.5. Syntenin and eIF5A gene expression by quantitative RT-PCR assay Real-time PCR amplification curves for the three genes generated by the MX3000P™ (STRATAGENE®) were very reproducible and indicated that the primers were selective and effective in producing the specific PCR products (Fig. 6A,C,E). The accuracy of mRNA quantification, and sensitivity and linearity of SYBR Green-based Q-RT-PCR were examined using a 10-fold serial dilution of each DNA standard. The relationship between threshold cycle (CT) and the log copy number of the DNA standard was linear with r2 N 0.97 for the three genes, indicating that the CT values changed
proportionally with serial dilution of the samples. The reproducibility of the techniques within and between assays was tested, using serial dilutions of eIF5A, syntenin and α2M cDNA standards. By reference to the standard curves, expression of eIF5A and α2M was high in GNIS but low in DIS when compared to NS (Fig. 7A). The increased expression in GNIS when compared to NS was 2.7-fold for eIF5A and 4.6-fold for α2M. However, both then decreased by 3.2fold and 4.7-fold, respectively in DIS. By contrast, syntenin expression appeared to increase gradually as the disease progressed, reaching 1.4-fold that of NS in DIS. A more detailed time-course examination of eIF5A and syntenin expression during the course of WSSV infection (Fig. 7B), revealed general trends for eIF5A expression. An initial rise was followed by a decline as the shrimp approached death, while syntenin expression fell initially and finally rose sharply as the shrimp approached death.
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4. Discussion The yeast two-hybrid assay is a powerful method for identifying protein–protein interactions that we have previously used to successfully identify several proteins binding to known bait proteins (Tonganunt et al., 2005; Senapin and Phongdara, 2006). After protein identification, we can further investigate and characterize the function of these binding proteins. Here, we have shown that eIF5A derived from a cDNA library of hemocytes from WSSV-infected shrimp can specifically bind with syntenin, a suggested adapter protein previously identified in shrimp hemocytes. Although eIF5A binding with syntenin has been previously reported by Li et al. (2004), ours is the first report of the phenomenon from the cDNA library of a marine animal. We earlier reported that syntenin was upregulated at the acute stage of WSSV infections in shrimp (Bangrak et al., 2002) and similar results were obtained again here using both semi-quantitative and quantitative RT-PCR analysis. The initial increase in eIF5A expression during the period of infection, when no gross signs of disease are visible, indicates that eIF5A may play some role in slowing disease progression. This is supported by the fact that its expression falls as the shrimp approach death. It has been suggested (Li et al., 2004) that binding of syntenin and eIF5A may act to prevent apoptosis and apoptosis has been proposed as a cause of death in WSSV-infected shrimp (Flegel, 2006). Thus, we may hypothesize that eIF5A binding to syntenin prevents apoptosis during the early stages of WSSV infection but fails to do so as the expression of eIF5A declines with disease progression. Although the function of α2M has been discussed in our earlier work (Chotigeat et al., 2006), the function of eIF5A remains an open question. The results from our work and elsewhere indicates that it is responsible for supporting viral replication while keeping the cell viable. Recent research has revealed that eIF5A is not an initiator of protein translation (Kang and Hershey, 1994; Zuk and Jacobson, 1998) although it could be involved in the translation of a specific subset of mRNAs (Park et al. 1997). It has also been correlated with cell proliferation (Kang and Hershey, 1994). Li et al. (2004) demonstrated the binding of eIF5A and syntenin and proposed a new biological activity for eIF5A as a regulator of p53. Their data showed that the eIF5A interaction with syntenin prevented it from inducing increased expression of p53 protein. In addition, eIF5A expression itself is significantly increased during dendritic cell maturation, and hypusine formation appears to be essential for this process (Kruse et al.,
2000). Ling et al. reported that eIF5A was involved in the apoptosis of tumor cells induced by inhibition of ubiquitin proteasomes. These reports indicate that eIF5A may be involved in cell growth and apoptosis. The finding that eIF5A is a cellular cofactor of HIV-1 Rev and HTLV-I Rex transactivator proteins indicates that eIF5A functions as part of or provides access to a cellular nuclear-cytoplasmic RNA transport system and therefore supports viral replication (Bevec et al., 1996; Junker et al., 1996; Ruhl et al., 1993; Katahira et al., 1995; Schatz et al., 1998; Elfgang et al., 1999). Van Oers et al. (1999) reported that the S. frugiperda genome had a single copy of an eIF5A gene but that it was transcribed into 4 different transcripts. Infection of S. frugiperda cells with a baculovirus resulted in a strong decrease in the number of all four transcripts as soon as 12 h after infection. They suggested that eIF5A was an essential protein in insect species and that depletion of this factor towards the end of a baculovirus infection was likely to reduce cell viability. A perplexing aspect of the eIF5A protein is that high expression occurs only in grossly normal, infected shrimp (GNIS) but not in uninfected normal shrimp (NS) or in moribund/dead shrimp. Perhaps this is a result of the cell's attempt to survive viral infection. It is expected that future research will result in the identification of more components of both the viral and host genomes and that this will allow us to better understand how host cells survive viral infection. Acknowledgements This work was supported by National Research Council of Thailand, The Royal Golden Jubilee Graduate Program from the Thailand Research Fund to Ms. Yanisa Laoong-u-thai (PHD/0250/2546). cDNA library was constructed from the project: Genomic Researches for Increasing Culture Efficiency of the Black Tiger Shrimp (Penaeus monodon) Phase I and Phase II: (BT-B-06-SG-09-4603) supported by NSTDA, Thailand. We thank Prof. Dr. Kenichi Fujise, University of Texas Health Science Center at Houston, and Prof. Dr. Brian Hodgson, Prince of Songkla University for reading the manuscript and making valuable comments. References Bangrak, P., Graidist, P., Chotigeat, W., Supamattaya, K., Phongdara, A., 2002. A syntenin-like protein with postsynaptic density protein (PDZ) domains produced by black tiger shrimp Penaeus monodon in response to white spot syndrome virus infection. Dis. Aquat. Org. 49, 19–25.
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