1 Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, 178 Daxue Road, Xiamen 361005, China
2 Department of Biochemistry and Molecular Biology, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
Correspondence
Feng Yang
mbiotech{at}public.xm.fj.cn
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ABSTRACT |
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INTRODUCTION |
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Within the genome of WSSV, on the basis of the presence of highly conserved motifs, WSV067 encoding a putative thymidylate synthase (termed WSSV-TS) has been tentatively characterized (Yang et al., 2001). It is located at position 3109231958 bp in the genome and encodes a 289 aa protein (32·6 kDa).
Thymidylate synthase (TS) is essential for the de novo synthesis of deoxythymidine monophosphate (dTMP) in prokaryotic and eukaryotic organisms by catalysing the reductive methylation of 2'-deoxyuridylate (dUMP) by 5,10-methylenetetrahydrofolate to give dTMP and dihydrofolate. Consequently it plays a major role in the DNA replication of a cell or a DNA virus (Perryman et al., 1986) and has been used successfully as a therapeutic target for the treatment of proliferation diseases such as cancer (Danenberg, 1977
).
The catalytic mechanism of TS has been widely studied in the past, and much is known about the structure and function of the enzyme (Perry et al., 1990). In protozoans and plants, TS combines with dihydrofolate reductase (EC 1.5.1.3) to form a bifunctional dihydrofolate reductaseTS (DHFRTS). For DNA viruses, TS is only found in bacteriophage (Belfort et al., 1983b
; Kenny et al., 1985
), herpesvirus (Bodemer et al., 1986
; Richter et al., 1988
; Russo et al., 1996
) and three insect viruses including Chilo iridescent virus (Invertebrate iridescent virus 6; IRV6) (Muller et al., 1998
), Melanoplus sanguinipes entomopoxvirus (MSEV) (Afonso et al., 1999
) and Heliothis zea virus 1 (HzV-1) (Chen et al., 2001
).
In this work, homologous and phylogenetic analyses were performed to study the evolutionary relationship of WSSV-TS using known TS sequences in the SWISS-PROT database. The transcription and expression of WSSV-ts was identified with RT-PCR, rapid amplification of cDNA ends (RACE) and Western blot analyses. The WSSV recombinant TS protein (termed rTS) was expressed in Escherichia coli and was functionally identified by dUMPfolate-binding activity assay.
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METHODS |
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Homologous and phylogenetic analyses of WSSV-TS.
The amino acid sequences of TS from mammals, fungi, bacteria, protozoa and DNA viruses in the SWISS-PROT databases were used in homologous and phylogenetic analyses. The homologous analysis was performed using DNAMAN software (Lynnon BioSoft). Amino acid sequences of TS from human (TYSY_HUMAN), mouse (TYSY_MOUSE), rat (TYSY_RAT), human herpesvirus 8 (TYSY_KSHV), herpesvirus saimiri (TYSY_HSVSA), equine herpesvirus type 2 (TYSY_SHVE2), herpesvirus ateles (TYSY_SHVAT), varicella-zoster virus (TYSY_VZVD), fruitfly (TYSY_DROME), mushroom (TYSY_AGABI), baker's yeast (TYSY_YEAST), HzV-1 (TYSY_HzV-1), IRV6 (TYSY_IRV6), MSEV (TYSY_MSEV), Escherichia coli (TYSY_ECOLI), bacteriophage T4 (TYSY_BPT4), Crithidia fasciculata (DRTS_CRIFA), carrot (DRTS_DAUCA) and soybean (DRTS_SOYBN) were used in the multiple sequence alignment (Table 1).
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Transcriptional analysis of gene.
Total RNAs, extracted from the hepatopancreas of WSSV-infected crayfish at different times after infection (i.e. 0 to 72 h p.i.), were treated with DNase and reverse-transcribed. The cDNAs were subjected to PCR using ts-specific forward and reverse primers (5'-Ttaaccatcatcaatatg-3', 5'-cagcgattacaccatttctag-3'). The PCR cycles were as follows: 94 °C for 2 min, 30 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, followed by an elongation at 72 °C for 10 min. The crayfish -actin gene was used as the internal control for RT-PCR with a gene-specific primer set (5'-TCATCAGGGTGTGATGGT-3' and 5'-TCTGAGTCATCTTCTCAC-3'). Total RNA from healthy crayfish was used as the negative control.
RACE.
