Download Varicella-Zoster Virus Specifies a Thymidylate Synthetase

J. gen. Virol. (1987), 68, 1449 1455. Printedin Great Britain
1449
Key words: VZV/thymidylatesynthetase/gene conservation
Varicella-Zoster Virus Specifies a Thymidylate Synthetase
By R. T H O M P S O N , 1. R. W. H O N E S S , : L. T A Y L O R , 1 J. M O R R A N ~ AND
A. J. D A V I S O N l t
1MRC Virology Unit and Department of Virology, University of Glasgow, Church Street,
Glasgow G11 5JR and 2Division of Virology, National Institute for Medical Research, Mill Hill,
London NW7 1AA, U.K.
(Accepted 10 February 1987)
SUMMARY
A homology search of proteins predicted from the recently reported complete DNA
sequence of varicella-zoster virus (VZV) revealed that the product of gene 13 was
highly homologous to eukaryotic and prokaryotic thymidylate synthetases (TSs). The
VZV protein was shown to be a TS by three functional tests. Firstly, a plasmid designed
to express the native protein was able to complement a strain of Escherichia coil in
which the natural TS gene is deleted. Secondly, in an enzyme assay for TS, extracts of
the complemented strain were capable of releasing tritiated water from 2'-deoxy[53H]uridylate. Thirdly, these extracts contained a protein that bound isotopically
labelled 5-fluoro-2'-deoxyuridylate, a ligand specific for the active site of TS. In
addition, a novel ligand-binding protein was detected in human cells infected with
VZV.
Two common human diseases, chickenpox and shingles, are caused by varicella-zoster virus
(VZV). This virus is a member of the Alphaherpesvirinae, a subfamily of the Herpesviridae
typified by the most extensively studied human herpesvirus, herpes simplex virus type 1 (HSV1). Davison & Scott (1986) identified genes encoding 67 unique proteins from an analysis of the
complete sequence of the 125 kbp linear double-stranded DNA genome of VZV. Five
glycoprotein genes were proposed from considerations of primary amino acid sequence, and the
glycoprotein products of four have been detected (Ellis et al., 1985; Davison et al., 1985; Keller
et al., 1986, 1987). Functional assignments for 12 additional VZV proteins are dependent on
comparisons with available data for HSV-1 proteins of known function (Davison & Scott, 1986).
The functions of two of the remaining 50 VZV proteins may be proposed on the basis of
homology with non-herpesvirus proteins. One is related to several eukaryotic protein kinases
(McGeoch & Davison, 1986), and the other is homologous to prokaryotic and eukaryotic
thymidylate synthetases. In this paper, we describe the structural and functional identification
of the VZV thymidylate synthetase (TS) gene and the detection of a novel TS in VZV-infected
cells.
TS (5,10-methylenetetrahydrofolate : dUMP C-methyltransferase, EC 2.1.1.45) is a homodimeric enzyme which catalyses the reductive methylation of deoxyuridylate to thymidylate. As
the sole means for supplying thymidine nucleotides de novo, TS is crucial in DNA metabolism. A
particularly striking feature of this enzyme is its high degree of amino acid sequence
conservation. For example, the human and Escherichia coli proteins are 53% homologous
(Takeishi et al., 1985). A computer-aided homology search of the 67 VZV proteins revealed that
the product of gene 13 has a similar degree of homology to published TSs. The protein-coding
t Presentaddress: Laboratoryof Viral Diseases, National Institute of Allergyand InfectiousDiseases, National
Institutes of Health, Bethesda, Maryland 20892, U.S.A.
0000-7483 © 1987 SGM
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region of gene 13 extends from 18441 to 19343 bp in the VZV genome, and specifies a 301
residue protein with a molecular weight of 34531 (Davison & Scott, 1986). Fig. l shows an
alignment of the amino acid sequence of this protein with those of TSs from seven other
organisms. Thus, Fig. 1 contains data from two eukaryotic viruses [VZV, Herpesvirus saimiri
(HVS)], two eukaryotes (Homo sapiens, Leishmania major), two prokaryotes (Lactobacillus caseL
Escherichia coli) and two prokaryotic viruses (bacteriophages T4 and ¢3T). Herpesvirus saimiri is
a member of the Gammaherpesvirinae with a simian host, and thus is distantly related to VZV.
