1. No category

JOURNAL OF VIROLOGY, Nov. 1992, p. 6451-6460
Vol. 66, No. 11
0022-538X/92/116451-10$02.00/0
Copyright © 1992, American Society for Microbiology
Equine Herpesvirus 1 Glycoprotein D: Mapping of the
Transcript and a Neutralization Epitope
C. CLAY FLOWERS AND DENNIS J. O'CALLAGHAN*
Department of Microbiology and Immunology, Louisiana State University Medical Center,
Shreveport, Louisiana 71130-3932
Received 22 June 1992/Accepted 6 August 1992
DNA sequence analysis of the unique short sequence (Us)
of the short region of the equine herpesvirus 1 (EHV-1)
genome has revealed the presence of nine open reading
frames (ORFs) (1, 3, 9, 16, 19, 20). Five of these ORFs
encode potential glycoproteins, four being homologs of
herpes simplex virus type 1 (HSV-1) gG (9), gD (1, 16, 19),
gI, and gE (1, 16). The EUS4 ORF specifies a potential
glycoprotein that has no counterpart in any herpesvirus
sequenced to date (9). With the single exception of varicellazoster virus (10), the alphaherpesviruses HSV-1 (37, 64),
pseudorabies virus (50), and bovine herpesvirus type 1 (62)
and the gammaherpesvirus Marek's disease virus (55, 56)
encode a gD which appears to mediate key events in the
early phase of the replication cycle (60). Notable among the
gD polypeptides is the conservation of cysteine residues
located within the central portion of the surface-exposed
domain. Studies of HSV-1 gD have indicated that these
cysteines form intramolecular disulfide bonds that are essential for the structural integrity of the protein (34, 67). Studies
to elucidate the antigenic structure of HSV-1 gD revealed
that the location of discontinuous epitopes correlates with
the placement of cysteine residues, indicating that the central domain of gD possesses a highly ordered structure (39).
In contrast, most continuous epitopes lie near the amino
terminus and within the carboxyl one-third of HSV-1 gD
(39).
Previous studies have implicated HSV-1 gD in virus
penetration since anti-HSV-1 gD antibodies neutralize infectivity (7, 13-15, 21, 28, 38), retard fusion of infected cells (4,
28, 38, 41), and inhibit penetration without affecting adsorp*
Corresponding author.
tion (21, 28, 41). Direct evidence for the participation of gD
in penetration was obtained by the demonstration that
HSV-1 virions that lack gD could attach to cells but were
noninfectious as a result of a block in virus entry (31, 33).
Similarly, the HSV-1 gD homologs of pseudorabies virus
.(gpSO) and bovine herpesvirus type 1 (gIV) were shown to be
essential gene products that are involved in the process of
penetration (11, 18, 47, 53). Recently, Muggeridge et al. (40)
and Feenstra et al. (17), through the analysis of a series of
HSV-1 gD deletion mutations, identified a domain that is
essential for infectivity.
The HSV-1 gD gene is transcribed as a 3.0-kb mRNA that
is one of three transcripts of a 3'-coterminal nested set
mapping within the Us of HSV-1 (29, 37, 54). The gD
transcript and polypeptide can be detected as early as 2 h
after infection but do not reach maximal levels until after the
onset of viral DNA replication; therefore, the gD gene has
been assigned to the beta-gamma kinetic class (8, 29, 32, 46,
58).
In contrast to the extensive knowledge of HSV-1 gD, little
is known about the nature of EHV-1 gD. In the present
study, the gD gene of EHV-1 KyA strain was shown to be
transcribed as a 3.8-kb mRNA that belongs to the betagamma kinetic class. This finding agrees with our recent
observations that gD is not regulated as an early gene since
the requirements for transactivation of the EHV-1 gD promoter differ from those of the early EHV-1 thymidine kinase
(TK) promoter (59). The cis-acting promoter elements of the
gD gene, as well as the 5' terminus of the gD transcript, map
within the Us. However, the 3' terminus of the gD transcript
maps within the terminal inverted repeat (TR) component to
sequences downstream of a consensus polyadenylation signal, a termination site used by at least two other mRNAs that
6451
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Studies with molecular and immunological techniques identified and mapped the transcript encoding
glycoprotein D (gD) of equine herpesvirus 1 KyA, as well as two continuous gD antigenic determinants. Three
mRNA species of 5.5, 3.8, and 1.7 kb overlap the gD open reading frame and are transcribed from the DNA
strand encoding gD. Northern (RNA) blot hybridization with both DNA clones and riboprobes, as well as S1
nuclease analyses, showed the 3.8-kb mRNA to encode gD and to be synthesized as a late (beta-gamma)
transcript. The 3.8-kb gD mRNA initiates within the Us segment 91 and 34 nucleotides downstream of the
CCAAT and TATA elements, respectively, and encodes a potential polypeptide of 392 amino acids. The
termination site of this transcript maps within the terminal repeat at a site also used by the 5.5-kb mRNA and
the IR6-encoded 1.2-kb mRNA, such that these three transcripts form a 3'-coterminal nested set. The extended
size (2,250 nucleotides) of the 3' untranslated region of the gD transcript and its termination within the terminal
repeat may result from the deletion of 3,859 bp, which eliminates two consensus polyadenylation signals
downstream of the gD open reading frame of EHV-1 KyA. Use of antisera to synthetic peptides of 19 amino
acids (residues 4 to 22) and 20 amino acids (residues 267 to 285) in Western immunoblot analyses revealed that
gD is present in EHV-1 virions as a 55-kDa polypeptide. In addition, these antisera detected the 55-kDa protein
as well as 58- and 47-kDa polypeptides in infected-cell extracts at late times of infection. Residues 4 to 22 make
up a continuous neutralizing epitope of gD, since incubation of equine herpesvirus 1 with the anti-19-mer serum
prior to infection results in reduced numbers of plaques and reduced levels of vims-encoded thymidine kinase.
