Download Identification of human herpesvirus 6 uracil

Journal of General Virology (1994), 75, 2349 2354. Printed in Great Britain
2349
Identification of human herpesvirus 6 uracil-DNA glycosylase gene
Shigeo Sato, Takeshi Yamamoto, Yuji Isegawa and Koichi Yamanishi*
Department o f Virology, Research Institute f o r Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita,
Osaka 565, Japan
Uracil-DNA glycosylase encoded in many species
functions as a DNA repair enzyme that removes uracil
residues from DNA. To investigate the potential function
of uracil-DNA glycosylase encoded by human herpesvirus 6 (HHV-6), we sequenced a DNA clone (pSTY09),
identified an open reading frame of 765 bp and compared the putative amino acid sequence with other
uracil-DNA glycosylases, by computer analysis. The
amino acid sequence of HHV-6 had similarities to other
uracil-DNA glycosylases, with the highest degree of
similarity to those of human cytomegalovirus and
Epstein-Barr virus. Two strongly conserved regions in
uracil-DNA glycosylase of other species also existed in
HHV-6. The gene product which was expressed in
Escherichia eoli demonstrated uracil-DNA glycosylase
activity. This is the first report to identify and characterize the uracil-DNA glycosylase gene in HHV-6.
Introduction
et al., 1994). A nomenclature has been adopted desig-
Human herpesvirus 6 (HHV-6) was first isolated in 1986
from the peripheral blood of patients with lymphoproliferative disorders and AIDS (Salahuddin et al.,
1986). The distinct nature of HHV-6 compared with
other human herpesviruses was confirmed by molecular
and immunological analyses (Josephs et al., 1986). The
virus replicates predominantly in CD4 + lymphocytes
(Lusso et al., 1988; Takahashi et al., 1989) and may
establish latent infection in cells of the monocyte/
macrophagelineage (Kondo et al., 1991). Infection with
this virus causes exanthem subitum (ES) or roseola
infantum, a common illness of infancy (Yamanishi et al.,
1988). Nucleotide sequence analysis of the genome has
demonstrated that HHV-6 is more closely related to
human cytomegalovirus (HCMV), a betaherpesvirus,
than to the neurotropic alphaherpesviruses such as
herpes simplex virus (HSV) and varicella-zoster virus
(VZV) or to the lymphotropic gammaherpesviruses such
as Epstein-Barr virus (EBV) (Efstathiou et al., 1992;
Lawrence et at., 1990). Furthermore, two variants of
HHV-6 have been identified based on differences in
epidemiology, in vitro growth properties, reactivity with
monoclonal antibodies, restriction endonuclease profiles
and nucleotide sequence (Wyatt et al., 1990; Ablashi et
al., 1991 ; Aubin et al., 1991, 1993; Schirmer et al., 1991 ;
Chandran et al., 1992; Gompels et al., 1993; Yamamoto
Nucleotide sequence data reported in this paper will appear in the
GSDB, DDBJ, EMBL and NCBI nucleotidesequence databases with
the accessionnumber D25277.
0001-2338 © 1994 SGM
nating viruses HHV-6A (variant A) and HHV-6B
(variant B) (Ablashi et al., 1993). To characterize
biochemical properties, some enzyme assays have been
performed in HHV-6-infected cells (Williams et al., 1989;
Shiraki et al., 1989; Teo et al., 1991). In those reports, it
was described that there were significant increases in the
activities of DNA polymerase and DNase in extracts
from virus-infected cells when compared to those from
mock-infected cells; however, there was no significant
increase in the activities of thymidine kinase (TK),
dUTPase and uracil-DNA glycosylase. Uracil-DNA
glycosylase is a D N A repair enzyme that removes uracil
residues from DNA. Because it has been reported that
neurons, in which HSV reactivation takes place, lack not
only T K and D N A polymerases, but also uracil-DNA
glycosylase (Focher et al., 1990, 1993), it is expected that
uracil-DNA glycosylase may be a new target of antiviral
agents against HSV infection. In this study, to investigate
the potential function of uracil-DNA glycosylase
encoded by HHV-6, we analyse the sequence of the D N A
clone (pSTY09) and express the 765 bp open reading
frame (ORF) in Escherichia coll.
