1 Graduate School of Life Science, National Defense Medical Center, Taipei 114, Taiwan, Republic of China
2 Institute of Zoology, Academia Sinica, Taipei 115, Taiwan, Republic of China
3 Department of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan, Republic of China
Correspondence
Chi-Yao Chang
cychang{at}gate.sinica.edu.tw
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY598033.
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INTRODUCTION |
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Studies on PNPs have revealed that PNP deficiency in humans causes severe T-cell immunity impairment (Giblett et al., 1975). Therefore, development of PNP inhibitors is important, as the inhibitors could potentially be effective suppressors of T-cell proliferative diseases, such as T-cell lymphoma and T cell-related autoimmune diseases, and may also be useful for the suppression of organ-transplant rejection (Bantia et al., 2001
). Recently, due to its difference in substrate specificity from human PNP, PNP from Escherichia coli has also been studied, in order to develop a gene-directed enzyme prodrug therapy (GDEPT) for cancers; it shows a profound bystander killing effect on tumour cells (Hughes et al., 1998
; Secrist et al., 1999
; Gadi et al., 2000
). Because of their potential biomedical significance, PNPs isolated from various organisms with substrate differences from human PNP have drawn more attention in recent years. PNP genes have been cloned and studied from a variety of organisms, including humans (Williams et al., 1984
), cows (Bzowska et al., 1995
), mice (Jenuth et al., 1993
), yeast (Lecoq et al., 2001a
), Schistosoma mansoni (Pereira et al., 2003
) and E. coli (Mao et al., 1997
).
Iridoviruses are large, icosahedral, cytoplasmic DNA viruses that contain circularly permutated and terminally redundant double-stranded DNA genomes (Delius et al., 1984; Schnitzler et al., 1987
; Jakob et al., 2001
). Currently, viruses in the family Iridoviridae are subdivided into four genera: Iridovirus, Chloriridovirus, Lymphocystisvirus and Ranavirus (Williams et al., 2000
). Grouper iridovirus (GIV), a member of the genus Ranavirus, is isolated from yellow grouper (Epinephelus awoara) (Lai et al., 2000
). Sequence analysis of the GIV major capsid protein (MCP) shows that GIV is related closely to Frog virus 3 (the type species of the genus Ranavirus) (Murali et al., 2002
), whose genome is heavily methylated at CpG sequences by a virus-encoded methyltransferase (Willis & Granoff, 1980
; Tidona et al., 1996
). However, neither methylation nor a virus-encoded methyltransferase was found in the GIV genome (C.-T. Tsai & C.-Y. Chang, unpublished results). Recently, five complete genomic sequences of members of the family Iridoviridae from various host species have been analysed (Tidona & Darai, 1997
; He et al., 2001
, 2002
; Jakob et al., 2001
; Jancovich et al., 2003
). These analyses show that iridoviruses encode a number of cellular protein homologues that may be involved in nucleic acid metabolism, such as ribonucleotide reductase, thymidine kinase and thymidylate synthase. However, the actual function of these proteins in iridoviruses remains to be elucidated. Computer analysis of the GIV genome (Lai et al., 2000
) resulted in the identification of a novel ORF that encodes a putative PNP. This is the first time that the PNP gene has been identified in a virus. This tempted us to characterize this protein. So, in the present study, we have cloned, sequenced and analysed the newly identified virus PNP gene. Recombinant protein expression, subcellular localization and enzymic activity of this virus PNP are also demonstrated.
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METHODS |
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PCR amplification and plasmid construction.