Complement is not required for neutralization mediated by the anti-19-mer serum.
6452
FLOWERS AND O'CALLAGHAN
are 3' coterminal with the gD mRNA. The EHV-1 gD gene
product was identified, by use of peptide-specific antibodies,
as a 55-kDa protein present in purified virions and infectedcell extracts. Synthetic peptides used as immunogens correspond to residues 4 through 22 and 267 through 285 of the
predicted mature gD polypeptide. Each of these domains
aligns with continuous antigenic sites of HSV-1 gD, suggesting that these two gD polypeptides have common structural
features. Importantly, the synthetic peptide composed of
residues 4 through 22 elicits the production of complementindependent neutralizing antibodies, as is the case for the
analogous residues of HSV-1 gD (7, 13, 38). These data
suggest that EHV-1 gD, like its HSV-1 counterpart, plays a
role in penetration.
N.J.).
Labeling of DNA and RNA probes. EHV-1 cloned restriction fragments were 5' end labeled following digestion with
the appropriate restriction endonuclease, dephosphorylation
with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.), extraction with phenol-chloroform (24:1, vol/vol), and precipitation with ethanol. Dephosphorylated DNA fragments were 5' end labeled
with [.y-32P]ATP (3,000 Ci/mmol; Dupont NEN) and T4
polynucleotide kinase (Bethesda Research Laboratories, Bethesda, Md.). Unincorporated label was removed from the
reactions by spin chromatography with Sephadex-G50 columns. Dephosphorylated oligonucleotides were purchased
from Synthetic Genetics Inc., San Diego, Calif., and were 5'
end labeled as described above. Klenow fragment (Dupont
NEN) was used to 3' end label restriction fragments as
described previously (25-27). Labeling reactions took place
away,
directly in restriction endonuclease reaction mixtures by the
addition of [a-32P]dCTP (800 Ci/mmol; Dupont NEN); 10
mM each dATP, dGTP, and dTTP (Bethesda Research
Laboratories); and 1 U of Klenow fragment (Dupont NEN).
The reaction mixtures were incubated at 30°C for 15 min,
and spin columns were used to separate labeled DNA from
unincorporated label.
Strand-specific riboprobes were generated by using the
Riboprobe Gemini System (Promega, Madison, Wis.).
EHV-1 DNA restriction fragments cloned into the pGEM-3Z
plasmid vector were restriction digested so that runoff transcripts could be synthesized from either the T7 or SP6 phage
promoters (27). Linear recombinant plasmids were gel purified by using the GeneClean DNA elution kit (BIO 101 Inc.,
La Jolla, Calif.). Single-stranded RNA probes labeled to high
specific activity with SP6/T7 grade [a-32PJCTP (Amersham
Corp., Arlington Heights, Ill.) were generated by using
either SP6 or T7 polymerase as specified by the manufacturer (Promega).
S1 nuclease analysis. Termini of viral mRNAs were
mapped by S1 nuclease analysis as described by Berk and
Sharp (2). Restriction endonuclease fragments labeled at the
5' or 3' end were hybridized overnight to 3.0 ,ug of poly(A)
mRNA in buffer containing 40 mM PIPES [piperazine-N,N'bis(2-ethanesulfonic acid)] (pH 6.4), 0.4 M NaCl, and 1 mM
EDTA. The temperature for hybridization was varied to
ensure that protected bands were not due to aberrant S1
nuclease cleavage. DNA-RNA hybrids were digested at 37°C
with 60 U of S1 nuclease enzyme (Bethesda Research
Laboratories) in a reaction buffer containing 0.250 M NaCl,
0.030 M sodium acetate (pH 4.6), and 0.001 M ZnCl2. The
S1-resistant fragments were analyzed by electrophoresis
through 6.0% polyacrylamide-urea gels.