Methods
Virus. The virus strain used in this study was HHV-6 strain HST
which was isolatedfrom a patient with ES and belongsto the HHV-6B
group (Yamanishi et al., 1988). The virus was propagated in fresh
human peripheral mononuclear cells as described previously
(Yamanishi et al., 1988).
DNA sequencing and analysis. In our laboratory, the DNA
sequencingof HHV-6B strain HST is in progress for the identification
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S. Sato and others
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C T G C A G G A T A A C C C A G A G C A A C T A C G C A C A T T G T T T G C G C T G A T A G G G G A C C C A G A A T C T C C~;GACA A T TC-GC T A A A C T T TTTCAATGGC~'rC C A G A C A T G T T C G C C T T e C G T C G G ~ T A
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240
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GACAAC GATAGAGC TCATCTAACC GTC CAC CCC TCTTC GGATAAC GTC C AC GC C TGGAGTTT TTT GTGCAAAC C CAC CGATGTTAAAGTTGTGATTCTGGGACACGAT CCGTATC CC GAC
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960
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T G T T T A A A C T C C T G G T G T A G A G A A G G A G T C C T A C T G C T A A A C TC G A T A T T C A C T G T A G T T C A T G G A T T A C C A A T G T C C C A C G A G G C A T T T G G T T G C 4 Z A A A C A C T G A G C T A C A A G A T T A T C
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Fig. 1. Nucleotide sequence and predicted amino acid sequence of the PstI KpnI fragment of pSTY09. The 765 bp ORF starts with
an ATG at position 550 and ends with a TAG at 1315, indicated by underlining. The amino acid sequence is shown below the nucleotide
sequence. The sequence indicated by asterisks represents a candidate TATA box at position 501, the broken underline indicates a
putative Spl-binding site at position 444, GGGCGG, and the double underline indicates a consensus polyadenylation signal,
AATAAA, at position 1353.
of viral genes. Genomic DNA was purified as described elsewhere
(Martin et al., 1982), digested with a restriction enzyme PstI (Takara
Shuzo Co.) and cloned into pUC19 (Yanisch-Perron et al., 1985). The
5.5 kb PstI fragment was cloned and designated pSTY09. Unidirectional progressive deletions of pSTY09, which spanned approximately 200 bp. were prepared with a deletion kit for kilo-sequencing
(Takara Shuzo Co.) and used as a template in the dideoxynucleotide
chain termination method (Sanger et al., 1977). The sequence data were
then assembled using the ABI sequencing system and analysed for the
presence of ORFs by using the computer program DNASIS (Hitachi).
Comparisons of the predicted amino acid sequences of HHV-6 with
those of other species were made using the SWISSPROT and National
Biomedical Research Foundation Protein Identification Resource
databases. Comparisons of amino acid sequences were carried out
using arithmetic means (Higgins et al., 1992).
(100 ~tg protein) as described elsewhere (Williams et al., 1989). Reaction
mixtures were incubated at 37 °C for 1 h and terminated by chilling to
0 °C followed by the addition of 25 ml of sheared calf thymus DNA
solution (1 mg/ml) and 25 ml of 4 M-perchloric acid. After 10 min at
0°C, the samples were centrifuged at 1400g for 10 min and the
supernatants were placed in vials with scintillant and counted in a
scintillation counter (Aloka, LSC700). A unit of uraci~DNA glycosylase activity was defined as the amount of enzyme required to release
1 pmol of uracil as acid-soluble material per min at 37 °C, as described
elsewhere (Williams et al., 1989).
Tramformation. The N c o I - K p n I fragment of pSTY09 containing the
765 bp ORF was inserted into the prokaryotic expression vector
pTrc99A (Amann et al., 1988) and designated p99/UNG. E. coli strain
CJ236, which is defective in uraciNDNA glycosylase activity (Kunkel
et al., 1987), was transformed using the method of Hanahan (1985).
