Available online at www.sciencedirect.com

8) 118–126www.elsevier.com/locate/yviro

Virology 372 (200

Constitutive expression of thymidylate synthase from LCDV-C induces atransformed phenotype in fish cells

Zhe Zhao, Yan Shi, Fei Ke, Sun Wei, Jianfang Gui, Qiya Zhang ⁎

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences,Graduate School of Chinese Academy of Sciences, Wuhan 430072, China

Received 1 August 2007; returned to author for revision 3 September 2007; accepted 20 October 2007Available online 26 November 2007

Abstract

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumorformation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virusfrom China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fishcells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressedcells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4–5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth andcrisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fishcells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated withtransforming activity.© 2007 Elsevier Inc. All rights reserved.

Keywords: Thymidylate synthase; Lymphocystis disease virus from China (LCDV-C); Cell transformation; Foci formation; Anchorage-independent growth

Introduction

Thymidylate synthase (TS; EC 2.1.1.45) plays a key role inthe biosynthesis of thymidylate (dTMP) which catalyzes thereductive methylation of dUMP by transfer of a methylenegroup from CH2H4-folate to generate dTMP, which is furtherphosphorylated to dTTP, a direct precursor for DNA synthesis(Carreras and Santi, 1995). Because the TS-catalyzed enzymaticreaction provides the sole intracellular de novo source of dTMP,the inhibition of TS results in the cessation of cellular pro-liferation and growth (Navalgund et al., 1980; Liu et al., 2002).Consequently, it has been identified as an important target of avariety of anticancer drugs and high level expression is corre-

⁎ Corresponding author. Fax: +86 27 68780123.E-mail address: zhangqy@ihb.ac.cn (Q. Zhang).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.10.028

lated with poor prognosis in many human cancers (Bertino andBanerjee, 2003; DiPaolo and Chu, 2004).

Lymphocystis disease virus (LCDV) is the causative agent oflymphocystis disease, which is a chronic disease of fish char-acterized by the benign, tumor-like lesions (Chinchar et al.,2005; Williams et al., 2005). The disease affects over 100 dif-ferent wild and cultured fish species worldwide, causing seriouseconomic losses (Garcia-Rosado et al., 2004). Although greatadvances have been made in LCDV, the molecular mechanismof infection, replication and pathogenesis is not clearly knownbecause it lacks an efficient cell culture system for propagationof this virus (Tidona and Daral, 1999). Many attempts have beenmade to propagate the virus worldwide, but with limited success(Wolf et al., 1966; Walker and Hill, 1980; Perez-Prieto et al.,1999; Chang et al., 2001; Zhang et al., 2003). Thus, the study ofviral functional genes in vitro will be useful to elucidate themolecular mechanism of infection, replication and pathogenesisof LCDV. LCDV-C (LCDV isolated in China), a species of

Fig. 2. Confirmation for constitutive expression of LCDV-C TS in FHM cells by western blotting. (A) SDS–PAGE of expressed protein in pET28a-TS/BL21 (DE3).M: protein molecular weight marker; Lane 1: non-induced; Lane 2: induced. (B) Validation of anti-LCDV-C TS serum by western blotting. Lane 1: purified fusionprotein expressed in the induced pET28a-TS/BL21 (DE3). (C) Western blotting detection of LCDV-C TS constitutive expression by a polyclonal antiserum (left) andpre-adsorption experiment for proving the antiserum specificity (right). Lanes 1 and 3: FHM/pcDNA3.1; lanes 2 and 4: FHM/pcDNA3.1-TS. The internal control isshowed by detecting the cellular β-actin (D).

Fig. 1. Subcellular distribution of LCDV-C TS in FHM and CHSE-214 cells. (A) Two fish cells were transfected with pEGFP-N3-TS. Green fluorescence shows thelocalization of TS-EGFP fusion protein (left panel) and the red images show the localization of nucleus stained by PI (middle panel). (B) Two fish cells were transfectedwith empty vector pEGFP-N3. Green fluorescence shows the localization of EGFP protein (left panel) and the blue images show the localization of nucleus stained byDAPI (middle panel). Scale bars: 10 μm.

