Identification of the equine herpesvirus type 1 glycoprotein 17/18 as

Journal of General Virology (1992), 73, 1227-1233. Printed in Great Britain
1227
Identification of the equine herpesvirus type 1 glycoprotein 17/18 as a
homologue of herpes simplex virus glycoprotein D
Debra M. Elton, lan W. Halliburton, Richard A. Killington, David M. Meredith and
William A. Bonass*
Department o f Microbiology, University o f Leeds, Leeds L S 2 9JT, U.K.
The DNA sequence of the equine herpesvirus type 1
(EHV-1) gD gene homologue has been determined for
the strain Abl and compared with previously published
sequences. A portion of the gene has been located to a
region of the genome which also encodes homologues
of the herpes simplex virus type 1 genes for gE and gI
and is known to encode an epitope of the virion protein
gp17/18. Analysis of the EHV-1 strain Kentucky A
(KyA) by DNA hybridization showed the presence of a
gD gene homologue and established the absence of
genes for gI and gE. Western blot analysis, however,
showed that KyA virus particles contain gpl 7/18, thus
indicating that this protein is encoded by the gD gene
homologue. The KyA gp17/18 was found to be smaller
than that detected in other strains and this is accounted
for by a frameshift mutation in the KyA sequence
relative to Abl. The mutation in the KyA strain results
in an altered C-terminal sequence and could explain the
apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have
also expressed part of the Abl gD gene as a fusion
protein with glutathione S-transferase in Escherichia
coli and shown that this reacts with the MAb 5H6
originally used to map gp17/18. These experiments
establish that gp17/18 is encoded by the gD gene
homologue.
Introduction
1986; Longnecker et al., 1987); PRV also has four genes,
coding for gX, gp50, gp63 and gI (Rea et al., 1985;
Petrovskis et al., 1986a, b) and VZV has two, encoding
gplV and gpI (Davison, 1983). DNA sequence analysis
of EHV-1 has since confirmed the presence of three
potential glycoprotein genes in the region to which
gp17/18 was mapped (Audonnet et al., 1990; Elton et al.,
1991a, b). Similar analysis of the Kentucky A (KyA)
strain of EHV-1 has shown that no genes are located
between the gD gene and the homologue of HSV-1 US9
(Flowers et al., 1991), a region in which Audonnet et al.
(1990) and Elton et al. (1991a, b) have located genes
homologous to those of HSV-1 gI and gE. This suggests
that either a gene rearrangement has occurred or that the
gE and gI gene homologues have been deleted in the
KyA strain. It has been shown previously that gE and gI
of HSV-1 are non-essential for virus growth in tissue
culture (Longnecker et al., 1987); the extent of similarity
between these glycoproteins and their counterparts in
EHV-1 (Elton et al., 1991 a) would suggest that they may
have similar functions.
The aims of this study were to analyse the genome of
KyA to confirm the presence or absence of genes for gE
and gI, and to compare the structure of the Us region
with that of other strains of EHV-1. The nucleotide
sequence of the entire gD gene of strain Abl was
The glycoproteins of herpesviruses are potentially important in inducing cellular and humoral immunity in the
host. For this reason much effort has been made to
identify the glycoprotein genes and their products in
several herpesviruses including herpes simplex virus type
1 (HSV-1) (Frink et al., 1983; Gompels & Minson, 1986;
Highlander et al., 1988), pseudorabies virus (PRV)
(Robbins et al., 1987) and varicella-zoster virus (VZV)
(Keller et al., 1986; McGeoch & Davison, 1986). The
glycoproteins of equine herpesvirus type 1 (EHV-1) are
less well characterized than those of some of the other
herpesviruses, although some of their genes have been
located (Allen & Yeargan, 1987) and sequenced (Allen &
Coogle, 1988; Whalley et al., 1989; Audonnet et al.,
1990; Elton et al., 1991 b). Initially only one glycoprotein
(gp17/18) was mapped to the short unique (Us) region of
EHV-1 DNA (Allen & Yeargan, 1987). This is in
contrast to other alphaherpesviruses analysed, some of
which have several glycoprotein genes in Us. HSV-1 has
at least four glycoprotein genes in this region, coding for
gG, gD, gI and gE (McGeoch et al., 1985; Frame et al.,
The EHV-1 gD sequencehas been deposited with GenBank and
assigned the accessionnumber M60946.