Table 1 lists the degree of homology between each pair of proteins, based on the alignment in
Fig. 1. The conservation of 56 residues in all eight proteins clearly illustrates the strong selective
constraints on the structure of TS.
No information on the kinetic class and structure of the m R N A specified by VZV gene 13 is
yet available, but the D N A sequence indicates that the protein-coding region of the gene
contains no introns, and that the m R N A is polyadenylated about 70 nucleotides downstream
from the translational stop codon. Honess et al. (1986) reported that the HVS gene lacks introns
in the protein-coding region and is transcribed abundantly at late times in infection. The L.
major, L. caseL E. coli and bacteriophage ~b3T TS m R N A s are also unspliced. The splicing status
of the human gene is unknown, as the D N A sequence was obtained from a cDNA clone
(Takeishi et al., 1985). There is evidence, however, that the TS gene from another vertebrate, the
mouse, may contain several introns (Jenh et al., 1985); the arrangement of exons in mouse
genomic D N A is unknown. The presence of a single intron in the bacteriophage T4 TS gene
constitutes the first known example of a spliced m R N A specified by a prokaryotic structural
gene (Chu et al., 1984, 1985; Belfort et al., 1985).
Most organisms contain separate genes for TS and dihydrofolate reductase (DHFR), which
uses as a substrate one of the products generated by TS. In contrast, several protozoa, including
L. major, specify a bifunctional protein containing a D H F R domain in the amino-terminal
portion and a TS domain in the carboxy-terminal portion (Beverley et al., 1986; Grumont et al.,
1986). None of the VZV proteins is significantly homologous to the D H F R s of other organisms.
In order to demonstrate directly that VZV gene !3 encodes a TS, the native protein was
expressed in E. coli. The expression vector pKK240-11 (Amann & Brosius, 1985) provides the
strong trp-lac fusion promoter and the lacZ ribosome binding site positioned correctly with
respect to an ATG codon located within an unique NcoI restriction site (CCATGG). The ATG
initiation codon of gene 13 is located similarly within an NcoI site, allowing the reading frame to
be positioned in the vector downstream from the prokaryotic transcription and translation
signals, and thus ensuring synthesis of the native gene 13 product. An NcoI-HindlII fragment
was isolated from a plasmid containing VZV EcoRI m and ligated to appropriately cleaved
pKK240-11. The ligated D N A was used to transform E. coli strain X2913 (obtained from Dr B.
Bachmann, E. coli Genetic Stock Centre) to ampicillin resistance. The host strain cannot grow
on minimal agar medium lacking thymine because the TS gene (thyA) is deleted, but all of the
colonies which contained the VZV fragment were able to grow under these conditions, thus
indicating that VZV gene 13 encodes a TS. One of the transformed colonies, carrying a plasmid
designated pGL271, was chosen for further study, and the plasmid structure was verified by
sequencing across the NcoI site.
Roberts (1966) assayed TS activity in crude homogenates of mouse cells by measuring release
of tritiated water from 2'-deoxy[5-3H]uridylate and so this method was used to assay TS in E. coli
strains. Mid-log phase cells were harvested by centrifugation, washed in 50 mM-Tris-HC1 pH
7.4, 50 mM-NaC1, 5 mM-EDTA and resuspended in 2 packed cell volumes of 50 mM-Tris-HC1
pH 7.4, 10 mM-dithiothreitol, 0.1% (v/v) Triton X- 100. Cells were disrupted by probe sonication,
and debris was removed by centrifugation at 10000g for 10 min. The resulting clarified extracts
contained approximately 15 mg/ml protein, and were stored at - 7 0 °C. Samples of 1 to 2 ~1
extract were incubated for 15 min at 37 °C in 40 gl reaction volumes containing 129 mM-TrisHC1 pH 7.5, 64 mM-NaF, 22 raM-sucrose, 0.15% (w/v) bovine serum albumin, 0.125 mMdithiothreitol, 19 mM-formaldehyde, 900 gM-(+)-L-5,6,7,8-tetrahydrofolate and 110 gM-2'deoxy[5-3H]uridylate (10.6 Ci/mmol). The reactions were stopped, and unreacted substrate was
adsorbed to charcoal. The results shown in Table 2 demonstrate significant levels of TS activity
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1
2
3
4
5
6
7
8
MCD L S C W T K V P G F T L T G E LQY L K Q V D D I L R Y G V R K R . . . . . . . . . .