119Z. Zhao et al. / Virology 372 (2008) 118–126

120 Z. Zhao et al. / Virology 372 (2008) 118–126

lymphocystis disease virus, was sequenced and elucidated(Zhang et al., 2004). Genome analysis show that the openreading frame (ORF) 11L of LCDV-C encodes a potential TSgene (Zhao and Zhang, 2004; Zhang et al., 2004). In addition,the increasing numbers of TS have also been identified in otherlarge DNA viruses, such as Kaposi's sarcoma-associated her-pesvirus (KSHV), Melanoplus sanguinipes entomopoxvirus(MSEV), White spot syndrome virus (WSSV) and Chilo iri-descent virus (CIV) (Afonso et al., 1999; Jakob et al., 2001;Gaspar et al., 2002; Li et al., 2004), but until now, few data havebeen available about their subcellular location and biologicalfunction. So, in the present study, we presented the subcellularlocation of LCDV-C TS and its biological effect on cell cycleprogression and cellular transformation in fish cells.

Fig. 3. Cell cycle analysis by flow cytometry. (A) Cell proportion of FHM/pcDNA3.1(B) Comparison of G1/ (S+G2) ratio in FHM/pcDNA3.1-TS and control cells at 24analysis was performed by using the Student's t-test. Column bars with asterisks ar

Result

Subcellular location of LCDV-C TS

The distribution of LCDV-C TS in fish cells was investigatedby detecting the fluorescence distribution of TS-EGFP in FHMand CHSE-214 cells. Fig. 1A showed that strong green fluo-rescence was predominantly presented in the cytoplasm withgranular appearance in pEGFP-N3-TS transfected FHM orCHSE-214 cells under fluorescence microscope. As control, thevector-expressed EGFP was distributed in both the cytoplasmand the nucleus of the two fish cells (Fig. 1B). The resultindicated that LCDV-C TS was exclusively a cytoplasmicprotein in fish cells. Moreover, there was no difference in the

-TS and FHM/pcDNA3.1 cells in G0/G1, S and G2/M phases at 24, 36 and 48 h., 36 and 48 h. The data are expressed as means±standard error (SE). Statisticale significantly different with the corresponding controls at Pb0.05.

Fig. 4. Growth analysis and morphology of LCDV-C TS expressing cells. (A) Growth curves of FHM/pcDNA3.1-TS (circles) compared to FHM/pcDNA3.1 cells(squares); results shown represent the average from triplicate wells and error bars represent±SE. Statistical analysis was performed by using the Student's t-test(Pb0.05). (B) The morphology of FHM/pcDNA3.1-TS and FHM/pcDNA3.1 cells under phase contrast microscope. Scale bars: 100 μm.

121Z. Zhao et al. / Virology 372 (2008) 118–126

subcellular location of two fish cells, revealing that LCDV-C TSlocalization is independent of the cell type.

Constitutive expression of LCDV-C TS in fish cells

To prepare anti-LCDV-C TS serum, a strong band wasobserved in the induced pET28a-TS/BL21 (DE3) with mole-cular weight of about 37.5 kDa corresponding to His·Tag-TSfusion protein (Fig. 2A). The antiserum was generated byimmunizing rabbit with the fusion protein and western blottingshows that it could recognize the fusion protein (Fig. 2B). Toinvestigate the biological role of LCDV-C TS, FHM cells weretransfected with a TS expression plasmid (pcDNA3.1-TS) andstable G418-resistant clones were selected and pooled andsubsequently confirmed by western blotting analysis. As shownin Fig. 2C, a specific protein band about 32.7 kDa was detectedin stable LCDV-C TS tranfected cells, whereas there were noproducts in the control cells. Furthermore, the specificity of theantiserum was confirmed by a pre-adsorption experiment. Asshow in Fig. 2C, the same antiserum, adsorbed with the purifiedfusion protein, can only recognize a quite weak protein band instable LCDV-C TS tranfected cells. The data indicated thatLCDV-C TS has been constitutively expressed in the trans-fected FHM cells.

LCDV-C TS promotes cell cycle progression into S and G2/Mphase in fish cells

The effect of LCDV-C TS expression on cell cycle progres-sion was investigated by flow cytometry analysis. As shown inFig. 3A, there were no obvious differences in cell cycle distri-bution between the FHM/pcDNA3.1-TS and control cells at 24h. However, there was an obviously higher percentage of S andG2/M phase in the FHM/pcDNA3.1-TS cells compared withthe control cells at 36 h and 48 h, respectively (Fig. 3A). Forexample, the proportion of S phase and G2/M phases were15.4% and 26.3% in the FHM/pcDNA3.1-TS cells, whereas forthe control cells were only 5.4% and 14.9% at 36 h (Fig. 3A). Asa result, the total percentage of S and G2/M phase has asignificant increase in the FHM/pcDNA3.1-TS cells comparedwith the FHM/pcDNA3.1 (Pb0.05) (Fig. 3B). Therefore, thedata illuminated LCDV-C TS expression can promote cell cycleprogression into S and G2/M phase and consequently couldenhance proliferative ability of transfected fish cells.