Based on the nucleotide sequence of WSV067, the 5' and 3' ends of the cDNA encoding WSSV-TS were obtained by 5'- and 3'-RACE using a commercial 5'/3'RACE kit (Roche), according to the manufacturer's recommendations. The RNA samples used in this study were extracted from WSSV-infected crayfish 24 h p.i. and then treated with RNase-free DNase. For 5'-RACE, the first strand cDNA was synthesized using the ts-specific primer TSsp1 (5'-CACAACTCCTCTCCAGAAAAT-3') and then a poly(A) tail was added to the cDNA products using terminal transferase in the presence of dATP. The primer TSsp2 (5'-GGTAGTGAGAACTGGAATAGT-3') and an oligo(dT) anchor primer supplied with the kit were used for PCR. For 3'-RACE, first-strand cDNA was synthesized using an oligo(dT) anchor primer. The primer TSsp3 (5'-GAGGGAGAACATCAATATTTG-3') and an anchor primer supplied with the kit were used for PCR. The PCR products from 5'- and 3'-RACE were each purified on a 1·5 % agarose gel and subcloned into the pMD18-T vector (TaKaRa). Arbitrarily selected clones were sequenced and compared with the genomic DNA sequence of WSSV.
Expression and purification of His-tagged WSSV-TS in E. coli.
The WSSV-ts gene was amplified from WSSV genomic DNA using the forward primer (5'-AGTCGGATCCGAGGGAGAACATCA-3') and the reverse primer (5'-ACTGAAGCTTCCTTAACATGATTC-3') that contained recognition sequences for BamHI and HindIII restriction enzymes (underlined). The amplicon was cloned into the pQE30 vector (Qiagen). The recombinant plasmid was transformed into E. coli BL21(DE3) cells. Liquid cultures were grown in a shaking incubator (200 r.p.m.) at 37 °C until the OD600 reached 0·5 and these were then induced with 0·5 mM IPTG for 8 h at 28 °C. The cells were harvested by centrifugation at 4000 g for 5 min. The recombinant WSSV-TS (termed rTS) was purified by NiNTA affinity chromatography under native conditions following methodology in the QIAexpressionist handbook (Qiagen). The E. coli cells containing pQE30 vector were also induced with IPTG and total protein extracts were applied to the NiNTA column as described above. Final eluates were collected and used as the negative control (termed NC).
Preparation of antibody.
The purified rTS was used as an antigen to immunize mice by intradermal injection once every 10 days. Antigen (100 µg) was mixed with an equal volume of Freund's complete adjuvant (Sigma) for the first injection. The subsequent three injections were conducted using 100 µg antigen mixed with an equal volume of Freund's incomplete adjuvant. Four days after the last injection, mice were exsanguinated and the antisera were collected. The antiserum titres were determined by ELISA using horseradish peroxidase-conjugated goat anti-mouse IgG (Promega). For a negative control, antigen was replaced with 1xPBS.
Western blot.
Total proteins, extracted from hepatopancreas of infected crayfish at various times (i.e. 0, 2, 4, 6, 12, 24, 48 and 72 h p.i.), were separated by SDS-PAGE. These proteins were transferred onto a PVDF membrane (Amersham Pharmacia). The membrane was then immersed in blocking buffer (2 % BSA, 20 mM Tris, 150 mM NaCl, 0·1 % Tween 20, pH 7·5) at room temperature for 30 min, followed by incubation with anti-rTS serum (diluted 1 : 2000) for 1 h. Following this, alkaline phosphatase-conjugated goat anti-mouse IgG (Promega) was used as the secondary antibody. Detection was performed using Western Blue Stabilized Substrate for Alkaline Phosphatase (Promega).
UV difference spectroscopy analysis.
This was performed as described (Lockshin et al., 1984). In brief, 5 µl of 0·424 mM dUMP was added to 500 µl buffer (50 mM TES pH 7·4, 25 mM MgCl2, 1·0 mM EDTA, 5·0 mM DTT) containing 5 µl purified rTS and 10 µl of 36·6 µM folate (Sigma). The reaction was conducted in a sample cuvette at room temperature in a spectrophotometer thermostatic chamber. The absorbance was recorded within the 250360 nm range and the final difference spectra, representing the rTSdUMPfolate complex, were obtained. In the negative control, the same dUMP dose was added to the sample cuvette containing the same buffer but substituting the NC fraction mentioned above.