Peptide-specific antibodies. The IBI Pustell software package, using the program of Jameson and Wolf (30), was used
to predict highly immunogenic regions of EHV-1 gD from
the amino acid sequence (19). Two regions, both of which
are predicted to localize to the surface-exposed domain of
gD, were selected for the synthesis of peptides. A 19-aminoacid peptide (19-mer; CEKAKRAVRGRQDRPKEFP) represents residues 4 to 22 of the predicted mature polypeptide.
A peptide of 20 residues (20-mer; EITQNKTDPKPGQADP
KPNC) represents a 19-amino-acid (a cysteine residue was
added for the coupling reaction) region located at residues
267 to 285. The peptides were synthesized by LSUMC
Core Laboratories, New Orleans, La.) and were coupled to
keyhole limpet hemocyanin (United States Biochemicals,
Cleveland, Ohio) by using glutaraldehyde or m-maleimidobenzoyl-N-hydroxysuccinimide ester (24). As a primary
immunization, New Zealand White rabbits were injected
intramuscularly with 0.5 mg of peptide-carrier conjugate
emulsified in complete Freund's adjuvant (1:1 [vol/vol];
Sigma). For subsequent immunizations, administered at
4-week intervals, 0.25 mg of peptide-carrier conjugate emulsified in incomplete Freund's adjuvant (Sigma) was used.
Peptide-specific antibodies were readily detected after the
second immunization by their reactivity to uncoupled peptide in a dot blot assay (24). Peptide-specific antibodies were
purified by affinity chromatography on columns containing
the appropriate peptide coupled to activated CH Sepharose
4B (Pharmacia LKB Biotechnology, Piscataway, N.J.). Column elution profiles were monitored by measuring the A280.
Immunoblot analysis. Solubilized proteins from EHV-1
virions or infected cells were analyzed by Western immunoblot analysis as previously described (6, 61). EHV-1 virions
were purified from supernatants of infected cultures by
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MATERIALS AND METHODS
Virus and cell culture. The EHV-1 Kentucky A (KyA)
strain was serially propagated by infection of LM cell
suspension cultures at low multiplicity of infection (0.05
PFU per cell [42]). Virus infectivity was quantitated by
plaque titration (42, 48).
Isolation of RNA and Northern blot analysis. For the
isolation of RNA, LM cells were infected with 15 PFU per
cell to ensure that at least 99% of the cells were infected.
Immediate-early RNA was isolated at 4 h postinfection from
cells pretreated with cycloheximide (final concentration, 100
p,g/ml; Sigma Chemical Co., St. Louis, Mo.) for 1 h before
infection. Early RNA was isolated at 8 h postinfection from
cells pretreated with 100 ,g of phosphonoacetic acid (PAA;
Abbott Laboratories, Chicago, Ill.) per ml for 1 h before
infection. Caughman et al. (6) have shown previously that
viral protein and DNA syntheses are inhibited effectively by
these concentrations of cycloheximide and PAA, respectively. For late RNA, no metabolic inhibitors were used and
the infected cells were harvested at 6, 8, and 10 h after
infection. The poly(A) fraction of total RNA was obtained
from infected cells by using the Fast Track mRNA isolation
kit (Invitrogen Corp., San Diego, Calif.). Virus-specific
transcripts were detected by Northern (RNA) blot analysis
in which 2 to 3 p,g of poly(A) RNA per lane was fractionated
on a 1.2% formaldehyde-agarose gel by standard techniques. After transfer of RNA to nitrocellulose (Schleicher
& Schuell, Keene, N.H.), the filters were probed with
[32P]dCTP- and [32P]dGTP-labeled cloned restriction fragments as described previously (22, 23, 25, 26). Molecular
size standards used in Northern blot experiments included
28S (4.8-kb) and 18S (1.9-kb) calf liver rRNA and 23S
(2.9-kb) and 16S (1.5-kb) E. coli rRNA (Pharmacia, Piscat-
J. VIROL.
VOL. 66, 1992
differential centrifugation, polyethylene glycol precipitation,
and dextran-10 rate velocity centrifugation (48). For the
preparation of infected-cell extracts, LM cells were infected
at a multiplicity of infection of 15 PFU per cell and harvested
10 h postinfection. Purified virions or infected cells were
solubilized in 1% sodium dodecyl sulfate (SDS) at 100°C for
1 min and sonicated for 30 s, and the extracts were clarified
by centrifugation at 50,000 rpm in a TLA 100.3 Beckman
RESULTS
Identification and kinetic class of transcripts overlapping
the EHV-1 gD ORF. To identify and characterize the mRNA
species transcribed from the gD coding sequences, we
performed Northern blot hybridization analyses with RNA
isolated from EHV-1-infected LM cells. Clone pSZ-4, which
contains the entire gD ORF (Fig. 1), (19) was P labeled by
nick translation, and the labeled DNA was hybridized to
poly(A) RNA isolated from untreated cells at 2-h intervals
between 2 and 10 h postinfection or isolated from cells
treated with metabolic inhibitors (see Materials and Meth-
6453
us
A.