DRTG I G T L S L F G M Q A R Y
M S T H T EEQHG E H Q Y L SQVQH I L N Y G S F K N . . . . . . . . . .
DRTGTGTLS] FGTQSRF
MPVACS E LPRRPLPPAAQERDAEPRPPHGE
L Q Y L G Q I QH ] L R C G V R K D . . . . . . . . . .
DRTGTGTLSVFGMQARY
• ............................
E R Q Y L E L I D R I M K T G I V K E. . . . . . . . . .
DRTGVGT I SLFGAQMRF
M L E Q P Y LD L A K K V L D E G H F K P . . . . . . . . . .
DRTHTGIYS1FGHQMRF
MKQYLELMQKVLDEGTQKN ..........
D R T G T G T LS I FGHQMRF
MKQYQC) L I K O I F ENG'/ET O. . . . . . . . . .
DR TGT GT I A LFGSK LRW
MTQFDKQYNSI I K D I INNGISDEEFDVRTKWDSDGTPAHTLSVMSKQMRF
1
2
3
4
5
6
7
8
NLRNE-FPLLTTKRVFWRAVVEELLWFIRGST-DSKEL
.......
AAKDIHIWDIYGSSKFLNRNGFHKRHT--SLENE-FPLLTTKRVFWRGVVEELLWFIRGST-DSKEL
.......
SAAGVHIWDANGSRSFLDKLGFYDRDE--S L R D E - F P L L T T K R V F W K G V L E E L L W F I K G S T N - A K E L. . . . . . .
SSKGVKIWDANGSRDFLDSLGFSTREE--SLRDNRLPLLTTKRVFWRGVCEELLWFLRGETS-AQLL. . . . . . .
ADKDIHIWDGNGSREFLDSRGLTENKE--DLSKG-FPLLTTKKVPFGLIKSELLWFLHGHTN-IRFL
.......
LQHRNHIWDEWAFEKWVKSDEYHGPDMTDF
NLQDG-FPLVTTKRCHLRSIIHELLWFLQGDTN-IAYL
.......
HENNVTIWDEWADEN ...............
D L T K G - F P A V T T K K L A W K A C ] A E L I W F L S G S T N - V N D L R L I Q H D S L I Q G K T V W D E N Y E N Q A K D L G Y H.S. . . . . .
D N S E - - V P I L T T K K V A W K T A I K E L L W I W Q L K S N D V T E L. . . . . . .
NKUGVHIWDQWKQED . . . . . . . . . . . . . . .
1
2
3
4
5
6
7
8
...................................
GDLGPIYGFQWRHFGAEYKDCQSNYLQQGIDQLQTVIDTI
...................................
GDLGPVYGFQWRHFGAEYKCVGRDYKGEGVDQLKQLIDTI
...................................
GDLGPVYGFQWRHFGAEYRDMESDYSGQGVDQLQRVIDTI
...................................
UDLGPVYGFQWRHFCADYKGFEANYDGEGVDQIKLIVETI
GHRSQKDPEFAAVYHEEMAKFDDRVLHDDAFAAKYGDLGLVYGSQWRAWH
........
TSKGDTIDQLGDVIEQI
...................................
GDLGPVYGKQWRAWP ........
TPDGRHIDQITTVLNQL
...................................
GELGPIYGKQWRDFG .............
GVDQIIEVIDRI
...................................
GTIGHAYGFOLCKKN .......
RSLNGEKVDQVDYLLHQL
folote-binding
site
< .............