0001-0742 © 1992 SGM
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1228
D. M. Elton and others
determined as the previously published sequences (Audonnet et al., 1990; Flowers et al., 1991) differ in the
predicted amino acid sequence at the C terminus. These
experiments, in conjunction with Western blot analysis
and prokaryotic gene expression, were designed to
establish which of the three candidate genes in EHV-I
Us codes for gp17/18.
Methods
Cells and media. RK13 cells were grown as monolayers in
autoclavable Dulbecco's modified Eagle's medium containing 10~ calf
serum.
Viruses. EHV-I strains Abl and Ab4, both low passage isolates, were
kindly provided by Dr J. Mumford, Animal Health Trust, Newmarket,
U.K. KyA, a high passage laboratory strain, was kindly supplied by Dr
H. Marsden, Institute of Virology, Glasgow, U.K. with the permission
of Dr D. J. O'Callaghan. Plaque-purified Ab4 was kindly supplied by
Dr A. Davison, Institute of Virology, Glasgow, U.K. All strains were
grown in RK13 ceils and virus stocks were prepared as previously
described (Meredith et al., 1989). DNA was extracted from cellassociated virus using the method of Robbins et aL (1988).
Southern blotting and DNA hybridization. Virus DNA was digested
with restriction endonucleases as recommended by the supplier (GibcoBRL). Approximately 1 ~tg samples of DNA were loaded onto 0.8%
agarose gels, subjected to electrophoresis at 8 V per cm in E buffer (0.04
M-Tris base, 0.002 M-EDTA, adjusted to pH 7.9 with glacial acetic acid)
and then stained in ethidium bromide. DNA fragments were
transferred to a GeneScreen hybridization membrane (New England
Nuclear) using vacuum blotting (Hybaid). The blots were air-dried,
baked at 80 °C and hybridized with radioactively labelled DNA
fragments (Rigby et al., 1977) using the membrane manufacturer's
recommended procedures.
Western blotting. Virus proteins were separated on 10~ acrylamide
gels (Laemmli, 1970) cross-linked with N,N'-diallyltartardiamine.
Samples were prepared by boiling purified virus for 3 min in a loading
buffer containing 0.125 M-Tris-HCI pH 6.8, 2 ~ SDS, 0.1~ bromophenol blue and 10~ glycerol, either with or without the addition of
DTT to 5 mM. Prokaryotic fusion proteins were prepared by
centrifugation of 1.5 ml of induced or uninduced culture for 1 min in an
Eppendorf centrifuge, washing the pellet in PBS and then resuspending
the pellet in 300 Ixl PBS. An aliquot was mixed with loading buffer
containing DTT as described above. Proteins were transferred from
acrylamide gels to nitrocellulose as described by Towbin et al. (1979).
Ascitic fluid containing the monoclonal antibody (MAb) 5H6, used by
Allen & Yeargan (1987) to map the gp17/18 gene, was diluted 1/500 in
10~ serum in PBS prior to use. Binding was detected using sheep antimouse lgG-peroxidase conjugate (Dako) diluted to 1/1000. The
peroxidase substrate was 0.6 mg/ml chloronaphthol, 0-01 ~ HzO z in
PBS.
DNA sequencing. Recombinant plasmids, containing EHV-1 DNA
cloned into pUCI8, were constructed using standard techniques
(Maniatis et al., 1982). Overlapping deletions were prepared using
unidirectional digestion with exonuclease III (Henikoff, 1984),
recircularized, then used to transform Escherichia coli strain TGI.
Plasmid D N A was then sequenced using T7 DNA polymerase
according to the manufacturer (Pharmacia). Nucleotide and derived
amino acid sequences were analysed using DNASIS and PROSIS
software packages (Hitachi).
