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Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260
Correspondence
Choy Leong Hew
dbshewcl{at}nus.edu.sg
or
dbshead{at}nus.edu.sg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary tables showing SGIV primers, partial cDNA sequences of 
-actin and GAPDH, primers for real-time PCR, and SGIV genes with temporal expression are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Chinchar et al., 2005; Williams et al., 2005), a novel iridovirus of the genus Ranavirus, is a large, icosahedral, cytoplasmic DNA virus. The virus, which causes sleepy grouper disease (SGD), has resulted in significant economic losses in marine net-cage farms in Singapore. It was isolated successfully in 1998 from diseased brown-spotted grouper, Epinephelus tauvina (Chua et al., 1994; Qin et al., 2001). The entire SGIV genome consists of 140 131 bp, and 162 open reading frames (ORFs), encoding polypeptides varying from 41 to 1268 aa, were predicted from the sense and antisense DNA strands (Song et al., 2004). These viruses are causative pathogens of serious systematic diseases in farms of both feral and cultured groupers. So far, genomic sequences of two grouper iridoviruses have been published: SGIV (Song et al., 2004) and grouper iridovirus (GIV) (Tsai et al., 2005), with whole-genomic sequence similarity of >90 %. Willis et al. (1977) designated 10 ‘early’ RNAs (of 47 mRNAs), expressed from 1 to 1.5 h after frog virus 3 (FV-3) infection of fathead minnow cells by using isotopic labelling of virus-specific RNA. The RNA transcriptional map of the Wiseana iridescent virus (WIV) has been studied by using a combination of [35S]methionine pulse-labelling and Northern blotting with WIV DNA probes (McMillan & Kalmakoff, 1994). Similarly, the transcriptional map and temporal cascade of Chilo iridescent virus (CIV) have been studied by carrying out Northern analyses with several putative CIV gene-specific probes (D'Costa et al., 2001, 2004). However, at present, the transcriptional program of viral genes in SGIV is still unclear. DNA microarrays provide a potential tool for the simultaneous measurement of gene expression in all of these viral genes. In this study, we constructed, for the first time, a viral DNA microarray covering 127 predicted ORFs of the SGIV genome. By using this SGIV DNA microarray, the transcriptional program of SGIV was uncovered. The temporal expression of SGIV genes was further confirmed by real-time RT-PCR. By using cycloheximide (CHX) and aphidicoline as inhibitors, the immediate-early (IE), early (E) and late (L) viral genes were uncovered. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were cultured in Eagle's minimum essential medium containing 10 % fetal bovine serum, 0.116 M NaCl, 100 IU penicillin G ml–1 and 100 µl streptomycin sulfate ml–1. Culture media were equilibrated with HEPES to a final concentration of 5 mM and adjusted to pH 7.4 with NaHCO3.
Positive controls for the SGIV DNA microarray.
Given that there is no grouper cDNA library available, we partially sequenced cDNAs of -actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the GE cells and designed unique amplicons for -actin and GAPDH in the SGIV DNA microarray as positive controls. The partial cDNA sequences of grouper -actin and GAPDH are shown in Supplementary Table S1, available in JGV Online. -Actin was also used for data normalization.
Preparation of amplicons for the SGIV DNA microarray.
One hundred and sixty-two SGIV ORFs were predicted on the basis of the published SGIV sequence (Song et al., 2004). Two rounds of PCR were used to generate the amplicons for the microarray. In the first round, specific primers with sizes ranging from 18 to 22 bp were generated on the basis of the SGIV full-length genome [8 bp universal sequences (TGACCATG), added to the 5' terminal of the forward primers, were designed; see Supplementary Table S2, available in JGV Online]. The amplicon sizes varied from 200 to 400 bp. Amplicons whose BLAST scores against other ORFs exceeded 400 were excluded. The genomic DNA of SGIV was used as template in the first round of PCR. In the second round, the DNA fragments from the first round were used as template and 5'-amino-modified universal primer 5'-GCTGAACAGCTATGACCATG-3' and ORF-specific reverse primer were applied. AmpliTaq DNA Polymerase (Applied Biosystems) was used in both rounds of PCR. Each PCR fragment was confirmed to be a single band and of the correct size by running on a 2 % agarose gel (data not shown). The final 129 amplicons, representing 127 viral ORFs and two host housekeeping genes, -actin and GAPDH (purified with a QIAquick 96 PCR Purification kit; Qiagen), were spotted onto lysine-coated slides in duplicate.