The complete GIV PNP ORF was amplified by PCR from GIV genomic DNA, using oligonucleotide primers with designed restriction enzyme cleavage sites. One forward primer, PNP(F) (5'-CGCGGATCCACCATGACGGATTACGATTTG-3', with a BamHI cleavage site), and two reverse primers, PNP(R1) (5'-TTTACAAGCTTTCGCGGAAGCTCG-3', with a HindIII cleavage site) and PNP(R2) (5'-TTTACGAATTCCTCGCGGAAGCTCG-3', with an EcoRI cleavage site), were designed based on the sequence of the GIV genome. Restriction enzyme cleavage sites are underlined. PCR was carried out under the following conditions: 5 min at 95 °C for 1 cycle; 1 min at 94 °C, 2 min at 55 °C and 3 min at 72 °C, for 35 cycles; 10 min at 72 °C for 1 cycle. The amplified fragments were digested by the designed restriction enzymes and subcloned into vectors that were previously cleaved by the same enzymes, except for pEGFP-N1, which was digested by BglII and EcoRI. PNP gene fragments, amplified by paired primers PNP(F)/PNP(R1), PNP(F)/PNP(R2), PNP(F)/PNP(R1) and PNP(F)/PNP(R2), were subcloned into vectors pBluescript SK() (Stratagene), pSecTag2B (Invitrogen), pcDNA3.1() (Invitrogen) and pEGFP-N1 (Clontech), respectively, to obtain plasmids pBluescript/PNPi, pSecTag2/PNPi, pcDAN3.1/PNPi and pEGFP-N/PNPi. These constructs were confirmed by restriction enzyme digestion and nucleotide sequence analysis.
Sequence and phylogenetic analysis.
Database similarity searches were performed by using the National Center for Biotechnology Information (NCBI) BLAST server (Altschul et al., 1997). Sequence alignment and phylogenetic analysis were carried out by using the BioEdit Sequence Alignment Editor (North Carolina State University) and CLUSTAL_X (Thompson et al., 1997
) programs, respectively.
Northern blot hybridization.
Northern blot hybridization was performed with 10 µg total RNA collected from GIV-infected GK cells at an m.o.i. of 10. Total RNA was extracted with RNAzol B reagent (TEL-TEST) following the manufacturer's instructions. RNA samples were separated on a 1·2 % formaldehyde agarose gel and transferred onto a Hybond-N nylon membrane (Amersham Biosciences). The membrane was hybridized at 42 °C overnight with a [32P]UTP-radiolabelled antisense PNP RNA probe, which was synthesized by T7 RNA polymerase from the BamHI-digested plasmid pBluescript/PNPi. After hybridization, the membrane was washed with a solution containing 0·1 % SDS and 0·1x SSC and exposed to Biomax X-ray film (Kodak) for signal detection. Control RNA was collected from mock-infected GK cells after 20 h.
Coupled in vitro transcription/translation reaction.
Plasmids pcDNA3.1/PNPi and pSecTag2/PNPi, containing the complete GIV PNP gene, were constructed for the coupled in vitro transcription/translation reaction in a cell-free system with rabbit reticulocyte lysate. Both autoradiography and immunoblot analyses were performed to detect GIV PNP protein expression. For autoradiography, template plasmids and [35S]methionine were added to TNT Quick Master Mix (Promega). The mixture was incubated at 30 °C for 90 min, followed by 10 % SDS-PAGE separation. Signals were detected by autoradiography. For immunoblot analysis, the same procedure was followed, but the mixture was incubated with cold methionine. After incubation, the reaction mixture was subjected to 12 % SDS-PAGE and transferred onto a Hybond-P membrane (Amersham Biosciences). The membrane was incubated with 5 % non-fat dry milk in TBST (25 mM Tris-buffered saline, 0·1 % Tween 20) at 4 °C overnight, then hybridized with a 1 : 5000-diluted anti-Myc antibody (Invitrogen) at room temperature for 1 h. After TBST washes, the membrane was incubated with 1 : 2000-diluted alkaline phosphatase (AP)-conjugated goat anti-mouse immunoglobulins (DAKO) at room temperature for 1 h. Signals were visualized by 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro-blue tetrazolium chloride (NBT) mixture staining.
Western blot hybridization.