Results
Comparison of gD sequences from Abl and KyA
The presence in EHV-1 of a gene homologous to HSV-1
gD was confirmed by sequence analysis of the DNA
upstream of the gI and gE genes (Audonnet et al., 1990;
Elton et al., 1991 b). The complete nucleotide sequence of
the EHV-1 strain Abl gD homologue was obtained from
both strands of the D N A by sequencing a 1602 bp Sinai
to HindlII region from the Us region of the genome
(GenBank accession number M60946). This sequence
was almost identical to that obtained by Audonnet et al.
(1990) for the Kentucky D (KyD) strain of EHV-1,
although there were a number of differences in the region
upstream of the predicted coding sequence of gD.
However, comparison of the Abl sequence with that
published for KyA by Flowers et al. (1991) identified a
frameshift at nucleotide 1798 in the KyA sequence,
which was due to a loss of two A residues. The derived
amino acid sequences for Abl and KyD were identical,
with the exception of a transition at position 195 from
glutamine in Abl to arginine in KyD, and the features
have been described for KyD by Audonnet et al. (1990).
The amino acid sequence for the KyA gD homologue
was ten amino acids shorter than that of Abl and the
terminal 13 amino acids were different, as shown in Fig
1. The difference between the C termini of these
polypeptide sequences resulted in a reduction in the
predicted Mr of the primary translation product from
45.2K for Abl or KyD to 44-0K for KyA. From these
data it can be predicted that the gD homologue of the
KyA strain would be 1.2K smaller than that of the other
two strains. The frameshift also resulted in the appearance of an extra cysteine residue which is predicted to fall
in the cytoplasmic portion of the glycoprotein.
Analysis of the glycoprotein genes in the Us region of the
KyA strain of EHV-1
A comparison of the published restriction analysis data
for the KyA strain of EHV-1 with that of other strains for
which data are available shows that KyA has a shorter
Us region (Whalley et al., 1981 ; Henry et al., 1981 ; Elton
et al., 1991a). Fig. 2 shows a comparison of the
arrangement of the Us regions of strains Abl (Elton et
al., 1991a, b) and KyA (Flowers et al., 1991). The data
suggest that KyA may have lost the genes for gE and gI.
To investigate this possibility, Southern blot analysis was
carried out on KyA genomic D N A digested separately
with the enzymes BamHI and EcoRI and compared with
D N A from the EHV-1 strains Abl and Ab4, the latter of
which contains a deletion of about 250 bp in Us (Fig. 3).
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1229
Glycoprotein D o f EHV-1
Abl
V G IS V G L G IA G L V L V G V IL Y V C L R R K K E L K K S A Q N G L T R L R S T F K D V K Y T Q L P
(a)
1 2 34
5 6 7 8
(b)
1 2 34
5 6
7
8
Fig. 11 Comparison of the deduced amino acid sequences of the Cterminal portions of the gD homologues from EHV-1 strains Abl and
KyA. Identical amino acids are indicated by colons. The position of an
additional cysteine residue in KyA is shown by an asterisk.
3858 bp
Abl
B
I
US9',',gE
i
HEH
,~
gI ,gD,
H
t
BE
\x~,
KyA
",~H
(~)US9 gD
Fig. 2. Diagram showing the layout of some of the genes in the Us
portions of Ab I and KyA. The position and size of the deletion in KyA
was determined by comparison of the nucleotide sequences from Abl
(Elton et al., 1991b) and KyA (Flowers et aL, 1991).
The digested DNA was blotted onto a nylon membrane
and probed sequentially with 32p-labelled EHV-1 DNA
probes comprising total Ab 1 DNA, gE coding sequence,
gI coding sequence or gD coding sequence.
Fig. 3 (a) shows that the KyA DNA hybridizes to the
total Ab 1 probe. Comparison of the KyA profiles in Fig.