LCDV-C TS enhances proliferation of transfected fish cells

Based on the above detections, the possibility that LCDV-CTS would have an effect on the growth of FHM cells was

122 Z. Zhao et al. / Virology 372 (2008) 118–126

investigated. Data in Fig. 4A showed that the number of FHM/pcDNA3.1-TS cells is significantly higher than that of controlcells (Pb0.05), demonstrating that constitutive expression ofLCDV-C TS enhanced the proliferation of FHM cells. Inaddition, the LCDV-C TS-expressed cells were morphologi-cally distinguishable from control cells, showing a small, slimand spindle shape and displayed multilayered growth (Fig. 4B).

LCDV-C TS induces foci formation and anchorage-independentgrowth in fish cells

To determine whether expression of LCDV-C TS results in atransformed phenotype of the FHM cells, we tested the ability ofFHM/pcDNA3.1-TS to form foci in monolayer cultures. The

Fig. 5. Focus assay. (A) Cellular foci were formed in LCDV-C TS expressing cells anlight microscopy. These cells lost contact inhibition and display multilayered growth a(C) Foci were quantified from triplicate wells and the data are presented as mean nuStudent's t-test (P b 0.001).

results in Fig. 5 showed that FHM cells with LCDV-C TSexpression gave rise to obvious foci 4–5weeks after inoculation,as compared with the occasional much smaller foci observed incells transfected with empty vector over the same period of time.The average number of foci was approximately 1270 in the TS-expressed cell pool from three independent experiments andis 140-fold higher than that in the control cells (Pb0.001)(Fig. 5C). Microscopic examination of the TS-induced focirevealed that those transfectants lost contact inhibition, resultingin piling up and crisscross morphology characteristic oftransformed cells (Fig. 5B).

Loss of anchorage dependence as measured by growth in softagar is also an important characteristic of the transformedphenotype. So we assessed the effect of LCDV-C TS expression

d visualized by staining with 0.4% crystal violet. (B) Magnification of foci undernd crisscross morphology characteristic of transformed cells. Scale bars: 100 μm.mbers of transformed foci±SE. Statistical analysis was performed by using the

Fig. 6. Anchorage-independent growth assay. (A) Growth of LCDV-C TS transformed cells in soft agar. Photograph of representative agar colonies from LCDV-C TSexpressing cells and vector control cells. Scale bars: 100 μm. (B) The colony forming efficiency of LCDV-C TS transformed cells in agar growth. The data arecalculated by counting colonies in agar from three independent experiments.

123Z. Zhao et al. / Virology 372 (2008) 118–126

on cell transformation using a soft agar colony formation assay.The result showed that the FHM/pcDNA3.1-TS cells hadacquired an anchorage-independent phenotype and could growand form large numerous colonies in 0.3% agar (Fig. 6A) whilethe control cells failed to produce any anchorage-independentcolonies and persisted as single cells over the same period oftime. The quantitative efficiency for transformation was ap-proximately 0.846% in soft agar by counting colonies with adiameter of N100 μm from triplicate plates (Fig. 6B). These datafrom both foci and soft agar assays suggest that constitutiveexpression of LCDV-C TS is sufficient to induce a transformedphenotype in fish cells in vitro.

Discussion

LCDV-C, a new species belonging to the genus Lympho-cystivirus, has the largest genome in vertebrate iridoviruses.Comparative genome analysis showed that there were signifi-cant differences between LCDV-C and LCDV-1 in gene content,gene organization and gene order (Tidona and Darai, 1997;Zhang et al., 2004). For example, some potential genes inLCDV-C were not found in LCDV-1, such as thymidylatesynthase (TS). Moreover, TS was not also present in the genomeof other vertebrate iridoviruses, except that tiger frog virus(TFV) and ambystoma tigrinum virus (ATV) have a shorthomologue sequence lacking the folate-binding site (He et al.,

2002; Jancovich et al., 2003, Zhao and Zhang, 2004; Williamset al., 2005; Eaton et al., 2007). Thus, it would be tempting tospeculate that TS may be related to some specific roles duringLCDV-C infection of cells.