Each culture broth (25 ml) of E. coli CJ236 containing plasmids
p99/UNG or pTrc99A was grown at 37 °C to an A600 of 0.8 in LB
medium with 50 mg/ml of ampicillin, and IPTG was added to a final
concentration of 1 mM. After further incubation for 2 h, the cells were
washed by centrifugation in general extraction buffer (0.15 M-NaCI,
1 mM-EDTA, 50 mM-Tris-HC1 pH 7.5) and suspended in the same
buffer. The cell suspensions were frozen and thawed four times in a dry
ice and ethanol bath and then subjected to sonication. The samples
were centrifuged to remove precipitated material and used as cell-free
extracts for enzyme assays.
Results
Sequence analysis of the uracil-DNA glycosylase locus
E n z y m e assays. Activated calf thymus DNA was labelled with [5'3H]dUTP (15 Ci/mmol) and [methyl, I',2'-3H]dTTP (90Ci/mmol)
(Amersham), respectively, as described elsewhere (Caradonna &
Cheng, 1980). The uracil-DNA glycosylase reaction mixture contained,
in a total volume of 0.2 ml, 50 mM-Tri~HCI pH 7.5, 10 mg BSA, 2 mMDTT, 10 mM-EDTA, labelled DNA (104 d.p.m./l~g) and the cell extract
Protein determinations. Protein was estimated by using the Pierce
BCA Protein Assay Reagent (Pierce Chemicals), using BSA as the
standard.
From the results of DNA sequencing, it was observed
that there were several ORFs in pSTY09. We were
particularly interested in the 765 bp ORF, because it had
a high similarity to HCMV UL114 which is thought to
encode uracil-DNA glycosylase. There were also ORFs
upstream and downstream of the 765 bp ORF and they
showed similarities to HCMV U L l l 5 and ULll3,
respectively (data not shown). A region of DNA
sequence in pSTY09 (1697 bp), numbered from positions
1 to 1697, is shown in Fig. 1. The 765 bp ORF starts with
an ATG at position 550, ends with a TAG at position
1315 and was expected to encode 255 amino acids, a
polypeptide of approximately 29K. The sequence surrounding the initiation codon, CCACCATGG, is identical to Kozak's optimal ATG context, CCPuCCATGG
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Uracil-DNA gtycosylase of HHV-6
(Kozak, 1989), and is preceded by a candidate TATA
box at position 501, CATAAA, which is identical to the
TATA box of the HSV TK gene and the HHV-6 DNA
polymerase gene (McGeoch et al., 1988; Teo et al., 1991).
Furthermore, at position 444, there was a putative
binding site for Spl, GGGCGG, one of several transcription factors, and the stop codon TAG at position
1315 was followed by a consensus polyadenylation
signal, AATAAA, at position 1353. From these aspects,
it was expected that the 765 bp ORF could encode a
protein.