>
site
FdUMP-binding
1
2
3
4
5
6
7
8
KTNPESRRMIISSWNPKDIPLMVLPPCHTLCQFYVAN--GELSCQVYQRSGDMGLGVPFN]AGYALLTYIVAHVT
KTNPTDRRMLMCAWNVSD]PKMVLPPCHVLSQFYVCD--GKLSCQLYQRSADMGLGVPFNIASYSLLTCMIAHVT
KTNPDDRRIIMCAWNPRDLPLMALPPCHALCQFYVVN--SELSCQLYQRSGDMGLGVPFNIASYALLTYMIAHIT
KTNPNDRRLLVTAWNPCALQKMALPPCHLLAQFYVNTDTSELSCMLYQRSCDMGLGVPFN1ASYALLTILIAKAT
KTHPYSRRLIVSAWNPEDVPTMALPPCHTLYQFYVND--GKLSLQLYQRSADIFLGVPFNIASYALLTHLVAHEC
KNDPDSRRIIVSAWNVGELDKMALAPCHAFFQFYVAD--GKLSCQLYQRSCDVFLGLPFNAISYALLVHMMAQOC
KKLPNDRRQIVSAWNPAELKYMALPPCHMFYQFNVRN--GYLDLQWYQRSVDVFLGLPFNIASYATLVHIVAKMC
KNNPSSRRHITMLWN~DDLDAMALTPCVYETQWYVKQ--GKLHLEVRARSNDMALGNPFNVFQYNVLQRMIAQVT
1
2
3
4
5
6
7
8
GLKTGDLIHTMGDAHIYLNHIDALKVQLARSPKPFPCLKIIRNVTDINDF
............
KWDDFQLDGYNPH
NLVPGEFIHT]GDAH1YVDH]DALKMQLTRTPRPFPTLRFARNVSCIDDF
............
KADDIILENYNPH
G L K P G D F I H T L G D A H I Y L N H I E P L K I Q L Q R E P R P F P K L R I L R K V E K I D D.F
...........
KAEDFQIEGYNPH
GLRPGELVHTLGDAHVYRNHVOALKAQLERVPHAFPTLIFKEERQYLEDY
............
ELTDUEVIDYVPH
GLEVGEFIHTFGDAHLYVNHLDQIKEQLSRTPRPAPTLOLNPDKHDIFDF
............
DMKDIKLLNYDPY
DLEVGDFVWTGGDTHLYSNHMDQTHLQLSREPRPLPKLIIKRKPESIFDY
............
RFEDFEIEGYDPH
NLIPGDLIFSGGNTHIYMNHVEQCKEILRREPKELCELVISGLPYKFRYLSTKEQLKYVLKLRPKDFVLNNYVSH
GYELGEY]FNIGDCHVYTRH]DNLKIQMEREQFEAPELWlNPEVKDFYNF
............
TVDDFKLINYKHG
•
•
, =
•
•
•
•
•
PPLKMEMAL
PI IKMHMAV
PTIKMEMAV
FA]KMEMAV
PAIKAPVAV
PGIKAPVA]
PP]KGKMAV
DKLLFEVAV
Fig. 1. Alignment of the TSs of VZV (1 ; Davison & Scott, 1986), HVS (2; Honess et al., 1986), H. sapiens
(3; Takeishi et al., 1985), L. major (4; Beverley et al., 1986), L. casei (5; Maley et al., 1979), E. coli (6;
Belfort et al., 1983) and bacteriophages T4 (7; Chu et al., 1984) and q~3T (8; Kenny et al., 1985). Amino
acid sequences are shown in one-letter code, dashes denoting blank characters inserted to align the
sequences. The TS domain of the bifunctional DHFR-TS of L. major is shown; dots serve to indicate
that the DHFR domain occupies the amino-terminal portion of the complete protein. The TS domain of
the DHFR-TS ofL. tropica differs from that of L. major in only a few residues (Grumont et al., 1986),
and is not included. Asterisks indicate residues conserved in all eight proteins. The conserved cysteine
residue which is covalently bound to FdUMP in the binary complex (Maley et al., 1979; Belfort et al.,
1983) and the proposed region for binding folate and its analogues in the ternary complex (Maley et al.,
1982) are shown.