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RESULTS |
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Expression of WSSV-ts in E. coli
For the convenience of protein purification and identification, full-length WSSV-TS was expressed in E. coli as a fusion protein with an N-terminal His tag. The induced (plus IPTG at 37 °C) and non-induced samples were analysed by 14 % SDS-PAGE (Fig. 4a). A band (about 32 kDa) corresponding to rTS protein was observed in the induced sample when compared to the sample without induction. The soluble rTS was purified by NiNTA affinity chromatography under native conditions, and was found to match the theoretical molecular mass of 32·6 kDa. Purified rTS was used for antibody preparation and the identification of function.
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To investigate expression of WSSV-ts in vivo, samples extracted at various times up to 72 h p.i. from hepatopancreas of infected crayfish were analysed by Western blotting. An apparent band (about 32 kDa) corresponding to the theoretical molecular mass of WSSV-TS (32·6 kDa) was observed (Fig. 4b). WSSV-TS was first detected at 6 h p.i., maximally at 12 h p.i. and remained fairly constant thereafter. This result, fairly consistent with that of RT-PCR, suggested that WSSV-ts was a genuine and early virus gene.
dUMPfolate-binding activity assay
As shown in Fig. 4(c), the UV difference spectra, which were obtained following addition of dUMP to a buffer containing rTS and folate, revealed two maximum absorbances at 263 nm and 322 nm and a minimum absorbance at 295 nm. These are characteristic of typical dUMPTSfolate complexes, and A322 increased linearly with the increased dUMP concentration. Since the function-related motifs in TS from different origins are all highly conserved, it was essential to rule out any influence of contamination from E. coli TS during rTS purification. In the negative control, only one maximum absorbance at 263 nm was detectable (Fig. 4d
) suggesting that there was no interference due to contaminating TS from E. coli and that the UV difference spectrum results were reliable. The kinetic data obtained by spectroscopic analysis indicated that the Km of dUMP was 4·7±0·5 µM and that the Km of folate was 7·6±0·3 µM.
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DISCUSSION |
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It has been reported that dUMP and folate are attached to the highly conserved TS binding sites to form a ternary complex during the catalytic reactions. The major amino acid residues for which a functional role has been reported are present within these binding sites. X-ray analysis of the TS crystal structure reveals that several key residues found in these motifs are essential for this reaction (Carreras & Santi, 1995). For instance in E. coli TS, the residues N177 and Y181, which are located in a consensus sequence PFNIASY of the dUMP-binding site (motif 3), are involved in determining pyrimidine specificity and in dUMP-binding activity (Hardy & Nalivaika, 1992
; Schiffer et al., 1995
). In addition, the aspartate residue (D221) in human TS, in the consensus sequence DMGLGVP in motif 3, is involved in folate cofactor binding and catalysis (Chiericatti & Santi, 1998
). By analysing the primary sequence of WSSV-TS, as shown in Fig. 1
, the same consensus regions (i.e. containing N201, Y205 and D193 residues) were also found in WSSV-TS. These highly conserved motifs and residues imply that WSSV-TS has a similar or even the same structure as other TS, and consequently make it possible for it to perform the same biochemical functions.
It has been reported that dUMP binding to, and folate cofactors of, TS cause a major conformational change that converts the enzyme from the open form to a closed form of the ternary complex and results in spectroscopic changes (Carreras & Santi, 1995) with maximum absorbance at
330 nm and
265 nm and minimum absorbance at
295 nm (Lockshin et al., 1984
). UV difference spectroscopic analysis using rTS revealed a maximum absorbance at 322 nm and a minimum absorbance at 295 nm, which corresponded to the characteristics of the ternary complex. This presumably reflected changes of chromophores of folate and TS. This difference spectrum suggested that the WSSV-TS protein had the capacity to form a ternary complex in the presence of dUMP and folate.
The TS of WSSV plays a key role in the virus dTTP synthetic system by providing sufficient dTMP for dTTP production. Consequently it has a close relationship with viral DNA replication and proliferation by regulating the balanced supply of dTTP for normal DNA metabolism in collaboration with dUTPase (WSV112), ribonucleotide reductase (WSV172) and thymidylate kinase (WSV395), which are all encoded by the WSSV genome and expressed at the early stage of infection (Tsai et al., 2000; Van Hulten et al., 2000
; our unpublished data). The identification of WSSV-TS thus provides us with a new research direction in the prevention and treatment of white spot syndrome disease.
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ACKNOWLEDGEMENTS |
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Received 19 February 2004;
accepted 11 March 2004.