I
PK
B.
-.--
go
IR6
EUS4
D
-.
V USS
-'
-
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I
fI
C.
X
pCF3 K
I
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-
pCF9
P p1-101
S
~ ~II
~
~~~~I
2224
pCF5 pCF6
gEUS4
B
~
1
3576
4735
pCF7
gD
US9
IR6
5.5
3.8
D.
1.7
11.2Ib
FIG. 1. Cloned restriction fragments, ORFs, and mRNAs mapping to Us and TR sequences of the EHV-1 KyA genome (43, 45).
(A) The structure of the viral genome is shown with the UL and Us
segments depicted by solid lines and the internal (solid box) and
terminal (striped box) IRs represented by rectangles. (B) Expanded
map of the Us and part of the IR and TR showing the positions of
ORFs of EHV-1 KyA that have been identified by DNA sequence
analysis. Within the IRs only the IR6 ORF is shown, while in the Us
the ORFs encoding the HSV-1 homologs of US2, PK, gG, gD, and
US9 are shown. The predicted product of the EUS4 ORF may be
unique to EHV-1. The open triangle between the gD and US9 ORFs
represents the 3,859-bp deletion of U. sequences encoding gI, gE,
and a unique 10-kDa ORF from the EHV-1 KyA genome (20). (C)
Expanded map of portions of the U. and TR. The designations of
cloned restriction fragments used in transcription mapping are noted
above the lines. The nucleotide positions of restriction cleavage
sites located between the Kpnl (K) site (nucleotide 1) and the SmaI
(S) site (nucleotide 4735) are noted below the lines. B, BamHI; X,
XbaI; P, PvuII). Arrows denote the position and direction of ORFs
encoding gEUS4 (9), gD (19), US9 (20), and IR6 (3). (D) Positions of
viral mRNAs as determined by Northern blot and Si nuclease
analyses. Arrows represent the position and direction of mRNAs. A
vertical bar denotes the map position of the 5' terminus, and a
dashed line indicates that the precise map position of the 5' terminus
was not determined. Analysis of transcripts mapping within the IR
has demonstrated that the diploid IR6 gene is transcribed as a 1.2-kb
mRNA (3). As predicted, the 1.2-kb mRNA was also detected within
the TR.
ods). No hybridization signal was observed with mockinfected RNA (Fig. 2, lane 1) or with RNA isolated at 2 and
4 h postinfection (lanes 2 and 4, respectively). At 6 h
postinfection (lane 5), a 3.8-kb mRNA was detected, and by
8 and 10 h postinfection (lanes 6 and 8, respectively), two
additional mRNAs of 5.5 and 1.7 kb were observed. All three
mRNAs accumulated to high levels by 10 h postinfection
(lane 8). No transcripts were detected when RNA isolated
under immediate-early conditions (cycloheximide block) of
infection was used (lane 4). Under early conditions of
infection (PAA block [lane 7]), the 3.8-kb species was barely
detectable and the 5.5- and 1.7-kb mRNAs were not observed. These data suggest that the 3.8-kb mRNA is a
member of the beta-gamma kinetic class since this mRNA
was detected, albeit at low levels, under early conditions of
infection and accumulated to significant levels in untreated
cells at 6 h postinfection (2 h after the onset of EHV-1 DNA
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rotor. Buffer containing protease inhibitors was added to the
extracts, resulting in final concentrations of 50 mM Tris-Cl
(pH 7.4), 190 mM NaCl, 0.5 mM EDTA, 0.2% SDS, 1%
Triton X-100, 50 ,ug of aprotinin per ml, 50 ,ug of leupeptin
per ml, and 20 ,ug of phenylmethylsulfonyl fluoride (Sigma)
per ml. Samples were electrophoresed through 9% acrylamide gels (acrylamide/N,N'-methylenebisacrylamide ratio,
30:0.8 [vol/vol]), and the separated proteins were electrophoretically transferred to nitrocellulose. Nonspecific protein-binding sites were blocked by incubation with bovine
serum albumin (3% in 166 mM NaCl-10 mM Tris-Cl [pH
7.4]) for 1 h. Following incubation of filters with preimmune
sera or peptide-specific antibodies, 1"I-protein A was used
to detect bound antibodies (61). The molecular weights of
viral polypeptides were determined by comparison with
'4C-labeled molecular weight markers (Bethesda Research
Laboratories) separated on the same gel.
Virus neutralization assays. Neutralization of EHV-1 infectivity by gD-specific sera was monitored by assaying
virus-specific TK activity. LM cell monolayers in 96-well
microtiter plates were infected with virus which had been
preincubated for 1 h with serum (preimmune or peptidespecific serum diluted 1/160 with phosphate-buffered saline).