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Table 1. Numbers o f amino acid residues shared by TSs
VZV
HVS
VZV
HVS
H. sap~ns
L. major
L. casei
E. coli
301
192
294
204
207
313
160
171
173
286*
138
148
139
133
316
124
130
141
126
156
264
H. sapiens
L. major
L. casei
E. coli
Phage T4
Phage ~b3T
Phage
T4
Phage
~3T
120
114
129
116
122
126
286
101
107
105
97
103
96
92
279
* TS domain. The complete DHFR-TS protein contains 520 residues.
Table 2. TS activity o f E. coli strains measured by the tritium release assay
Host strain
X2913
Z2913
Z2913
Plasmid
pGL271
pKK 240-11
None
Phenotype
TS activity*
Thy+
ThyThy-
1.220
0-009
0.012
* Assays were performed in duplicate on three independent extracts and mean activities are expressed as nmol
thymidylate formed per ~tg protein.
only in the strain containing pGL271, which expresses the VZV TS gene, and not in the host
strain alone or in the host strain containing the vector plasmid without the TS gene. A similar
result was obtained using an independent clone containing the VZV TS gene on a smaller N c o I XbaI fragment (data not shown).
The inhibitor 5-fluoro-2'-deoxyuridylate (FdUMP) has been used successfully to identify the
TS protein in crude extracts of human cells (Lockshin et aL, 1979) and mouse cells (Ayusawa et
al., 1981) and, by virtue of the difference in molecular weight between the cellular and viral
proteins, the HVS TS in infected simian cells (Honess et al., 1986). The F d U M P binds
irreversibly to the conserved cysteine residue indicated in Fig. 1, and use of the 32p-labelled
compound provides a sensitive and very specific means of radiolabelling the TS polypeptide.
Samples of the E. coli extracts (50 gg protein) were incubated with [32p]FdUMP and 5,10methylenetetrahydrofolic acid, as described by Honess et al. (1986), in order to allow formation
of the ternary complex. Reaction mixtures were subjected to SDS-polyacrylamide gel
electrophoresis in a gel containing 12 ~o polyacrylamide crosslinked with N,N'-diallyltartardiamide. Figure 2 (a) shows that a single FdUMP-binding polypeptide with an apparent molecular
weight of 32500 was present in extracts from the strain expressing the VZV TS gene, but not in
extracts of either the host strain alone or the host strain containing the vector plasmid without
the TS gene. For comparison, Fig. 2(a) also includes results for E. coli strain C600, which
expresses the host TS gene. The ternary complex containing the E. coli TS polypeptide migrated
slightly faster than the VZV TS complex, an observation consistent with the smaller predicted
molecular weight of the E. coli TS subunit (30441 ; Belfort et al., 1983).
The FdUMP-binding assay was also employed to detect TS polypeptides in VZV-infected
cells. Human foetal lung cells were infected with the VZV strain described by Dumas et al.
(1981). When 20 to 3 0 ~ of cells showed a cytopathic effect, the monolayers were washed in
phosphate-buffered saline, and the cells were resuspended at approximately 107 cells/ml in
50mu-Tris-HC1 pH 7.5, 80 mM-KCI, 10 mM-2-mercaptoethanol, 1 mM-EDTA and 0.1 ~ (v/v)
Triton X-100 and sonicated. An uninfected cell extract was prepared similarly. The extracts
were centrifuged, and samples of 5, 10 and 50 ~tl of the supernatants were assayed for
[32p]FdUMP_binding activity as described above. Figure 2 (b) shows that a polypeptide with an
apparent molecular weight of 35 000, corresponding to the ternary complex of human TS, was
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(a)
1453
(b)
"7
¢xl
t'q
t~
t~
Uninfected cell
extract (p.1)
5
10
50
VZV-infected
cell extract (~tl)
5
10
50
000
-32500
- 35
32500-
Fig. 2. Autoradiographsof electrophoretically separated extracts of (a) E. coli strains or (b) uninfected
and VZV-infected human foetal lung ceils labelled with [32p]FdUMP in the presence of 5,10methylenetetrahydrofolate.The apparent molecular weights of the ternary complexesof VZV (32500)
and human (35000) TSs are indicated.
present in both uninfected and infected cells. The additional polypeptide, present only in
infected cells, had an apparent molecular weight of 32 500, and thus corresponded in size to the
ternary complex of VZV TS expressed in E. coli.