Subcellular location of TS has been characterized by differenttechniques and cells and produced variable localization patterns,such as the nucleus, the cytoplasm or themitochondria (Johnstonet al., 1991; Samsonoff et al., 1997; Gribaudo et al., 2000). Inthis study, we revealed that LCDV-C TS had a predominantlygranular cytoplasmic appearance in fish cells by virtue oftransfecting with an expression vector carrying the LCDV-C TSgene. In addition, to avoid the bias of fish cells, we also observedthe same localization of LCDV-C TS in mammalian cells, babyhamster kidney cell (BHK) (data not shown). Previously,Cinquina et al. (2000) reported that a viral TS from Kaposi'ssarcoma-associated herpesvirus (KSHV) was also exclusively acytoplasmic protein.

TS have been studied extensively as an important target forcancer chemotherapeutic agents since it plays a critical role in cellcycle regulation and cell proliferation (Ju et al., 1999; Kastanoset al., 2001; Liu et al., 2002; Bertino and Banerjee, 2003). Morerecently, studies have documented that ectopic overexpression ofhuman TS not only can induce a transformed phenotype inmammalian cells in vitro, but also accelerate the development ofhyperplasia and tumors of the endocrine pancreas in vivo(Rahman et al., 2004; Voeller et al., 2004; Chen et al., 2007).

124 Z. Zhao et al. / Virology 372 (2008) 118–126

However, there were no reports concerning the biologicalfunction of viral TS. In this study, our current interest wasfocused on the transforming activity of LCDV-C TS in vitro, andwe cloned LCDV-C TS gene into an expression vectorpcDNA3.1, which is under the control of the strong eukaryoticCMVpromoter and developed a fish cell lines where LCDV-CTScan be constitutively expressed. The results demonstrated thatconstitutive expression of LCDV-CTS results in an increase in thepercentage of S and G2/M phase in transfected FHM cells andconsequently promotes cell proliferation faster comparedwith thecontrol cells. Moreover, the constitutive expression led cells toform foci in monolayer cultures and acquire an anchorage-independent growth. Although there is no corresponding fishmodel to test the ability of LCDV-C TS-transformants to formtumors in vivo like nude mice model system, constitutiveexpression of LCDV-C is sufficient to induce a transformedphenotype in fish cells in vitro as manifested by foci assay andsoft agar assay. Therefore, this works provide evidence as wellsupport the role of TS in cellular transformation from otherspecies besides human. To our knowledge, this is also the firstreport on viral TS sequence associated with transforming activityin fish transfected cell system.

Materials and methods

Plasmid construction

Three pairs of primers, P1/P2 (P1, 5′-AAGCTTTAAAAA-TGGCTAATGAA-3′, HindIII; P2, 5′-CTCGAGGGTTAAATA-GATAAATCTA-3′, XhoI), P1/P3 (P3, 5′-GGATCCAATAGATAAATCTAACGAG-3′ BamHI) and P4/P2 (P4, 5′-AAGCTT-GCCGCCACCATGGCTAATGAA-3′ HindIII), were used forplasmid construction. The entire LCDV-CTS gene, amplified fromLCDV-C genomic DNA using the above paired primers, wasrespectively subcloned into prokaryotic vector pET28a-c (+)(Novagen) and eukaryotic vectors pEGFP-N3 (Clontech) andpcDNA3.1 (+) (Invitrogen). These different constructs, namedpET28a-TS, pEGFP-N3-TS and pcDNA3.1-TS, were confirmed byrestriction enzyme digestion and DNA sequencing.

Prokaryotic expression, protein purification and antibodypreparation

The pET28a-TS plasmid was transformed into E. coli BL21(DE3), and the bacteria were induced for 4 h by 1mM IPTG at37 °C to express a fusion protein. The fusion protein present inthe inclusion body was purified under denatured conditionsusing HisBind Purification Kit (Novagen) and used to generatepolyclonal rabbit anti-LCDV-C TS serum (Zhao et al., 2007).The antiserum was validated by western blotting analysis andthe performance was done as described in the previous study(Zhang et al., 2006).