3 (a) (lanes 1 and 5) with the other strains (lanes 2 to 4 and
6 to 8) shows that the KyA strain differs extensively from
the others. This is particularly evident with the EcoRI
profiles (lanes 5 to 8). Fig. 3(b) clearly shows that
whereas Abl and Ab4 contain DNA which hybridizes
with the EHV-1 gE probe, the KyA DNA shows no
hybridization at all, indicating that this gene is not
present in this strain. Fig. 3 (c) shows an identical result
obtained by reprobing with the EHV-1 gI coding
sequence. As expected from the map location of the gE
and gI gene homologues (Elton et al., 1991 a), both probes
hybridize to the B a m H I D and EcoRI B and C fragments,
as defined by the restriction maps of Whalley et al.
(1981). The Southern blot was then probed with a
fragment of DNA encoding the complete gD sequence
(Fig. 3d). This blot shows that all the strains contain
DNA to which the gD probe hybridizes, as expected
from DNA sequence analysis. The B a m H I fragment to
which the gD probe hybridizes in KyA is, however,
considerably smaller than that found in the other strains.
This confirms the smaller size of the B a m H I fragment in
the centre of the KyA Us region from the EHV-1
restriction map of Henry et aL (1981), compared to that
of Whalley et al. (1981). Analysis of the EcoRI fragments
(lanes 5 to 8) shows that in KyA, Abl and plaquepurified Ab4 the gD probe hybridized to three frag-
(c)
1 2 3 4 5
6 7 8
(d)
1 2 3 4
5 6 7 8
Fig. 3. Autoradiograms from a Southern blot of DNA from EHV-1
strains digested with BamHI (lanes 1 to 4) and EcoRI (lanes 5 to 8).
Lanes 1 and 5, KyA; lanes 2 and 6, Abl; lanes 3 and 7, non-plaquepurified AM; lanes 4 and 8, non-plaque-purified Ab4. The Southern
blot was sequentially probed with (a) Ab 1 genomic DNA, (b) gE coding
sequence, (c) gI coding sequence and (d) gD coding sequence. The
closed triangle in (d) indicates the position of the KyA BamHI M
fragment. The open triangle indicates the difference in the EcoRI K
fragments of Abl and plaque-purified Ab4. The non-plaque-purified
Ab4 contains an equimolar mix of both sized EcoRI K fragments.
ments, all of which were smaller in KyA. In Abl these
bands correspond to EcoRI B, C and K. The additional
smaller fragment seen in Fig. 3 (d), compared to (b) and
(c), hybridizes to the gD probe because this gene spans
the EcoRI site within the B a m H I D fragment. Lane 8 of
Fig. 3 (d) shows that the non-plaque-purified isolate of
Ab4 contains a mixed population of molecules in
approximately equal proportions, as the gD probe
hybridizes to a doublet band corresponding to the single
EcoRI K fragment of Abl. This is the result of a deletion
of approximately 250 bp within the B a m H I D fragment.
The upper band of the doublet is the same size as the
EcoRI K fragment in Abl, and the single band in the
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D. M. Elton and others
1230
1
2
3
4
5
6
1
2
3
4
5
6
-l16K
--97K
--66K
--45K
--46K
•
~..ii~~.
•~ ~:
....
-30K
Fig. 4. Western blot of purified virus reacted with MAb 5H6. Lanes 1
and 4, Abl ; lanes 2 and 5, Ab4 (plaque-purified); lanes 3 and 6, KyA.
Lanes 1, 2 and 3 non-reduced; lanes 4, 5 and 6 are thiol-reduced. The
positions of Mr markers are indicated.
plaque-purified Ab4 preparation is the same size as the
lower band of the doublet, indicating that this isolate
contains only D N A with the deletion in U s.