Virus infection and CHX and aphidicoline treatments.
GE cells were mock-infected or infected with SGIV at an m.o.i. of 3 p.f.u. per cell. To investigate the temporal expression of viral genes, total RNA was harvested from mock-infected and SGIV-infected GE cells at 0, 1, 4, 8, 16, 32, 48, 72 and 96 h post-infection (p.i.). CHX, a protein-synthesis inhibitor that prevents de novo protein synthesis by preventing translation, was used to study the transcription of viral IE genes. To assess IE gene transcription, SGIV mock-infected and SGIV-infected cultures were treated with different concentrations of CHX (50, 100, 200 or 500 µg ml–1) 1 h before infection. Aphidicoline is a specific inhibitor of DNA polymerase 
. In the presence of aphidicoline, viral DNA replication is inhibited. Given that the L genes were expressed after viral DNA replication, the expression of L viral genes would be downregulated compared with those without aphidicoline treatment. To examine the viral E genes, the transcriptomes from the cultures with aphidicoline treatment and SGIV infection at 3 p.f.u. per cell were compared with those from the culture with mock aphidicoline treatment and SGIV infection at 3 p.f.u. per cell. In the aphidicoline treatment, aphidicoline at a final concentration of 30 µg ml–1 was added to the culture 1 h prior to SGIV infection.
Total RNA preparation, reverse transcription and labelling.
Total RNA was extracted and purified by using a Qiagen RNeasy Mini kit. RNAsin (10 units; Promega), 100 units DNase I and 10 µl enzyme buffer 3 (Roche) were added to the total RNA solution, mixed well and incubated at room temperature for 20 min. The RNA samples were later purified with a Qiagen RNeasy column and stored at –70 °C. For reverse-transcription reactions, 10 mM dATP, 10 mM dGTP, 10 mM dCTP, 2 mM dTTP (Invitrogen) and 8 mM aa-dUTP (Ambion) were used. For each reverse-transcription reaction, 10 µg total RNA was reverse-transcribed by using PowerScript Reverse Transcriptase (BD Clontech) with random primers [d(N)6, 0.5 µg µl–1] (Life Technologies). After reverse transcription, the unused aa-dUTPs were removed with Microcon YM-30 columns (Amicon). The cDNAs were coupled with mono-functional NHS-ester Cy dyes (Amersham Biosciences). After removing unincorporated/quenched Cy dyes with a QIAquick PCR purification kit (Qiagen), the mixtures were hybridized to the SGIV DNA chip by using the MAUI hybridization system (BioMicro Systems) and incubated overnight at 42 °C. The hybridizations were repeated on duplicate arrays with independently prepared RNA. The data obtained from the different arrays were consistent. The mean correlation coefficient of 127 viral elements of duplicates was 0.9865. The mean correlation coefficient of 127 viral elements between repeats was 0.9750.
Real-time PCR.
In order to validate the DNA microarray data, semi-quantitative real-time RT-PCR was applied and -actin was used as the control. The specific primers for real-time RT-PCR were checked after PCR and showed a single, specific band after running on 2 % agarose gel. Information on the real-time PCR primers is provided in Supplementary Table S3, available in JGV Online. The total RNA samples were reverse-transcribed using PowerScript Reverse Transcriptase (BD Clontech) with random primers [d(N)6, 0.5 µg µl–1]. cDNA (50 ng) was subsequently subjected to real-time PCR by using a QuantiTect SYBR Green PCR kit (Qiagen) in the Lightcycler 2.0 system (Roche). The real-time data were collected and analysed with the 2–
CT method (Livak & Schmittgen, 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tsai et al., 2005). The specificity of the arrays was validated with cDNA probes prepared from mock-infected and SGIV-infected GE cells. The cDNA probes from uninfected cells detected only -actin and GAPDH, while cDNA probes from infected cells detected all SGIV DNA targets, as well as -actin and GAPDH (data not shown).
Temporal gene-expression analysis of the SGIV genome
Total RNA was harvested from mock-infected cells and SGIV-infected cells at 0, 1, 4, 8, 16, 32, 48, 72 and 96 h p.i.