Three separate monolayers were infected at each multiplicity of infection ranging from 0 (mock infected) to 10, and the
infected cells were harvested 16 h after infection. The
method of Wolcott and Colacino (68) was used to monitor
EHV-1-specific TK activity in infected-cell lysates. The
protocol is based on the selective phosphorylation of 5'[12'I]iodo-2'-deoxycytidine (2,000 Ci/mmol; Dupont NEN)
followed by lanthanum chloride precipitation of phosphorylated compounds. TK activity is represented by the radioactivity present in the precipitate. Each determination of TK
activity represents the average of three independent TK
assays performed for each cell lysate.
Plaque reduction assays were also used to assess EHV-1
neutralization. To inactivate complement, preimmune or
peptide-specific sera were incubated for 1 h at 56°C. Virus
inoculum containing 3,000 PFU was incubated with serial
dilutions of preimmune or peptide-specific sera, and the
virus-antibody mixtures were incubated at 37°C for 1 h.
Virus infectivity was measured by plaque assay in LM cell
monolayers (48). Neutralization activities represent the average of three infected cultures and were calculated as the
percentage of plaques that had been neutralized.
mRNA AND EPITOPES OF EHV-1 gD
6454
J. VIROL.
FLOWERS AND O'CALLAGHAN
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synthesis [6]). The 5.5- and 1.7-kb mRNAs were not detected when viral DNA synthesis was inhibited with PAA
but were detected in unblocked infections at 8 h postinfection, suggesting that these mRNAs belong to the gamma
kinetic class.
Direction of transcription and fine mapping of the 5.5-, 3.8-,
and 1.7-kb mRNAs. To determine which of the three overlapping mRNA species encodes gD, we conducted Si nuclease analyses as well as Northern blot hybridization experiments with riboprobes and a series of cloned fragments
containing Us sequences. To generate riboprobes, clone
pCF6 was used since it contains potential cis-regulatory
elements for gD expression and sequences encoding the
amino-terminal portion of the gD protein (Fig. 1) (19). Clone
pCF6 was inserted into pGEM-3Z such that transcription
from the T7 phage promoter yielded a riboprobe complementary to the gD coding strand while transcription from the SP6
phage promoter yielded a riboprobe complementary to the
noncoding strand of gD. As shown in Fig. 3A (lane 4), the
T7-generated riboprobe hybridized to all three mRNAs (5.5,
3.8, and 1.7 kb). The hybridization to the 5.5- and 3.8-kb
mRNAs was readily apparent by 15 min of autoradiographic
exposure, whereas the 1.7-kb mRNA required a longer
exposure for its detection. The SP6-generated riboprobe
failed to hybridize to any of the three mRNAs (Fig. 3A, lane
2), even after prolonged periods of autoradiographic exposure. These results demonstrate that the 5.5-, 3.8-, and
1.7-kb mRNAs are transcribed from the gD coding strand.
To determine the map location of each mRNA, we performed Northern blot analyses with a series of contiguous
clones containing Us and/or terminal repeat (TR) sequences
as probes (Fig. 1). Clones pCF3, pCF5, pCF6, and pCF7
(Fig. 1), which contain only Us sequences, hybridized to
mRNAs of 5.5, 3.8, 2.9, and 1.7 kb (Fig. 3B). The 2.9-kb
mRNA terminates within clone pCF3 since this mRNA was
not detected with clone pCF5. The 1.7-kb mRNA was
detected with probe pCF6 but not with pCF7, indicating that
the 3' terminus of the 1.7-kb mRNA lies within clone pCF6.
Since the 3.8-kb mRNA was detected with clone pCF6 but
not with clone pCF5, the 5' terminus of this mRNA appears
1
2
3
4
1
2
3
4
5
6
FIG. 3. Mapping of viral mRNAs transcribed from Us and TR
sequences by Northern blot hybridization analyses. (A) Determination of the direction of transcription of the 5.5-, 3.8-, and 1.7-kb
mRNAs. A riboprobe complementary to the gD coding strand was
generated with T7 RNA polymerase and clone pCF6 (Fig. 1)
template DNA. An SP6 RNA polymerase-generated riboprobe was
used to detect mRNAs complementary to the gD noncoding strand.
The probes were hybridized to poly(A) RNA isolated at late times of
infection (10 h) from mock-infected (M; lanes 1 and 3) and EHV-1
infected (L; lanes 2 and 4) cells. The hybridization signal for the
1.7-kb mRNA is apparent upon longer autoradiographic exposure.
