Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein.

Abstract

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

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Rana grylio virus thymidine kinase gene: an early gene
of iridovirus encoding for a cytoplasmic protein
Zhe Zhao Æ Æ Fei Ke Æ Æ Yan Shi Æ Æ Guang-Zhou Zhou Æ Æ
Jian-Fang Gui Æ Æ Qi-Ya Zhang
Received: 28 May 2008/Accepted: 18 December 2008/Published online: 6 January 2009
? Springer Science+Business Media, LLC 2009
Abstract
feature of many large DNA viruses. Here, a TK gene
homologue was cloned and characterized from Rana grylio
virus (RGV), a member of family Iridoviridae. RGV TK
encodes a protein of 195 aa with a predicted molecular
mass of 22.1 kDa. Homologues of the protein were present
in all the currently sequenced iridoviruses, and phyloge-
netic analysis showed that it was much close to cellular TK
type 2 (TK2), deoxycytidine kinase (dCK) and deoxygua-
nosine kinase (dGK). Subsequently, Western blotting
revealed TK expression increased with time from 6 h post-
infection in RGV-infected cells. Using drug inhibition
analysis by protein synthesis inhibitor (cycloheximide) and
DNA replication inhibitor (cytosine arabinofuranoside),
RGV TK was classified as the early expression gene during
in vitro infection. Subcellular localization by TK–GFP
fusion protein expression and immunofluorescence staining
showed RGV TK was an exclusively cytoplasmic protein
in fish cells. Collectively, current data indicate that RGV
TK was an early gene of iridovirus which encoded a
cytoplasmic protein in fish cells.
The presence of thymidine kinase (TK) is a
Keywords
Thymidine kinase ? Early viral gene ? Cytoplasmic protein
Rana grylio virus (RGV) ? Iridovirus ?
Introduction
Iridoviruses are large DNA viruses that replicate in the
cytoplasm of infected cells [1]. The viral genome is both
circularly permutated and terminally redundant and is a
double-stranded DNA genome ranging from 103 to 212 kbp
in length [2]. Based on the Eighth Report of the Interna-
tional Committee on Taxonomy of Virus (ICTV), the
family Iridoviridae has been subdivided into five genera:
Iridovirus, Chloriridovirus, Ranavirus, Lymphocystisvirus,
and Megalocytivirus [3]. To date, 12 genome sequences of
iridoviruses including at least one from each genus have
been completely sequenced and analysed [2, 4–14]. These
analyses show that iridoviruses encode a number of cellular
protein homologues that may be mostly involved in nucleic
acid metabolism, such as ribonucleotide reductase (RR),
thymidylate synthase (TS), deoxyuridine triphosphatase
(DUT), and purine nucleoside phosphorylase (PNP) [15].
The presence of the cellular homologue genes may be of
significant advantage for iridoviruses that independently
replicate in the cytoplasm of the host cell [16].
TK is also a key nucleotide metabolism enzyme that
catalyses the phosphorylation of thymidine to thymidine
monophosphate (dTMP) and plays a role in the salvage
pathway of pyrimidine synthesis [17]. The enzyme has been
intensively studied over the past few decades because of its
roles in cell proliferation, apoptosis and antiviral drug
activation [18]. TK is not only widely distributed in
eukaryotes and prokaryotes, but also present in a number of
large DNA viruses, such as herpesviruses, poxviruses, asf-
aviruses, and whispoviruses [19–22]. However, viral TKs
also belong to different subfamilies based on the sequence
homology and substrate specificity [19, 20, 23]. For
example, herpesvirus TKs have relatively broad substrate
specificity such that they can phosphorylate deoxycytidine
The sequence reported here has been deposited in the GenBank
database under accession no EU747722.
Z. Zhao ? F. Ke ? Y. Shi ? G.-Z. Zhou ? J.-F. Gui ?
Q.-Y. 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
e-mail: zhangqy@ihb.ac.cn
123
Virus Genes (2009) 38:345–352
DOI 10.1007/s11262-008-0318-x

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(dC) [24] and thymidine monophosphate (TMP) [25]. The
herpesvirus TKs can be considered functionally equal to the
cellular TK2 and dCK. By contrast, poxvirus and asfavirus
TKs are very similar to mammalian TK1: they have a strict
substrate specificity for dT [26, 27]. It has been reported that
viral TKs are responsible for virus-efficient replication in
some specific cell type, virus virulence, pathogenesis and
reactivation from latency [20, 28]. As for the iridoviruses,
TK is conserved within the genome sequences of all the
sequenced members in the family Iridoviridae and belongs
to the core genes of iridoviruses [15]. Furthermore, TK
activity has also been shown in Bohle iridovirus [23].
However, no work has been carried out on other aspects of
iridovirus TK, such as temporal expression pattern and
subcellular localization.