Comparison of the predicted amino acids constituting
putative uracil-DNA glycosylases of HHV-6 and other
herpesviruses
Comparison of amino acid sequences of uracil-DNA
glycosylases from humans, yeast, E. coli and other
human herpesviruses revealed a striking similarity between all these proteins. The alignment of the uracilDNA glycosylases of different species demonstrated that
they were highly conserved regions of various sizes,
reported previously as consensus sequences (Mullaney et
al., 1989), and Fig. 2 shows that the HHV-6 protein also
contained these regions. Two regions of the HHV-6
protein, in particular amino acids 87 to 107 and 146 to
169, were highly conserved in the sequences of the
different species, and it was expected that these consensus
regions were VVI-GqDPYh--gqahGLaFsv and GVLL1Nt-lTV-rg .... SH---GW, respectively. The amino acids
indicated with capital letters are conserved among all
eight uracil-DNA glycosylases for which sequences are
published, and those indicated with lowercase letters are
conserved among more than six species. The amino acid
sequence identities (Higgins et al., 1992) between uracilDNA glycosylase of HHV-6 and those of six other
Table 1. Amino acid identity between uracil-DNA
glycosylase of HHV-6 strain H S T and those of other
herpesviruses, E. coli, yeast and humans*
Origin
Identity (%)
EBV
HCMV
VZV
HSV-1
HSV-2
EHV- 1
30.5
30.0
t 9-9
13.2
14.1
13-2
24.1
12.1
19-5
E. coli
Yeast
Human
*** • ***
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**
******
HHV-6
(87-107)
VVILGHDPYP-DGRGCGLAFGT
HCMV
(85-105)
VVIVGQDPYC-DGSASGLAFGT
EBV
(85-105)
VVILGQDPYHG-GQANGLAFSV
VZV
(142-163)
VVIIGQDPYPTAGHAHGLAFSV
HSV-I
(172-193)
VVIIGQDPYHHPGQAHGLAFSV
HSV-2
(93-114)
VVIIGQDPYHHPGQAHGLAFSV
EHV-I
149-170)
VVIVGQDPYHAPGQAHGLAFSV
Human
139-160)
VVILGQDPYHGPNQAHGLCFSV
Yeast
156-177)
WIIGQDPYHNFNQAHGLAFSV
E.coli
(57-78)
VVILGQDPYHGPGQAHGLAFSV
Consensus
WI-GqDPYh--gqahGLaFsv
******
**e***
*
**
**
HHV-6
146-169 )
GVLLLNS I F T V V H G L P M - S H E A F G W
HCMV
144 - 167 )
GVLLLNTVFTVVHGQPG-SHRHLGW
EBV
144-167 )
G V L L L N T ILTVQKGKPG- S HAD IGW
VZV
202-225 )
GVLLLNTTLTVRRGTPG- SHVYLGW
HSV-I
232 -255 )
GVLLLNTTLTVKRGAAA-SHSRIGW
HSV-2
153 - 176 )
G V L L L N T T L T V K R G A A A - S H SKLGW
EHV-I
209 - 232 )
GVLL INTTLTVARGKPG- S H A T L G W
Human
199- 222 )
GVLLLNAVLTV-RAHQANSHKERGW
Yeast
217 -240 )
G V L L L N T S L T V -RAHNANSH SKHGW
E.coli
117-140 )
GVLLLNTVLTV -RAGQARSHASLGW
Consensus
G V L L I N t - I T V - r g .... S H - - - G W
Fig. 2. Conserved regions of amino acid sequences of uracil-DNA
glycosylases. Amino acid residues 85 to 107 and 146 to 169 from HHV-6
uracil-DNA glycosylase were aligned with amino acid residues
derived from uracil-DNA glycosylases of HCMV (Chee et al., 1990),
EBV (Baer et al., 1984), VZV (Davison and Scott, 1986), HSV-1
(McGeoch et al., 1988), HSV-2 (McGeoch et al., 1991), EHV-1
(Telford et al., 1992), humans (Olsen et al., 1989), yeast (Percival et al.,
1989) and E. co//(Varshney et al., 1988). Gaps in the protein sequences
were introduced to yield maximal alignment and are indicated by
dashes ( ) . In the consensus sequence, the amino acids conserved
amongst all eight species are indicated by capital letters and those
conserved in more than six species are indicated by lower case letters.
The residues identical between HHV-6 and HCMV are indicated by
asterisks.
herpesviruses, humans, E. coti and yeast, ranged between
12.1% and 30.5 % (Table 1). Hydropathy profiles of the
HCMV and HHV-6 proteins were very similar,
suggesting that these proteins have similar structures
(data not shown).
Expression of uracil-DNA glycosylase in E. coli
* The alignments were done separately for each herpesvirus and
other species and the location and the number of gaps were different for
each pairwise comparison. Amino acid residues for uraciNDNA
glycosylase of HHV-6 were aligned with those derived for other species
which are listed in Fig. 2.