Confirmation of the identity of the product of the gD gene
homologue
It has already been shown that the gene for gp 17[ 18 maps
to a 4.7 kb BamHI to EcoRI fragment (Allen & Yeargan,
1987) in which we, and others, have located the genes for
gE and gI homologues (Audonnet et al., 1990; Elton et
al., 1991 a, b). We have also shown that this fragment of
D N A contains a significant portion of the gD gene
homologue. To identify the gene coding for gplT/18 we
have used Western blot analysis of purified virus from
EHV-1 strains KyA, A b l and Ab4 and a MAb, 5H6,
originally used to m a p the gene for gp17/18 (Allen &
Yeargan, 1987). Fig. 4 shows the reaction of the antibody
on purified virus proteins under both reducing and nonreducing conditions. In lanes 1 to 3, which contain
samples treated by boiling without D T T , bands are
clearly present in all three lanes showing that all three
Fig. 5. Western blot analysis of glutathione S-transferase-gD fusion
protein expression. Lanes 1, 3 and 5, non-induced; lanes 2, 4 and 6,
induced with 0.5 mM-IPTG.Lanes 1 and 2, pDE405/2; lanes 3 and 4,
pDE405/4; lanes 5 and 6, pDE406. Both isolates of pDE405 have the
gD gene in the correct orientation for expression; the insert in pDE406
is in the reverse orientation. The last lane shows Mr markers.
strains contain gp17/18. In A b l and Ab4 (lanes 1 and 2)
5H6 reacts with a band o f 5 0 K ; with K y A there is a weak
reaction with a similar band, but a much stronger band
of 102K is also present. In lanes 4 to 6, containing
reduced samples, 5H6 reacts with a species of 54K in
A b l and Ab4 and a slightly smaller protein of 53K in
KyA.
Fusion protein expression
Part of the gD gene o f E H V - 1 (nucleotides 1071 to 1602)
was subcloned into the prokaryotic expression vector
p G E X 1 (Smith & Johnson, 1988). Ligation of D N A
fragments, in frame, into this vector allows synthesis of
foreign proteins fused to the C terminus ofglutathione Stransferase. Expression is isopropyl fl-D-thiogalactopyranoside (IPTG)-inducible, controlled by the lacZ promoter. Recombinant plasmids were constructed which
had the gD sequence in either orientation; the fragment
was inserted into the EcoRI site of pGEX1 and
corresponded to the 3' end of the gene located within the
4.7 kb BamHI-EcoRI fragment to which gp17/18 had
been m a p p e d (Allen & Yeargan, 1987).
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Glycoprotein D o f E H V - 1
Western blot analysis of bacterial lysates from induced
and non-induced cultures of E. coli harbouring the
recombinant plasmids was performed (Fig. 5), using the
anti-gpl7/18 MAb 5H6. Fig. 5 shows that this antibody
reacted with a polypeptide of approximately 45K in
cultures containing pDE405/2 and pDE405/4 (two
independently isolated plasmids which gave identical
restriction enzyme profiles, indicating that the gD
fragment was in the correct orientation for expression),
but gave no reaction with pDE406 (a plasmid containing
the gD fragment in the reverse orientation for expression). Expression of the fusion protein was significantly
enhanced by the IPTG induction. The predicted Mr of
the glutathione S-transferase-gD fusion protein is 44K
which is in agreement with the observed data. Together
with the hydridization data, this provides conclusive
evidence that gp17/18 is a homologue of HSV-1 gD.
Discussion
The gp17/18 gene was originally mapped to the Us region
of the EHV-1 genome by Allen & Yeargan (1987). We,
and others, have sequenced the D N A from this region
(Audonnet et al., 1990; Elton et al., 1991 a, b) and have
located complete open reading frames for gI and gE gene
homologues and part of an open reading frame potentially coding for a gD gene homologue. We have
completed the sequence of the remaining portion of the
putative EHV-1 gD gene for the strain Abl and shown
that it shares significant identity with the gD of HSV-1.