Rana grylio virus (RGV) is a pathogen of cultured pig
frog (Rana grylio) and bears significant similarity to frog
virus 3 (FV3), the type species of the genus Ranavirus in
the family [29–31]. Recently, individual proteins of RGV
have been investigated to understand their roles in virus
infection [32–34]. As a continuing effort, here, we cloned
the TK from RGV genome and further characterized its
feature in cultured fish cells.
Materials and methods
Cells and virus
Fathead minnow (FHM) and grass carp ovary (GCO) cells
were cultured in TC199 medium supplemented with 10%
fetal bovine serum (FBS) at 25?C. RGV was propagated as
described previously [29].
Gene cloning and computer-assisted analysis
According to the conserved TK sequences of FV3 [10], a
pair of primers (50-GTCACTCTCGGTAAAGGAAC-30;
50-GAATAGACAACACAAGACGC-30) located in the 50
and 30flanking regions of the TK ORF was designed and
used to amplify RGV TK from the genomic DNA. The
fragment was cloned and sequenced, and the sequence data
were compiled and analysed using DNASTAR software.
The nonredundant protein sequence database of the
National Center for Biotechnology Information (National
Institutes of Health, MD, USA) was searched using
BLASTP, and iterative searches were performed using PSI-
BLAST [35]. Multiple sequence alignments were con-
structed using ClustalX 1.83 and edited using GeneDoc.
Phylogenetic analysis was done using MEGA4.0 by the
neighbor-joining (NJ) method with Poisson correction for
distance calculation [36].
Prokaryotic expression, protein purification and
antibody preparation
The TK ORF was amplified from RGV genomic DNA
using primers (50-GTCGAATTC ATGAGCATCCCTAC
AGT-30;50-GTCAAGCTTCGTACCGCACATTTC-30)
and ligated into the prokaryotic vector pET-32a (Novagen).
The recombinant plasmid, named pET32a-TK, was trans-
formed into Escherichia coli BL21 (DE3), and the bacteria
were induced for 5 h by 1 mM IPTG at 37?C to express the
fusion protein. The fusion protein was purified from
inclusion bodies under denaturing conditions using a His-
Bind Purification Kit (Novagen), mixed with an equal
volume of Freund’s adjuvant (Sigma) and used immunize
mice by hypodermal injection once every 7 days. After the
fifth immunization, anti-TK serum was collected and tested
by Western blotting with lysates from virus-infected cells.
Western blotting and drug inhibition assay
Total protein was isolated from GCO cells infected with
RGV at an m.o.i. of one at various times post-infection
(p.i.) (2, 4, 6, 8, 12, 16, 24, 36 and 48 h) or mock infected,
and was subjected to Western blot analysis as described
previously [31]. Anti-TK serum was used as the primary
antibody at a dilution of 1:500, followed by alkaline
phosphatase-conjugated goat anti-mouse IgG (H?L) anti-
body at a dilution of 1:1000 (Vector laboratories inc) as
the secondary antibody. Internal control was carried out
simultaneously by detecting cellular b-actin protein.
Cycloheximide(CHX),asproteinsynthesisinhibitor,and
cytosine arabinoside (AraC), as DNA replication inhibitor,
were used to classify the transcription class of RGV TK.The
treatment and RT-PCR analysis was performed according to
the previous study [33]. The specific primers TK-F/TK-R
(TK-F, 50-ACAGTCATAGCGTTTAGCG-30; TK-R, 50-
ACAGTCATAGCGTTTAGCG-30) were used to detect
RGVTKtranscripts.Ascontrol,twopairsofprimers,ICP-F/
ICP-R (ICP-F, 50-AAGCCTACCTGTGCGACTC-30; ICP-
R,50-CCGTCAGTCTCCAGGTTTT-30)andMCP-F/MCP-
R (MCP-F, 50-GACTTGGCCACTTA TGAC-30; MCP-R,
50-GTCTCTGGAGAAGAAGAA-30), were used to detect
the transcripts of the known immediate-early (IE) gene,
ICP18gene[37],andlate(L)gene,majorcapsidproteingene
(MCP) [38], respectively.
Subcellular localization
The subcellular localization of RGV TK was performed by
GFP fusion protein expression and immunofluorescence
staining. For GFP fusion protein expression, a recombinant
eukaryotic vector pEGFP-TK was constructed by cloning
the entire TK ORF into pEGFP-N3 (Clontech) using
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primers
50-TCTGGTACCCGATATTTGCCGCACA-30). This vec-
tor expressed RGV TK with a C-terminal fusion green
fluorescent protein (GFP) tag. Plasmid pEGFP-TK was
transiently transfected into GCO and FHM cells, and the
cells were fixed at 48 h post-transfection and stained with
propidium iodide (PI, Sigma) as described previously [39].