Varicella-zoster virus and HVS, the only two herpesviruses shown to date to have a TS gene,
belong to different subfamilies of the herpesviruses (the Alpha- and Gamma-herpesvirinae,
respectively), and this gene is apparently absent from other members of each family. The
published D N A sequence of Epstein-Barr virus (EBV), a member of the Gammaherpesvirinae,
clearly contains no TS homologue (Baer et al., 1984), and Honess et al. (1986) were unable to
detect novel TS activities in cells infected with HSV-1 or pseudorabies virus, members of the
A lphaherpesvirinae. Proof that HSV-1 lacks a TS gene must await determination of the complete
HSV-1 D N A sequence, but the hypothesis is supported by three additional lines of evidence.
Firstly, no potential TS gene transcript has been mapped in the region of the HSV-1 genome
corresponding to the portion of the VZV ge'nome containing gene 13 (Wagner, 1985). These two
viruses, VZV and HSV-1, are very similar in gene layout, such that genes immediately adjacent
to VZV gene 13 have similarly arranged counterparts in HSV-1. However, the region of the
HSV-1 genome corresponding to VZV gene 13 instead contains a smaller gene in the opposite
orientation (Frink et al., 1983). The proposed product of this gene is not homologous to the TS
family. Secondly, D N A hybridization experiments showed that this region of the HSV-I
genome is only weakly homologous to the corresponding region of the closely related HSV-2
genome (Draper et al., 1984), which also contains a smaller gene in the opposite orientation from
the VZV TS gene (Dowbenko & Lasky, 1984). The presence of a TS gene in this region of both
genomes would have resulted in a region of marked homology. The possibility that HSV-1 has a
TS gene at a different genome location is rendered unlikely by the third line of evidence, that no
significant hybridization was detected between D N A fragments containing VZV gene 13 and
fragments representing the entire HSV-1 genome (Davison & Wilkie, 1983).
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The presence of a TS gene in one member of the Alphaherpesvirinae (VZV) and its apparent
absence from another (HSV-1), and its presence in one member of the Gammaherpesvirinae
(HVS) and absence from another (EBV), raise important questions regarding the origins and
roles of this gene in the herpesviruses. The VZV and HVS enzymes clearly belong to the
eukaryotic group, as both are closely related to each other and to the human TS (Table 1).
However, the origins of the VZV and HVS TS genes remain uncertain, as there is currently no
method to determine unambiguously whether the TS gene is an ancestral feature lost during the
divergences leading to contemporary herpesvirus subfamilies, or whether it has been acquired
after divergence.
In addition to a TS, VZV and HVS specify a thymidine kinase (TK; Doberson et al., 1976;
Davison & Scott, 1986; Honess et al., 1982), which catalyses the phosphorylation of thymidine to
thymidylate. Thus, these viruses supplement the cellular pathways for providing thymidylate de
novo (using TS) and by thymidine salvage (using TK). Little is known about the expression of
these virus TS and TK genes, but it seems plausible that the two enzymes are used at different
stages of pathogenesis to provide thymidylate for D N A synthesis. It is not yet known whether
TS is essential for growth of VZV in normal tissue culture. The viability of TK-negative mutants
of VZV and HSV-1 in tissue culture (Shiraki et al., 1983; Dubbs & Kitt, 1974) indicates that a
virus-coded function for provision of thymidylate is not required, and therefore that TS also may
be dispensable. Nevertheless, the presence of a TS gene in VZV suggests that this enzyme may
provide a suitable target for selective antiviral chemotherapy.
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