(B) Cloned restriction fragments (Fig. 1) were used as probes in
Northern blot analysis to identify the positions of viral mRNAs
isolated at 10 h postinfection. The Us/TR junction lies within clone
pCF9 between nucleotides 2730 and 2731 (Fig. 1).
to lie within clone pCF6. All four clones (pCF3, pCF5,
pCF6, and pCF7) which map within the Us hybridized to the
5.5-kb mRNA. The 5.5- and 3.8-kb mRNAs were also
detected with clones pCF9 and pl-101 (Fig. 3B). The Us/TR
junction is located within clone pCF9, 507 bp from the
BamHI cleavage site at position 2224 (Fig. 1) (20); therefore,
the 5.5- and 3.8-kb mRNAs are transcribed from both Us
and TR sequences. Clones mapping to the right of pl-101 do
not hybridize to the 5.5- and 3.8-kb mRNAs (data not
shown), suggesting that the 3' termini of the 5.5- and 3.8-kb
mRNAs map within clone pl-101. Additional mRNA species
of 2.3 and 1.2 kb were detected with clones pCF9 and pl-101
(Fig. 3B) and correspond to the mRNAs encoded by the
EHV-1 US2 and IR6 genes, respectively, which map at the
junction of the internal IR and the Us segment (3).
Northern blot analysis with probes which overlap the US9
ORF detected only the 5.5- and 3.8-kb mRNAs (data not
shown), suggesting that the 3,859-bp deletion of Us sequences (20) adversely affected expression of the US9 gene.
Audonnet et al. (1) noted that a TATA-like element was
located 140 nucleotides upstream of the US9 ORF in the
genome of the KyD strain of EHV-1; however, in the KyA
strain this potential US9 promoter element is not present
since the deletion maps 27 bp upstream of the TATA-like
sequence (20).
SI nuclease analysis. Northern blot analysis indicated that
the 5' terminus of the 3.8-kb mRNA was positioned within
clone pCF6. Indeed, the DNA sequence of pCF6 (19)
revealed CCAAT and TATA boxes and a possible cap site
within nucleotides 538 to 889 (Fig. 1). To determine more
precisely the transcription start site of the 3.8-kb mRNA, we
used S1 nuclease analysis. Clone pCF6 was digested with
NcoI (position 704 relative to the KpnI site [Fig. 1]), labeled
at the 5' terminus with y-32P, and hybridized to RNA
Downloaded from http://jvi.asm.org/ on December 29, 2014 by guest
FIG. 2. Northern blot analysis of poly(A) mRNA isolated from
EHV-1-infected LM cells at various times after infection in the
presence or absence of metabolic inhibitors. Clone pSZ-4 (Fig. 1)
was 32p labeled by nick translation and hybridized to RNA isolated
from mock-infected cells (lane 1), EHV-1-infected cultures at 2 h
(lane 2), 4 h (lane 3), 6 h (lane 5), 8 h (lane 6), and 10 h (lane 8) after
infection. Immediate early RNA (lane 4) was isolated at 4 h
postinfection from cells treated with cycloheximide, while early
RNA was isolated at 8 h postinfection from cells treated with PAA
(lane 7) as described in Materials and Methods.
1 .2
VOL. 66, 1992
mRNA AND EPITOPES OF EHV-1 gD
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FIG. 4. Si nuclease analysis of the 5' terminus of the 3.8-kb
mRNA. (A) Clone pCF6 was restriction digested and 5' end labeled
at the NcoI site at position 704 (relative to the KpnI site). The probe
was hybridized to RNA isolated from mock-infected (lane 2) and
EHV-1-infected ([ATE refers to 10 h postinfection [lane 3]) RNA at
58°C. Following digestion with Si nuclease, the DNA-RNA hybrids
were fractionated on a 6% polyacrylamide-urea gel. The diagram
below the gel in panel A outlines the positions of the transcripts
relative to the labeled probe (indicated by an asterisk). Lanes 4 to 7
show the M13mpl8 sequence used to determine the size of the
protected fragments. Additional molecular weight markers (in thousands) are shown in lane 1. (B) Si nuclease analysis of potential
transcription initiation sites downstream of the TATA box at position 328 (Fig. 1). Clone pCF5 end labeled at the 5' terminus of the
HpaI cleavage site (nucleotide 538) was used as probe in Si nuclease
analysis. Early RNA (lane 3) was isolated 8 h postinfection from
cells treated with PAA 1 h prior to infection. Late RNA (lane 4) was
isolated 10 h postinfection from uninhibited infections. The Siresistant fragment observed when late RNA (lane 4) was used is
approximately 538 nucleotides and corresponds to the size of the
probe.
isolated from mock-infected or EHV-1-infected cells at late
times of infection. The DNA-RNA hybrids were subjected to
digestion with Si nuclease, and the protected fragments
were analyzed on a 6% urea sequencing gel. Two Siresistant fragments of 166 and 115 nucleotides were observed when 10-h-postinfection RNA was used (Fig. 4A,
lane 3). The 166-nucleotide fragment corresponds to the size
of the probe and indicates full protection of the probe by the
5.5-kb transcript. The partially protected fragment of 115
nucleotides (Fig. 4A, lane 3) maps the 3.8-kb mRNA transcription initiation site 6 nucleotides downstream of the
predicted cap site at nucleotide 594. These data indicate that
transcription initiation of the 3.8-kb mRNA occurs 34 nucleotides downstream of the TATA box (position 561) and 68
nucleotides upstream of the second in-frame ATG (position
661) of the gD ORF. Thus, these findings suggest that the
3.8-kb mRNA encodes the gD gene product and that cisacting transcriptional regulatory elements located upstream
of the gD mRNA cap site direct expression of the gD gene.