For immunofluorescence localization, all performances
were done according to the previous study [33]. Anti-TK
serum was used as the primary antibody at a dilution of
1:100, followed by FITC-conjugated goat anti-mouse
antibody at a dilution of 1:50 (Pierce) as the secondary
antibody. All the samples were examined under a Leica
DM IRB fluorescence microscope.
(50-TCTAAGCTTCAATGAGCATCCCTAC-30;
Results
Identification and sequence analysis of RGV TK
Using the designed primers, a 649-bp fragment was
amplified from the RGV genome. Sequence analysis
revealed that this fragment contained the complete ORF of
RGV TK (GenBank accession no.EU747722). The ORF
was 588 bp and encoded a peptide of 195 aa with a pre-
dicted molecular mass of 22.1 kDa. Homologues of the
protein were present in all the sequenced iridoviruses to
date, and database searches showed it had the highest
identity to zebrafish TK type 2 (TK2) and deoxycytidine
kinase (dCK) among noniridoviruses using a position-
specific iterative BLAST search. An alignment of amino
acid sequence of RGV TK with cellular TK2 and dCK is
shown in Fig. 1, and overall the proteins showed striking
similarity. Upon the alignment, it was also revealed that
RGV TK contained the conserved nucleotide binding
motif, but lacked the signal peptide targeting mitochondrial
at the N-terminus of TK2 (Fig. 1).
Phylogenetic analysis
To better understand the pattern of TK isoenzymes in
iridoviruses, and to compare it with the situation in other
large DNA viruses, a phylogenetic tree was constructed
with overall amino acid sequences of 40 TK isoenzymes
from viral and cellular sources. As shown in Fig. 2, RGV
TK was grouped together with that of FV3 with a 100%
bootstrap, and they were further clustered with other
members of iridovirus. Moreover, iridovirus TKs were
clustered within a monophyletic clade of cellular TK2,
dCK, and deoxyguanosine kinase (dGK) kinases, and
appeared more closely related to them than to the cellular
TK1 kinases. In contrast, poxvirus and asfavirus TKs
appeared to associate more closely with the cellular TK 1
kinases.Figure 3 showedthe deduced aminoacid
Fig. 1 Multiple alignments of
RGV TK amino acid sequences
with several typical cellular
TK2 and dCK from mammals
and fish Multiple sequence
alignments are performed using
ClustalX 1.83 and edited using
GeneDoc. Amino acid
numbering is shown to the right.
Sequence gaps are represented
by dashes. Nucleotide binding
motif is highlighted in black
box. Invariant residues are
marked by black background
while the highly conserved
residues (more than 80%
identity) are indicated over a
gray background. Abbreviations
and accession numbers are
following: Homo sapiens,
HsTK2 (O00142) and HsdCK
(P27707); Mus musculus,
MmTK2 (NP_066356) and
MmdCK (NP_031858); Danio
rerio, DrTK2 (NP_001002743)
and DrdCK (NP_001014374)
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sequences of representative species from each genus of the
family Iridoviridae. Within the iridovirus family, the RGV
TK has the highest identity with FV3 TK, while ISKNV
TK appears the most divergent with respect to size and
nucleotide-binding motif sequence. The conserved nucle-
otide-binding motif (GXXXXGK) near the N-terminus of
the protein is boxed, and the consensus amino acid residues
are indicated by black background.
Expression and purification of RGV TK in prokaryotic
system
To prepare anti-RGV TK serum, pET32a-TK was trans-
formed into Escherichia coli BL21 (DE3) and expression
of the fusion protein was induced. As shown in Fig. 4,
the fusion protein was approximately 42.1 kDa, which
included the RGV TK (22.1 kDa) and the Trx/His/S
tag (20.0 kDa) (Lane 2), whereas no protein band was
found at the same position of the non-induced pET32a-
TK/BL21 (Lane 1). The fusion protein was purified
using Ni-IDA affinity chromatography (Fig. 4, lane 3)
and used to generate anti-RGV TK polyclonal antibody
in mice.