To determine whether or not the product of the putative
uracil-DNA glycosylase gene exhibits enzyme activity,
the 765 bp ORF was inserted into the prokaryotic
expression vector pTrc99A and designated p99/UNG.
pTrc99A and p99/UNG were then transferred into
uracil-DNA glycosylase-defective E. coli CJ236 and the
enzyme assay was performed with crude cell lysates as
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S. Sato and others
Table 2. Enzyme activities in E. coli containing
p 9 9 / U N G and pTrc99A*
Substrate for uracil-DNA glycosylaseassay
Cell lysate
CJ236-p99/UNG
CJ236-pTrc99A
Denatured
CJ236-p99/UNG
[3H]UTP-labelled
DNA
8-0
I-5
0-6
[3H]TTP-labelled
DNA
1-6
i-3
NDt
* Enzyme activity expressed as units/mg protein. A unit of
uracil-DNA glycosylaseactivitywas definedas the amount of enzyme
required to release 1 pmol of uracil as acid-solublematerial per rain.
t NO, not determined.
described in Methods. The data presented in Table 2
summarize the results of the uracil-DNA glycosylase
assay. When [3H]dUTP-labelled D N A was used as the
substrate, the amount of released label in the cell lysate
containing p 9 9 / U N G was significantly increased relative
to that in the lysate containing pTrc99A (8"0 unit/mg
compared to 1"5 unit/rag, respectively), and this activity
was completely lost by heat denaturation of the cell
lysates. Additionally, this activity depended on the
amount of lysate and the length of the incubation time
(data not shown). However when [3H]dTTP-labelled
DNA was used, there was little or no difference between
the amount of released label observed for the p99/UNGcontaining lysate and that for the lysate containing the
vector pTrc99A (1.6 unit/mg compared to 1.3 unit/mg,
respectively). These results showed that the release of
uracil from DNA was not a non-specific reaction such as
that catalysed by exonuclease, but a uracil-specific
reaction, suggesting that the gene product of the ORF in
pSTY09 was a functional uracil-DNA glycosylase. The
activity in the lysate containing pTrc99A indicated in
Table 2 may be caused by a small degree of contaminating exonuclease owing to a decrease in the
activity of heat-denatured lysates.
Discussion
Uracil-DNA glycosylase activity was first detected in
extracts of E. coli (Lindahl, 1974), and its gene has been
sequenced (Duncan & Chambers, 1984; Varshney et al.,
1988). Thereafter~he genes of yeast and human uracilD N A glycosylases were reported (Percival et al., 1989;
Olsen et al., 1989). Furthermore, the uracil-DNA
glycosylase encoded by HSV has been purified, its cDNA
was cloned and the locus mapped to UL2 in the viral
genome (Caradonna et al., 1987; Worrad & Caradonna,
1988; Mullaney et al., 1989). Homologous genes were
also found in other herpesviruses for which the genomes
had been sequenced: gene 59 in VZV (Davison & Scott,
1986; Davison & Taylor, 1987), BKRF3 in EBV (Baer et
al., 1984; Perry & McGeoch, 1988), U L l l 4 in HCMV
(Chee et al., 1990) and equine herpesvirus type 1 (EHV1) (Telford et al., 1992). It was reported that this enzyme
was highly conserved between humans, E. coli, yeast and
herpesviruses, and that no other protein was known to be
so strongly conserved from bacteria to humans (Olsen et
al., 1989). However it was reported that there was no
significant increase in the activities of TK, dUTPase and
uracil-DNA glycosylase in extracts from HHV-6A (GS
strain)-infected cells (Williams et al., 1989). We also
failed to detect an increase in enzyme activity in HHV-6B
(HST strain)-infected cells relative to mock-infected cells
(data not shown). The similarities of genomic configuration between HHV-6 and HCMV were expected because
it had been reported already that HHV-6 is closely
related to HCMV (Neipel et al., 1991; Teo et al., 1991 ;
Josephs et al., 1992; Liu et aI., 1993). As the two viruses
also exhibited similarities with respect to the genomic
region studied in this paper, the 765 bp ORF in pSTY09
was suggested to be a U L l l 4 (HCMV) homologue, in
HHV-6, i.e. its product may have the activity of
uracil-DNA glycosylase. To characterize the function of
the 765 bp ORF, sequence analysis of this region was
performed. As there were consensus signals for transcription and translation in the upstream and downstream regions of the 765 bp ORF, it was suggested that
the O R F could encode a protein. Moreover, the putative
amino acid sequence was compared to those of uracilDNA glycosylases already published. Table 1 shows that
HHV-6 protein was most similar to those of HCMV and
EBV among well characterized herpesviruses. In addition, the conserved regions among published uracilDNA glycosylases were also conserved in the putative
HHV-6 enzyme, as shown in Fig. 2. As two regions of the
HHV-6 protein (amino acids 87 to 107 and 146 to 169)
were most strongly conserved among those of different
species, it is possible that these regions may be part of the
catalytic site.