Western blot hybridization was performed with lysates that were collected from GIV-infected GK cells at an m.o.i. of 10. At 2 h intervals, the infected cells were harvested, rinsed with cold PBS and lysed in 2x SDS/sample buffer [100 mM Tris/HCl (pH 6·8), 200 mM dithiothreitol, 4 % SDS, 0·2 % bromophenol blue, 20 % (v/v) glycerol]. Cell lysates were subjected to 12 % SDS-PAGE and transferred onto a Hybond-P membrane (Amersham Biosciences). The membrane was incubated with 5 % non-fat dry milk in TBST at 4 °C overnight, then hybridized with a 1 : 3000-diluted anti-GIV PNP antiserum (J.-W. Ting & C.-Y. Chang, unpublished results) at room temperature for 1 h. After TBST washes, the membrane was incubated with 1 : 2000-diluted AP-conjugated goat anti-mouse immunoglobulins at room temperature for 1 h. Signals were detected by BCIP/NBT mixture staining. The control sample was collected from mock-infected GK cells after 24 h.
EGFP fusion protein expression in cells.
GK and HeLa cells were cultured in a six-well multidish (NUNC) overnight and transfected with plasmid pEGFP-N/PNPi by LipofectAMINE Plus (Invitrogen) following the manufacturer's instructions. After 24 h incubation, the transfected cells were observed to assess the expression of green fluorescence under a fluorescence microscope (IX 70; Olympus).
Immunocytochemistry.
GK cells were cultured on an eight-well Lab-Tek II chamber slide (NUNC) overnight and infected with GIV at an m.o.i. of 0·1 for 8 h. The infected cells were fixed in 4 % paraformaldehyde/0·1 % Triton X-100 for 30 min on ice. After cold PBS washes, the fixed cells were incubated with a 1 : 100-diluted anti-GIV PNP antiserum at room temperature for 1 h. The cells were then washed with PBS four times and incubated with 1 : 1000-diluted AP-conjugated goat anti-mouse immunoglobulins at room temperature for 1 h. Signals were visualized by BCIP/NBT mixture staining.
Enzymic activity analysis.
The reaction mixture (100 µl) contained 20 mM Tris/HCl (pH 7·5), 1 mM MgCl2, 0·5 mM KH2PO4, 10 µg purified recombinant GIV PNP protein (J.-W. Ting & C.-Y. Chang, unpublished results) and 1 µg tested purine (guanosine, adenosine and inosine) or pyrimidine (cytidine and uridine). Reactions were performed at room temperature for 30 min. The enzymic activity of GIV PNP was determined from disappearance of substrates and appearance of products, which were monitored by HPLC as described previously (Lecoq et al., 2001b) by using a Vydac 218TP C18-RP HPLC column at 260 nm.
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RESULTS |
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Subcellular localization of GIV PNP
To detect the subcellular localization of GIV PNP, GK cells were infected with GIV. At 8 h p.i., the cytopathic effect resulted in rounded, granular and refractile cells and the intercellular space was wider. Results of immunocytochemistry showed that a positive, dark-purple colour appeared in the cytoplasm of GIV-infected cells (Fig. 5a), whereas uninfected cells were morphologically normal and no specific signal was detected (Fig. 5b
).
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Enzymic assay of GIV PNP
To determine whether GIV PNP was functional, the recombinant GIV PNP protein was prepared in an E. coli expression system. Purified recombinant GIV PNP was assayed for its substrate specificity and ability to catabolize various purine and pyrimidine nucleosides. Results showed that GIV PNP could accept guanosine as its substrate and convert it to guanine (Fig. 6). The retention times of guanine and guanosine in HPLC were 5·1 and 13·4 min, respectively. Inosine and adenosine were also candidate substrates of GIV PNP. Although significant disappearance of substrates was observed, the corresponding products could not be detected in this system. Among the different substrates tested, GIV PNP showed the best metabolic activity on guanosine (31·2 %) and adenosine (21·2 %), and was less efficient on inosine (6·7 %). Pyrimidine nucleosides, including cytidine and uridine, were not metabolized by GIV PNP (data not shown).