Cell culture and transfection

Fathead minnow (FHM) and Chinook salmon embryo(CHSE-214) cells were maintained at 25 °C in TC199 medium

supplemented with 10% fetal bovine serum (FBS). Forsubcellular location, FHM and CHSE-214 cells were grownto 90% confluence prior to transfection and then transientlytransfected with pEGFP-N3-TS or empty vector pEGFP-N3using the Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions. For stable transfection, plasmidpcDNA3.1-TS were transfected into FHM cells using theabovementioned method. After transfection for 48 h, transfectedcells were split 1:3 and grown for 4 weeks in 400 μg/ml of G418(Amresco) selection medium. The stable transfectants weretermed FHM/pcDNA3.1-TS and confirmed by western blottinganalysis using anti-LCDV-C TS serum. The control cells stablytransfected with pcDNA3.1 had been obtained in the previousstudy and were termed FHM/pcDNA3.1 (Huang et al., 2007).To prove the specificity of polyclonal anti-LCDV-C TS serum,the antiserum was pre-adsorbed against its antigen (purifiedfusion protein) for 1 h at room temperature and subsequentlyused in western blotting under the same conditions.

Subcellular localization

Transiently transfected cells were grown on glass cover-slips. After 48 h, cells with pEGFP-N3-TS were fixed with70% ethanol at −20 °C for 2 h and then stained with 4 μg/mlpropidium iodide (PI) (Sigma) for 1 h, whereas cells withpEGFP-N3 were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS) for 15 min and then stainedwith 4′,6-diamidino-2-phenylindole (DAPI) for 10 min.Green fluorescence displayed the distribution of the targetprotein, and the cell nucleus was indicated by the red fluo-rescence of PI or the blue fluorescence of DAPI. Fluorescencesignal was detected under a Leica DM IRB fluorescencemicroscope.

Cell cycle analysis

For cell cycle analysis, FHM/pcDNA3.1-TS and control cellswere passaged into 6-well plates in triplicate at a density of5×105 cells per well in TC199 containing 5% FBS. Aftercultured for 24 h, 36 h or 48 h, cells were respectively collectedand fixed in 70% ethanol overnight at −20 °C. Cells were thenpelleted, washed and resuspended in PBS for 1 h containing50 μg/ml PI and 100 μg/ml RNase A (Sigma). Flow cytometryanalysis was performed in Epics Altra flow cytometer (Beck-man Coulter, USA) and the cell cycle analysis was done usingthe cell quest program by manual setting regions for G0/G1, Sand G2/M phases. Data from 10,000 cells were collected foreach data file.

Cell proliferation assay

For generating growth curves, FHM/pcDNA3.1-TS andcontrol cells were seeded into 24-well plates at an initialdensity of 3×104 cells per well in TC199 containing 5% FBS.Cells were harvested daily, and the cell numbers were countedin triplicate with a hemacytometer under light microscope untilday 8.

125Z. Zhao et al. / Virology 372 (2008) 118–126

Foci and anchorage-independent growth assays

Foci assays were performed by plating FHM/pcDNA3.1-TScells onto six-well dishes in TC199 containing 5% FBS. Aftergrown to confluence, several drops of fresh medium were addedto the cells every 3 days for 4–5 weeks until cellular foci wereobserved under the light microscope. Foci were stained with0.4% crystal violet in 30% methanol for 10 min and then werewashed several times with distilled water and photographed.The number of cellular foci was counted from triplicate wells todetermine mean and standard error (SE).

Anchorage-independent growth was evaluated by colonyformation in semi-solid medium. FHM/pcDNA3.1-TSand control cells were suspended at 1.2×105 cells per 60-mmdishes in 3 ml of TC199 medium with 10% FBS and 0.3%melted soft agar onto a 3 ml bottom layer of 0.6% agar medium.The cells were fed every 3 days with several drops of medium,and the cell morphologywas photographed after 3–4weeks. Thecolony forming efficiency was calculated by counting coloniesin soft agar from three independent experiments.

Acknowledgments

Grateful thanks for Ms. Jing Zhang who provides the help offlow cytometry analysis. This work is supported by grants fromthe National Major Basic Research Program (2004CB117403),the National 863High Technology Research Foundation of China(2006AA09Z445, 2006AA100309 and 20060110A4013), theNational Natural Science Foundation of China (30671616 andU0631008) and the Project of Chinese Academy of Sciences(KSCX2-YW-N-021) and the Key Technologies R&D Programof China (2006BAD03B05).