The assignment of the gD transcription start site at nucleotide 594 by Si nuclease analysis suggests that the CCAAT
and TATA boxes located in clone pCF6 are important in
transcription of the gD gene. However, CCAAT- and TATAlike elements map within clone pCF5 (nucleotides 243 and
FIG. 5. Determination of the 3' termini of the 1.7-, 5.5-, and
3.8-kb mRNAs. (A) Si nuclease analysis of the 3' terminus of the
1.7-kb mRNA. Clone pSZ-4 (Fig. 1) was digested and 3' end labeled
at the KpnI terminus (asterisk). The probe was hybridized to
mock-infected (lane 2) and late-infected (10 h [lane 3]) mRNAs at
58°C. The positions of the transcripts relative to the labeled probe
are outlined in the diagram at the bottom of panel A. Molecular
weight markers (in thousands) are shown in lane 1. (B) Si nuclease
analysis of the 3' termini of the 5.5- and 3.8-kb mRNAs. Clone
pl-101 (Fig. 1) was 3' end labeled at the StyI restriction site (position
3731) and was hybridized to mock-infected (lane 1), or early-infected
(lane 2) RNA isolated from cells treated with PAA 1 h prior to
infection, and late-infected RNA (lane 3) isolated at 10 h from an
uninhibited infection.
328, respectively [Fig. 1]); therefore, it was important to
determine whether an additional transcriptional start site for
the gD mRNA exists. Si nuclease analysis with clone pCF5
labeled at the 5' terminus of the HpaI site (position 538) was
used as the hybridization probe. The approximately 538nucleotide fragment observed in analyses with late RNA
corresponds to the full-length probe protected by the 5.5-kb
mRNA (Fig. 4B). No partially protected fragment was
observed, indicating that transcription initiation does not
occur within clone pCF5. Thus, these data indicate that the
gD transcript initiates solely at position 594 and encodes a
polypeptide of 392 amino acids whose synthesis initiates at
the second in-frame ATG.
Northern blot analysis revealed that the 3' terminus of the
1.7-kb mRNA was located within clone pCF6. Analysis of
the DNA sequence revealed a consensus polyadenylation
signal sequence at position 649 (AATAAA [52]), a CA
dinucleotide at position 672 that may serve as the site of
cleavage, and a G+T-rich region (nucleotides 692 through
703) similar to that known to bind components of the
cleavage-polyadenylation complex (66). When clone pSZ-4
was 3' end labeled at the KpnI site (Fig. SA) and used as a
probe in S1 nuclease analysis, a single Si-resistant fragment
of approximately 696 nucleotides was observed with late
RNA but no protected fragments were observed with mockinfected RNA (Fig. SA). These data localize the 3' terminus
of the 1.7-kb mRNA to within 25 nucleotides of the predicted
cleavage site at position 672.
The 3' termini of the 5.5- and 3.8-kb mRNAs were mapped
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1 2
6456
FLOWERS AND O'CALLAGHAN
A
B
MW X 103
-9 7
-6 8
-5 5
_4
4?
55
4-5
-4 3
-29
1
2
3
4
5
6
1
2
3
FIG. 6. Identification of the EHV-1 gD gene products by immunoblot analysis with peptide-specific antibodies. (A) Solubilized
proteins of EHV-1-purified virions were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose.
Filters were reacted with affinity-purified anti-19-mer antibody (lane
2) or anti-20-mer antibody (lane 5) and, as negative controls,
preimmune serum (lanes 1 and 4). 1"I-protein A was used to detect
bound antibodies. The specificity of binding of the anti-19-mer
antibody (lane 3) and the anti-20-mer antibody (lane 6) to the 55-kDa
protein was demonstrated by competition with the appropriate
peptide. (B) Western blot analyses of infected-cell extracts with the
anti-19-mer gD antibody. Proteins present in extracts prepared from
4 x 105 mock-infected (lane 1) or EHV-1-infected (lane 2; 12 h
postinfection) LM cells were subjected to Western blot analysis with
the anti-19-mer gD antibody. Viral proteins of purified virions were
analyzed for comparison (lane 3). Identical results were obtained
with the anti-20-mer gD antibody.
mer serum, anti-20-mer serum, or preimmune serum. At 12 h
postinfection (when EHV-1 TK levels are maximal [data not
shown]), the cell monolayers were harvested and solubilized
and cell extracts were prepared for analysis of TK activity.