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DISCUSSION |
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The complete genome sequences of several iridoviruses, such as fish lymphocystis disease virus (LCDV) (Tidona & Darai, 1997), Chilo iridescent virus (CIV) (Jakob et al., 2001
), mandarin fish infectious spleen and kidney necrosis iridovirus (ISKNV) (He et al., 2001
), tiger frog virus (TFV) (He et al., 2002
) and Ambystoma tigrinum virus (ATV) (Jancovich et al., 2003
), have been published. Although their putatively encoded proteins are not always the same, no PNP gene is found in any of these genomes. The presence of a PNP gene in GIV is therefore surprising. We propose the host and tissue specificity of GIV as possible reasons for the presence of GIV PNP. Viruses of the family Iridoviridae infect a wide range of hosts, both invertebrate and vertebrate (Williams, 1996
). In fish, outbreaks of iridovirus infection have been reported in salt-water species (Inouye et al., 1992
; Chou et al., 1998
; Jung & Oh, 2000
; Weng et al., 2002
; Chen et al., 2003
), freshwater species (Whittington et al., 1994
; McGrogan et al., 1998
; He et al., 2000
) and tropical freshwater ornamental fish (Sudthongkong et al., 2002
). If it is assumed that PNP's role is in the salvage pathway of purine substrates, it could be proposed that the host of GIV, the grouper, might have a nucleotide metabolism pathway that does not satisfy the purine supply for GIV; therefore, GIV has to carry its own PNP to fulfil its specific needs. Tissue specificity could be another possible reason. In histopathological studies, necrosis and enlargement of cells are commonly observed in the spleen and kidney of iridovirus-infected fish, and aggregation of virus particles in the cytoplasm of spleen and kidney cells is also detected by electron microscopy (He et al., 2000
; Jung & Oh, 2000
; Chen et al., 2003
). GIV, isolated from the spleen of iridovirus-infected yellow grouper (Lai et al., 2000
), propagates with a higher titre in GK cells than in others (Lai et al., 2003
). As spleen and kidney are haemopoietic organs in fish, it is assumed that PNP in these tissues is extremely active and that the nucleotide metabolism in these organs may affect iridovirus activity. Propagation in these organs might have made it easier for an ancestor of GIV to acquire the cellular PNP gene from its host through reverse transcription and recombination during evolution. However, further studies are needed to answer the question of whether the presence of GIV PNP is beneficial to GIV replication.
It is important for a large DNA virus (e.g. iridovirus), which replicates in the cytoplasm of the host cell, to acquire enough nucleic acid substrates for replication and transcription. To overcome this problem, most large DNA viruses gain cellular genes encoding enzymes that are involved in nucleic acid metabolism from their host during evolution (Tidona & Darai, 2000). These enzymes include two subunits of ribonucleotide reductase, thymidine kinase, thymidylate synthase, adenosine triphosphatase (ATPase), deoxyuridine triphosphatase (dUTPase) and nucleoside triphosphatase I. Ribonucleotide reductase is common in large, cytoplasmic DNA viruses. It is a central enzyme in DNA synthesis and catalyses the reductive synthesis of deoxyribonucleotides from the corresponding nucleoside diphosphates (Breidbach et al., 2000
). Thymidine kinase is reported in herpesviruses (Chen et al., 1998
) and poxviruses (Beaud, 1995
). The herpes simplex virus type 1-encoded thymidine kinase has multiple kinase activities that are usually performed by separate human enzymes. Its broad substrate specificity makes it an important target for the basis of cancer therapeutic drug design (Boehmer & Lehman, 1997
). Putative thymidylate synthases have been reported in TFV and CIV, whose sequences are conserved from bacteria to humans (He et al., 2002
). It is involved in the reductive methylation of deoxyuridylate (dUMP) to thymidylate (dTMP) and provides the only de novo source of dTMP (Liu et al., 2002
). In the case of ribonucleotide reductase, as some large DNA viruses, such as betaherpesviruses, only encode an ORF for one subunit of this enzyme (Lembo et al., 2004
), and some viruses, such as fowlpox virus, might have lost this gene during evolution (Binns et al., 1992
), it is suggested to be a non-essential gene product. PNP is not present in other iridoviruses that have been reported so far, and it could be possible that PNP may not be an essential enzyme for other iridoviruses, but may have some function that is specific to GIV. To determine whether the PNP gene is essential or non-essential to GIV, the investigation of a GIV PNP-deletion mutant is suggested.