The derived amino acid sequence of the gD homologue
was almost identical to that of KyD (Audonnet et al.,
1990) but differed significantly from the C terminus of
KyA gD, suggesting that the latter may be atypical of
EHV-1 strains. Analysis of the strain KyA using
Southern blot analysis and D N A hybridization has
confirmed that this strain has a smaller B a m H I fragment
in the centre of Us and lacks the genes for the EHV-1 gI
and gE homologues. Hybridization studies have shown
that the majority of natural isolates of this virus contain a
fragment whose size is indistinguishable from that of the
B a m H I D fragment of Abl (data not shown), implying
that they probably do contain genes for gI and gE. The
result for KyA indicates that these genes are not essential
for virus growth in tissue culture, which is known to be
the case in HSV-1 (Longnecker et al., 1987). This may
corroborate the suggestion that these glycoproteins have
a role in viral evasion of host immune response via Fcreceptor complex formation (Adler et al., 1978; Bell et
al., 1990; Hanke et al., 1990). As the KyA strain has been
passaged extensively in tissue culture (Perdue et al.,
1974) it is possible that these genes have been lost
through a deletion mutation arising in cell culture. This is
1231
consistent with these glycoproteins having a role only in
natural infection and there being no selective pressure to
maintain these genes in cell culture.
Analysis of the structural proteins of KyA using MAb
5H6 has confirmed the presence of gp17/18. This
provides strong evidence that this glycoprotein is a
product of the gD gene homologue, as this is the only
glycoprotein gene present in KyA in the region to which
gp17/18 was mapped. The difference in electrophoretic
mobility of gp 17/18 in KyA and Ab 1 can be explained by
the fact that the Abl strain, as with the KyD strain
(Audonnet et al., 1990), has a gp17/18 that is 10 amino
acid residues longer than that found in the KyA isolate.
The difference in both length and the amino acid
sequence at the C termini could account for the
difference in Mr of l'2K that was seen on the Western
blots. In the absence of reducing agent KyA virus
particles contained a protein with an apparent Mr of
102K, a size consistent with the formation of a gD dimer.
The C terminus of the KyA gD contains an additional
cysteine residue, compared to Abl, which may play a
part in the apparent dimerization seen on Western blots
of purified KyA. The complex was disrupted only by the
addition of DTT and not by boiling, as both non-reduced
and reduced samples were heated prior to electrophoresis, consistent with the involvement of disulphide
bridges. This may reflect the form in which the KyA gD
homologue is found in mature virus particles or may
result from the method used to prepare samples for
electrophoresis. This is currently under further study.
The expression of the C-terminal portion of the gD
gene in E. coli followed by the reaction of the product
with MAb 5H6 confirmed that gp17/18 is encoded by the
gD gene homologue. Other studies carried out on
gp 17/18 have shown that this glycoprotein shares similar
characteristics with gD of HSV-1, implying that in
addition to sequence homology these glycoproteins may
also have a similar function. We have previously shown
that anti-gp 17/18 MAbs can induce passive immunity to
EHV-1 in a small animal model (Stokes et al., 1991),
indicating that this glycoprotein carries epitopes important for recognition by the host immune system. We have
also shown that antibodies to gp17/18 neutralize virus
infectivity and inhibit virus penetration (Whittaker et
al., 1992). The C-terminal portion of HSV-1 gD may be
important in the induction of a virus-neutralizing
antibody response as three peptides from this region
were successfully used to raise neutralizing antisera
(Strynadka et al., 1988). It is possible that the EHV-1 gD
fusion protein described here may induce a neutralizing
response when inoculated into a small animal model,
because this protein contains the sequences equivalent to
the HSV-1 peptides used by Strynadka et al. (1988).
Studies are now in progress to evaluate the potential of
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D. M . Elton and others
the EHV-I gD fusion protein and other recombinant
f o r m s o f this g l y c o p r o t e i n to s t i m u l a t e a h o s t i m m u n e
response.
The authors would like to thank George Allen for providing the
MAb 5H6, Howard Marsden and Denis O'Callaghan for supplying the
KyA strain, Andrew Davison for the plaque-purified isolate of Ab4,
Jenny Mumford for the original Ab4 isolate and Clay Flowers for
helpful discussions. This work was supported by the Equine Virology
Research Foundation.
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(Received 19 November 1991; Accepted 6 January 1992)
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