References

Afonso, C.L., Tulman, E.R., Lu, Z., Oma, E., Kutish, G.F., Rock, D.L., 1999. Thegenome of Melanoplus sanguinipes entomopoxvirus. J. Virol. 73, 533–552.

Bertino, J.R., Banerjee, D., 2003. Is the measurement of thymidylate synthase todetermine suitability for treatment with 5-fluoropyrimidines ready for primetime? Clin. Cancer Res. 9, 1235–1239.

Carreras, C.W., Santi, D.V., 1995. The catalytic mechanism and structure ofthymidylate synthase. Ann. Rev. Biochem. 4, 721–762.

Chang, S.F., Ngoh, G.H., Kuch, L.F.S., Qin, Q.W., Chen, C.L., Lam, T.J., Sin,Y.M., 2001. Development of a tropical marine fish cell line from Asianseabass (Lates calcarifer) for virus isolation. Aquaculture 192, 133–145.

Chen, M., Rahman, L., Voeller, D., Kastanos, E., Yang, S.X., Feigenbaum, L.,Allegra, C., Kaye, F.J., Steeg, P., Zajac-Kaye, M., 2007. Transgenicexpression of human thymidylate synthase accelerates the development ofhyperplasia and tumors in the endocrine pancreas. Oncogene 26, 4817–4824.

Chinchar, V.G., Essbauer, S., He, J.G., Hyatt, A., Miyazaki, T., Seligy, D.,Williams, T., 2005. Iridovirade. In: Fauqet, C.M., Mayo, M.A.M.J.,Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy, VIIIth Report of theICTV. Elsevier/Academic Press, London, pp.145–162.

Cinquina, C.C., Grogan, E., Sun, R., Lin, S.F., Beardsley, G.P., Miller, G., 2000.Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus.Virology 268, 201–217.

DiPaolo, A., Chu, E., 2004. The role of thymidylate synthase as a molecularbiomarker. Clin. Cancer. Res. 10, 411–412.

Eaton, H.E., Metcalf, J., Penny, E., Tcherepanov, V., Upton, C., Brunetti, C.R.,2007. Comparative genomic analysis of the family Iridoviridae: re-annotating and defining the core set of iridovirus genes. Virol. J. 4, 11.

Garcia-Rosado, E., Castro, D., Cano, I., Alonso, M.C., Perez-Prieto, S.I.,Borrego, J.J., 2004. Protein and glycoprotein content of lymphocystis diseasevirus (LCDV). Int. Microbiol. 7, 121–126.

Gaspar, G., De Clercq, E., Neyts, J., 2002. Human herpesvirus 8 gene encodes afunctional thymidylate synthase. J. Virol. 76, 10530–10532.

Gribaudo, G., Riera, L., Lembo, D., De Andrea, M., Gariglio, M., Rudge, T.L.,Johnson, L.F., Landolfo, S., 2000. Murine cytomegalovirus stimulatescellular thymidylate synthase gene expression in quiescent cells and requiresthe enzyme for replication. J. Virol. 74, 4979–4987.

He, J.G., Lü, L., Deng, M., He, H.H., Weng, S.P., Wang, X.H., Zhou, S.Y., Long,Q.X., Wang, X.Z., Chan, S.M., 2002. Sequence analysis of the com-plete genome of an iridovirus isolated from the tiger frog. Virology 292,185–197.

Huang, Y.H., Huang, X.H., Zhang, J., Gui, J.F., Zhang, Q.Y., 2007. Subcellularlocalization and characterization of G protein-coupled receptor homologfrom lymphocystis disease virus isolated in China. Viral Immunol. 20,150–159.

Jakob, N.J., Muller, K., Bahr, U., Darai, G., 2001. Analysis of the first completeDNA sequence of an invertebrate iridovirus: coding strategy of the genomeof Chilo iridescent virus. Virology 286, 182–196.

Jancovich, J.K., Mao, J., Chinchar, V.G., Wyatt, C., Case, S.T., Kumar, S.,Valente, G., Subramanian, S., Davidson, E.W., Collins, J.P., Jacobs, B.L.,2003. Genomic sequence of a ranavirus (family Iridoviridae) associated withsalamander mortalities in North America. Virology 316, 90–103.

Johnston, P.G., Liang, C.M., Henry, S., Chabner, B.A., Allegra, C.J., 1991.Production and characterization of monoclonal antibodies that localizehuman thymidylate synthase in the cytoplasm of human cells and tissue.Cancer Res. 51, 6668–6676.