PNPs are grouped into low- and high-molecular-mass categories. No significant sequence similarity has been observed between these two PNP classes, but they catalyse virtually the same chemical reactions (Bzowska et al., 2000). They differ from each other in the structure of the molecule, molecular mass of the monomer and substrate specificity. GIV PNP resembles the low-molecular-mass PNPs in both size and sequence. Phylogenetic analysis showed that GIV PNP was more closely related evolutionarily to mammalian PNPs (Fig. 2b
). Mammalian PNPs are also classified in the low-molecular-mass PNP group. They are homotrimers with a molecular mass of approximately 80100 kDa. Their natural substrates are guanosine and inosine. Although GIV PNP, in this study, exhibited an enzymic activity to catalyse guanosine and inosine, like human PNP, it also catalysed adenosine, a natural substrate of bacterial PNPs. The difference in substrate specificity from that of mammalian PNPs may be due to the substitution of Lys244 with Val241 at the base active site of GIV PNP (Table 1
).
In the past decade, based on the differences in substrate specificity between PNPs from E. coli and humans, an anti-tumour suicide-gene approach, GDEPT, has been developed. As E. coli, but not mammalian, PNPs can accept adenosine as a substrate, the E. coli PNP gene, which could be introduced into and allowed to express in human tumours, converts a relatively non-toxic prodrug, such as 6-methylpurine-2'-deoxyriboside (an adenosine analogue), to a potent cytotoxic agent (6-methylpurine) and the E. coli PNP-transfected tumour cells, as well as the bystander cells, will be killed (Parker et al., 1997; Hughes et al., 1998
; Voeks et al., 2002
; Gadi et al., 2003
). As GIV PNP has the ability to catalyse adenosine phosphorolysis, like E. coli PNP, we suggest that it could be a promising alternative target for the development of GDEPT. Furthermore, PNP inhibitors have been applied to suppress T-cell proliferative diseases and rejection of organ transplants (Bantia et al., 2001
). Some PNP inhibitors have also been developed as antiparasitic drugs, as PNP inhibition leads to death of the parasite from lack of purine supply (Alvarez et al., 2002
; Kicska et al., 2002a
, b
). Therefore, it is possible that PNP inhibitors may have an effect on the control and treatment of iridoviral infection in fish.
In conclusion, a novel virus PNP gene has been cloned and characterized from grouper iridovirus. The amino acid sequence and phylogenetic analyses indicated that it is a mammalian PNP homologue. Comparison of the predicted amino acid active sites also revealed significantly high conservation between the human and GIV PNPs, suggesting that GIV PNP could play a role similar to that of the human PNP. GIV PNP is a non-structural protein that could not be detected in purified virions (data not shown) and is expressed only in the cytoplasm of host cells after infection. HPLC analysis revealed that this protein is a functional enzyme in purine nucleoside metabolism. Further studies that are currently in progress may provide essential information that is necessary for the control of iridovirus infection and to assess its potential as an anti-tumour application.
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ACKNOWLEDGEMENTS |
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Received 3 May 2004;
accepted 7 July 2004.