Temporal expression pattern of RGV TK during
in vitro infection
The temporal expression pattern of RGV TK was charac-
terized during RGV infection by Western blot analysis. As
shown in Fig. 5a, a specific protein band was detected by
anti-TK serum from 6 h p.i in RGV infected cells and its
level increased to a high level at 48 h p.i. Its molecular
Lymphocystis disease virus 1 (NP_078725)
Mus musculus dCK (NP_031858)
Rattus norvegicus dCK (NP_077072)
Gallus gallus dCK (NP_001006451)
Homo sapiens dCK (P27707)
Danio rerio dCK (NP_001014374)
Gallus gallus dGK (NP_001072968)
Danio rerio dGK (NP_001093561)
Homo sapiens dGK (Q16854)
Mus musculus dGK (NP_038792)
Rattus norvegicus dGK (NP_001100072)
Gallus gallus TK2 (NP_001026631)
Danio rerio TK2 (NP_001002743)
Homo sapiens TK2 (O00142)
Mus musculus TK2 (NP_066356)
Rattus norvegicus TK2 (NP_001099636)
Invertebrate iridescent virus 6 (NP_149606)
Invertebrate iridescent virus 3 (YP_654601)
Rana grylio virus (EU747722)
Frog virus 3 (YP_031664)
Human herpes virus 8 (AAB08396)
Epstein-Barr virus (ABB89277)
African swine fever virus (NP_042744)
Fowlpox virus (P10052)
Swinepox virus (NP_570223)
Myxoma virus (NP_051775)
Sheeppox virus (NP_659638)
Yaba-like disease virus (NP_073451)
Vaccinia virus (YP_232976)
Gallus gallus TK1 (NP_990229)
Homo sapiens TK1 (P04183)
Mus musculus TK1 (NP_033413)
Rattus norvegicus TK1 (NP_434687)
Varicella zoster virus (P09250)
Human herpes virus 1 (AAF72495)
Human herpes virus 6 (BAF93475)
Human cytomegalovirus (P16788)
100
100
85
90
97
85
97
99
98
95
91
89
98
83
71
100
97
100
92
90
95
88
92
100
71
0.2
Infectious spleen and kidney necrosis virus (NP_612254)
Cellular
dCK
Cellular
dGK
Cellular
TK2
Iridovirus
γ
Herpesvirus
Asfavirus
Herpesvirus
Poxvirus
Cellular
TK1
α / β
Fig. 2 Phylogenetic tree
derived from 40 TK isoenzymes
representing the viral and
cellular sources Phylogenetic
tree was constructed using
MEGA4.0 by the neighbor-
joining (NJ) method with
Poisson correction for distance
calculation. GenBank accession
numbers of each sequence are in
parentheses. The numbers given
are frequencies (%) at which a
given branch appeared in 500
bootstrap replications and only
bootstraps[60% were shown
for convenience. RGV TK is
indicated by an arrow
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mass was about 22.1 kD, which corresponded to the the-
oretical molecular weight of RGV TK. No signal was
observed in the control sample (Mock) that was collected
after 48 h. As an internal control, the protein levels of
b-actinexpressionwereconsistent throughout the
experiment. The data suggest that RGV TK might be an
early (E) viral gene.
To further verify RGV TK as an E gene, CHX and AraC
inhibition assay was performed. As expected, the IE gene
ICP18 was expressed in all RGV-infected cell samples
regardless of the absence or presence of CHX and AraC,
whereas the transcription of L gene MCP was inhibited in
the presence of CHX and AraC, and was only presented
in the sample infected with RGV at 48 h p.i. (Fig. 5b). In
contrast, the TK transcript was detected in the AraC-treated
sample infected with RGV for 48 h, and its content is less
than that in the untreated sample, but not in the sample
treated with CHX infected with RGV for 6 h (Fig. 5b), thus
indicating that RGV TK is an E viral gene during in vitro
infection.
RGV TK is a cytoplasmic protein in fish cells
The intracellular localization of RGV TK in fish cells was
first investigated by detecting the fluorescence distribution
of TK-GFP. Figure 6a showed that strong green fluores-
cence was predominantly presented in the cytoplasm of
pEGFP-TK transfected GCO and FHM cells under fluo-
rescence microscope. As control, the vector-expressed
EGFP was distributed in both the cytoplasm and the
nucleus of the two fish cells (data no shown). In addition,
the cytoplasmic localization of RGV TK was also con-
firmed by immunofluorescence staining. As illustrated in
Fig. 3 Multiple sequence alignment of TK orthologs in iridoviruses.
RGV TK and one representative sequence from each genus of the
family Iridoviridae are included in the alignment. FV3, Frog virus 3;
LCDV-1, Lymphocystis disease virus 1; CIV, Chilo iridescent virus;
IIV-3, Invertebrate iridescent virus 3; ISKNV, Infectious spleen and
kidney necrosis virus; the black-shaded regions are completely
conserved while the grey-shaded regions are partially conserved with
more than 80% identity. GXXXXGK motif was the nucleotide-
binding region and boxed at the top of the alignment. Gaps introduced
to maximize alignment (dashes) and amino acid numbering is shown
to the right
kDa
116
66.2
45
35
25
18.4
M123
Fig. 4 SDS-PAGE of expressed and purified RGV TK protein
Escherichia coli BL21 (DE3) with plasmid pET32a-TK was induced
by IPTG to express RGV TK fusion protein, and the fusion protein
was purified using a HisBind Purification Kit. Lanes: 1, pET32a-TK/
BL21, non-induced; Lane 2, pET32a-TK/BL21, induced; Lane 3,
purified tagged RGV TK protein. The target protein is indicated by an
arrow. M, Protein molecular mass markers
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