Of the 127 viral elements on the SGIV array, 16 (13 %) of the 127 investigated viral ORFs commenced expression at 1 h p.i., 106 (83 %) commenced expression at 4 h p.i. and five (4 %) ORFs commenced expression at 8 h p.i. (see Supplementary Table S4, available in JGV Online).
In our viral DNA microarray, 68 (53.5 %), 43 (34 %), two (1.5 %) and 14 (11 %) of the 127 investigated viral ORFs were detected to reach maximum expression at 32, 48, 72 and 96 h p.i., respectively (see Supplementary Table S4, available in JGV Online).
Hierarchical clustering, in which the expression of each gene at every time point was compared and grouped according to the similarity in gene-expression profiles, was applied to examine the relationship between the genes and their expression patterns. A coloured mosaic matrix, in which each column represents a time point and each row indicates the expression pattern of a single ORF, was used to feature the temporal viral gene-expression data generated from our viral DNA microarray (Fig. 1). The ordered and varied patterns of viral gene expression are illustrated in Fig. 1.
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-actin as controls. The results of real-time RT-PCR were consistent with the DNA microarray data (Fig. 2a–c). The consistency between the real-time RT-PCR and viral DNA microarray data supports the general applicability and utility of our array approach.
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). This phenomenon suggests that the expression of SGIV viral genes does indeed depend on the presence of one or more viral proteins. When the viral gene expression was analysed in the presence of 500 µg CHX ml–1, we found that 41 (32.3 %) ORFs displayed a 1.3-fold upregulation (listed in Table 1). These 41 ORFs were not sensitive to CHX treatment and were considered as strong IE gene candidates (Table 1). As expected, the two putative IE genes, namely ORF086R and ORF162L, were included in this candidate IE gene list (Table 1).
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). Viral genes that consistently displayed twofold downregulation from 16 to 48 h p.i. with aphidicoline treatment were considered to be L gene candidates. After analysing the aphidicoline treatment across time, we found that 50 (38.1 %) ORFs consistently displayed twofold downregulation and were considered to be candidates for viral L genes (Table 1
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), was analysed, ORF086R, a putative IE gene, commenced its expression as early as 1 h p.i., DNA replication- and transcription-related genes, for example DNA polymerase (ORF128R) and the two largest subunits of DNA-dependent RNA polymerase II (ORF073L and ORF104L), increased their expression levels at 4 h p.i. and the major capsid protein (ORF072R) commenced expression as late as 8 h p.i. (see Supplementary Table S4, available in JGV Online). These results indicate that SGIV replication may proceed through a strictly temporally ordered transcriptional program. These results are consistent with the notion, based on FV-3, that one or more IE proteins are needed to activate viral E gene transcription and that one or more viral E proteins are required to switch on viral L gene transcription (Willis et al., 1977; Williams et al., 2005).
Another interesting finding is that SGIV genes vary in their peak time (PT), which is defined as the time (h p.i.) at which the transcript of a viral gene accumulates to its maximum amount. The PTs of SGIV genes range from 32 to 96 h p.i. (see Supplementary Table S4, available in JGV Online). No relationship was found between the functions of SGIV genes and their PTs. Although IE and E genes were expressed earlier than L genes in the SGIV replication cycle, the abundances of all SGIV genes' transcriptomes in the host cell after 8 h p.i. were substantial. These results are consistent with the earlier observations in FV-3 and CIV (Chinchar & Yu, 1992; Chinchar et al., 1994; D'Costa et al., 2001).
When the gene tree was analysed, some of the viral ORFs encoding viral structural proteins clustered together at the top of the gene tree. These include ORF072R, encoding the viral major capsid protein, ORF019R, encoding a myristylated membrane protein, ORF141R, encoding a glycoprotein, and another two ORFs, ORF009L and ORF007L, encoding two proteins of unknown function that have been identified from the mature viral particles by mass spectrometry (Song et al., 2006). The clustering gene tree also shows a tendency for genes with similar functions, such as ORF029L and ORF131R, both of which encode homologues of the Ig-like domain, to be clustered together, despite being located apart from each other in the viral genome. In the SGIV genome, a number of viral genes are novel and their function is unknown. It has been reported that the co-expression of genes of known function with novel genes may provide a relatively simple means to postulate the functions of these poorly characterized ones (Eisen et al., 1998).