A related D N A sequence (Dambaugh et al; GSDB
accession number L14772) for which we have not yet
found an accompanying publication, aligns closely with
that of HHV-6B (strain Z29), showing 99"8 % nucleotide
sequence identity.
To determine whether or not the product of the 765 bp
O R F in pSTY09 can act as a uracil-DNA glycosylase,
the ORF was expressed in uraciLDNA glycosylasedefective E. coli strain CJ236. The following two reasons
led to the conclusion that the release of uracil from D N A
by the expressed protein was not a non-specific reaction,
such as those catalysed by exonuclease, but a uracilspecific reaction carried out by uracil-DNA glycosylase.
Firstly, when [3H]dUTP-labelled DNA was used as the
substrate, the amount of released radioactivity detected
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Uracil-DNA gtycosylase of HHV-6
in the cell lysate containing p 9 9 / U N G was five times that
in the lysate containing pTrc99A, but this activity was
completely lost by heat denaturation of the cell lysates.
Secondly, when [3H]dTTP-labelled D N A was used, there
was no difference between the amount of released
radioactivity observed for the p99/UNG-containing
lysate and that for the Trc99A-containing lysate. The
results of the enzyme assay and the similarity of amino
acid sequences indicated that the 765 bp ORF in pSTY09
had the uracil-DNA glycosylase function. Additionally,
the existence of highly conserved regions in amino acid
sequences among the different species indicates that this
enzyme has an important role in D N A replication.
Uracil-DNA glycosylase is thought a target for new
antiviral agents against herpesvirus infections, because it
was reported that adult neurons lack the activity of not
only TK, D N A polymerase ~, 7/e, but also of uracilD N A glycosylase (Focher et al., 1990, 1993). In the
case of HSV infection, virus replication depends on viral
enzymes in neurons in which reactivation takes place and
inhibitors of those enzymes may suppress viral reactivation and replication. Since it has been reported that
HHV-6 might invade the central nervous system and
cause neurological symptoms (Kondo et al., 1993), the
HHV-6 uracil-DNA glycosylase may also be a target of
new antiviral agents. Development of agents that
specifically inhibit virus-encoded enzymes has been
difficult because of the high degree of amino acid
conservation and biochemical similarities between human and virus uracil-DNA glycosylase. But several
inhibitors of the HSV uracil-DNA glycosylase, such as
bacteriophage PBS2-encoded uracil-DNA glycosylase
inhibitor and some uracil analogues, have been synthesized and screened for their capacity to discriminate
between the virus and human uraci~DNA glycosylase
(Wang & Mosbaugh, 1988; Winters & Williams, 1990;
Focher et al., 1993). To characterize the biochemical and
biophysical properties and to investigate the possibility
of developing anti-HHV-6 agents, it will be necessary to
overproduce and purify this enzyme and to develop a
method of enzyme assay that has higher sensitivity. The
D N A sequence data and the enzyme activities expressed
in E. coli described in this communication will be helpful
in further studies.
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