Ju, J., Pedersen-Lane, J., Maley, F., Chu, E., 1999. Regulation of p53 expressionby thymidylate synthase. Proc. Natl. Acad. Sci. U. S. A. 96, 3769–3774.

Kastanos, E.K., Zajac-Kaye,M., Dennis, P.A., Allegra, C.J., 2001. Downregulationof p21/WAF1 expression by thymidylate synthase. Biochem. Biophys. Res.Commun. 285, 195–200.

Li, Q., Pan, D., Zhang, J.H., Yang, F., 2004. Identification of the thymidylatesynthase within the genome of white spot syndrome virus. J. Gen. Virol. 85,2035–2044.

Liu, J., Schmitz, J.C., Lin, X., Tai, N., Yan, W., Farrell, M., Bailly, M., Chen, T.,Chu, E., 2002. Thymidylate synthase as a translational regulator of cellulargene expression. Biochim. Biophys. Acta 1587, 174–182.

Navalgund, L.G., Rossana, C., Muench, A.J., Johnson, L.F., 1980. Cell cycleregulation of thymidylate synthetase gene expression in cultured mousefibroblast. J. Biol. Chem. 255, 7386–7390.

Perez-Prieto, S.I., Rodriguez-Saint-Jean, S., Garcia-Rosaso, E., Castro, D.,Alvarez, M.C., Borrego, J.J., 1999. Virus susceptibility of the fish cell lineSAF-1 derived from gill-head seabream. Dis. Aquat. Org. 35, 149–153.

Rahman, L., Voeller, D., Rahman, M., Lipkowitz, S., Allegra, C., Barrett, J.C.,Kaye, F.J., Zajac-Kaye, M., 2004. Thymidylate synthase as an onco-gene: a novel role for an essential DNA synthesis enzyme. Cancer Cells 5,341–351.

Samsonoff, W.A., Reston, J., McKee, M., O'Connor, B., Galivan, J., Maley, G.,Maley, F., 1997. Intracellular location of thymidylate synthase and its stateof phosphorylation. J. Biol. Chem. 272, 13281–13285.

Tidona, C.A., Darai, G., 1997. The complete DNA sequence of lymphocystisdisease virus. Virology 230, 207–216.

Tidona, C.A., Daral, G., 1999. Lymphocystis disease virus (Iridoviridae). In:Granoff, A., Wevster, R.G. (Eds.), Encyclopedia Virology. Academic Press,New York, pp. 908–911.

Voeller, D., Rahman, L., Zajac-Kaye, M., 2004. Elevated levels of thymidylatesynthase linked to neoplastic transformation of mammalian cells. Cell Cycle3, 1005–1007.

Walker, D.P., Hill, B.J., 1980. Studies on the culture assay of infectivity andsome in vitro properties of lymphocystis virus. J. Gen. Virol. 51, 385–395.

Williams, T., Barbosa-Solomieu, V., Chinchar, V.G., 2005. A decade of advancesin iridovirus research. Adv. Virus Res. 65, 173–248.

Wolf, K., Gravell, M., Malsberger, R.G., 1966. Lymphocystis virus: isolationand propagation in centrarchid fish cell lines. Science 151, 1004–1005.

Zhang, Q.Y., Ruan, H.M., Li, Z.Q., Yuan, X.P., Gui, J.F., 2003. Infectionand propagation of lymphocystis virus isolated from the cultured

126 Z. Zhao et al. / Virology 372 (2008) 118–126

flounder Paralichthys olivaceus in grass carp cell lines. Dis. Aquat. Org. 57,27–34.

Zhang, Q.Y., Xiao, F., Xie, J., Li, Z.Q., Gui, J.F., 2004. Complete genome sequenceof lymphocystis disease virus isolated from China. J. Virol. 78, 6982–6994.

Zhang, Q.Y., Zhao, Z., Xiao, F., Li, Z.Q., Gui, J.F., 2006. Molecularcharacterization of three Rana grylio virus (RGV) isolates and Paralichthys

olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses. Aquaculture251, 1–10.

Zhao, Z., Zhang, Q.Y., 2004. Structure Analysis of thymidylate synthase genefrom LCDV-C. Virol. Sin. 19, 602–606.

Zhao, Z., Ke, F., Gui, J.F., Zhang, Q.Y., 2007. Characterization of an early geneencoding for dUTPase in Rana grylio virus. Virus Res. 123, 128–137.