It has been reported that the IE, E and L transcripts of FV-3 were synthesized in three coordinated phases (Willis et al., 1977; Willis & Granoff, 1978). Similarly, SGIV genes can be classified as IE genes, E genes and L genes. CHX-insensitive SGIV genes are suggested to be IE genes. Aphidicoline-sensitive SGIV genes are suggested to be L genes. When combining the results of CHX and aphidicoline treatments, the 127 SGIV elements on the microarray included 28 (22.1 %) IE genes, 49 (38.6 %) E genes, 37 (29.1 %) L genes and 13 (10.2 %) unclassified genes (Table 1).
E viral transcripts contain IE and E viral genes. It has been proposed that E transcripts in FV-3 encode regulatory proteins and key catalytic enzymes (Goorha et al., 1978; Goorha, 1982; Williams et al., 2005). Similar observations were made for SGIV. SGIV E transcripts contain replication-related genes, e.g. DNA polymerase (ORF128R), as well as transcription-related genes, such as the second-largest subunit of DNA-directed RNA polymerase II (ORF073L).
Although combining DNA microarrays and drug treatments can provide a wealth of information concerning the expression profile of different viral genes, the approach has some inherent limitations. For example, in the list of unclassified ORFs, ORF019R and ORF141L, which encode two structural proteins (a myristylated membrane protein and a glycoprotein, respectively), are insensitive to the CHX treatments, even at high concentrations (500 µg ml–1) and ORF146L, encoding NTPase/helicase, shows a high sensitivity to the aphidicoline treatment. The possible mechanisms behind drug sensitivity or drug resistance of these unclassified SGIV genes need further investigation.
When investigating the temporal expression of different-stage genes, we found that the IE genes commenced expression between 1 and 4 h p.i., most of the E genes commenced expression at 4 h p.i. and most of the L genes commenced expression between 4 and 8 h p.i. The expression of three of the E genes (ORFs 83R, 099R and 111R) and seven of the L genes (ORFs 008L, 010L, 021L, 055R, 089L, 116R and 154R) was found to increase as early as 1 h p.i. The functions of these viral E and L genes are still unknown.
We also found several interesting phenomena in SGIV. First, ORF030L, which was predicted to be a virus tegument protein (a structural protein), showed insensitivity to both CHX and aphidicoline. ORF030L might have other functions besides being a tegument protein of SGIV. Second, the SGIV genome contained: (i) ORF144R, encoding a homologue of the FGF (fibroblast growth factor) 22 of rat, a major active species of presynaptic organizing molecule (Umemori et al., 2004), (ii) ORF145R, encoding a homologue of the mouse FGF 10, which is related closely to FGF 22 (Tagashira et al., 1997; Okazaki et al., 2002; Strausberg et al., 2002; Umemori et al., 2004). FGF 22 and FGF 10 play important roles in presynaptic differentiation (Umemori et al., 2004). Expression of FGF homologues by SGIV may play an important role in forming the clinical symptoms of SGIV-infected groupers.
Although it has been reported that SGIV is an enveloped virus that enters cells by endocytosis to start the viral infection cycle and buds from the plasma membrane in the late infection phage (Qin et al., 2001), little is known about the processes that occur during SGIV infection. By combining the results of SGIV temporal gene-expression profiles and different-stage viral genes, the patterns of different-stage viral gene expression are uncovered. Our results should provide new insights into the processes of the SGIV infection cascade and the pathogenesis and replication strategies of SGIV. Our SGIV DNA microarrays coupled with global biochemical and genetic strategies might greatly accelerate the functional analysis of a number of functionally unknown genes in the SGIV genome. Given that most ORFs from GIV, as well as a number of ORFs from other iridovirus genomes, such as Ambystoma tigrinum virus (Jancovich et al., 2003), Chilo iridescent virus (Jakob et al., 2001), Infectious spleen and kidney necrosis virus (He et al., 2001), Lymphocystis disease virus 1 (Tidona & Darai, 1997), orange-spotted grouper iridovirus (Lu et al., 2005) and tiger frog virus (He et al., 2002), are homologous to those of SGIV (Song et al., 2004; Lu et al., 2005; Tsai et al., 2005), our results should also be valuable to research on these viruses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 20 May 2006;
accepted 9 June 2006.
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