EHV-1 TK activity was measured by the method of Wolcott
and Colacino (68), and ['"I]iododeoxycytidine was used as
the virus-specific TK substrate. Cells infected with untreated
virus and cells infected with virus preincubated with preimmune serum showed similar levels of TK activity over a
range of multiplicities of infection (Fig. 7A). TK levels in
cells infected at a multiplicity of 10 PFU per cell with
untreated virus or virus incubated with preimmune serum
were approximately fivefold greater than those in mockinfected cells. Incubation of virus with anti-19-mer serum
prior to infection at a multiplicity of infection of 10 PFU per
cell resulted in a greater than twofold reduction in TK
activity compared with TK levels induced by untreated virus
or virus incubated with preimmune serum. In contrast to
these findings for the anti-19-mer serum, no evidence of
neutralizing activity was obtained for the anti-20-mer serum
(data not shown). As a positive control for neutralization,
virus was preincubated with anti-EHV-1 virion serum previously shown to have potent neutralizing activity (61). This
antiserum caused a reduction in TK levels equivalent to the
reduction observed by the anti-19-mer serum.
To verify that the anti-19-mer serum has neutralizing
activity and to determine whether this activity is complement independent, we performed plaque reduction assays by
preincubating approximately 3,000 PFU of EHV-1 with
serial dilutions of anti-19-mer serum that was either untreated or heated at 56°C for 1 h to inactivate complement.
As shown in Fig. 7B, the anti-19-mer serum achieved 99%
inhibition of plaque formation at a serum titer of 80 relative
to preimmune serum. Even at a serum titer of 1,280, approx-
Downloaded from http://jvi.asm.org/ on December 29, 2014 by guest
by Northern blot analysis to TR sequences contained within
clone pl-101. A polyadenylation signal sequence (AAT
AAA, nucleotide 4069), a potential cleavage/polyadenylation
site (CA; position 4086), and a G+T-rich region (nucleotides
4096 to 4118) were identified within the DNA sequence of
clone pl-101 (3). To determine more precisely the locations
of the 3' ends of the 5.5- and 3.8-kb mRNAs, clone pl-101
(Fig. 1) was digested with StyI (position 3731), 3' end
labeled, and hybridized to mock, early, or late RNAs (Fig.
SB). A single protected fragment of approximately 360
nucleotides was observed with both early and late RNA (Fig.
SB, lanes 2 and 3, respectively). This fragment maps the 3'
termini of both the 5.5- and 3.8-kb mRNAs to lie within the
TR at position 4091, or 6 nucleotides downstream of the
predicted CA dinucleotide cleavage site.
Recently, the 3' terminus of the 1.2-kb IR6 mRNA (transcribed from both inverted repeats [IRs]) and the 3' terminus
of the 2.3-kb US2 mRNA (transcribed from the Us into the
IR) were localized to sequences within the internal IR
identical to sequences within the TR used for the termination
of the 5.5- and 3.8-kb mRNAs (3) (see above). Therefore,
two families of 3'-coterminal mRNAs use this termination
site within the IRs. One family consists of the 2.3-kb mRNA
of the US2 gene and the 1.2-kb mRNA of the diploid IR6
gene, which terminate within the IR, while the second family
includes the 5.5-kb, 3.8-kb (gD), and 1.2-kb (IR6) mRNAs
which terminate within the TR. The 360-nucleotide S1resistant fragment observed with early RNA (Fig. 5B, lane 2)
results from partial protection of the probe by the 1.2-kb IR6
transcript, since this mRNA is the only species of the
3'-coterminal families that is present at significant levels
during early times of infection (3, 22, 23).
Identification of the EHV-1 gD gene product. The protein
product of the gD gene was investigated by using gD-specific
antibodies directed to synthetic peptides that correspond to
immunogenic domains predicted from computer analysis of
the gD amino acid sequence (19). A 19-mer representing
amino acids 4 through 22 of the mature polypeptide and a
20-mer corresponding to residues 267 through 285 were
coupled to keyhole limpet hemocyanin, and the peptidecarrier conjugates were used to immunize rabbits. Peptidespecific antibodies present in rabbit sera were readily detected by dot blot analysis and were purified by affinity
chromatography (data not shown). In Western blot analyses
of purified EHV-1 virions (Fig. 6A), both the anti-19-mer
(lane 2) and anti-20-mer (lane 5) antibodies reacted with a
55-kDa virion protein. The specificity of each antibody was
confirmed by the demonstration that the peptide used as
immunogen blocked the reaction of its respective antibody
with the 55-kDa EHV-1 virion protein (Fig. 6A, lanes 3 and
6). Western blot analysis was also used to identify gD
polypeptides present in EHV-1-infected cells harvested at
late times of infection (12 h postinfection [Fig. 6B]). Both the
anti-19-mer (Fig. 6B, lane 1) and the anti-20-mer (data not
shown) antibodies failed to react with proteins present in
mock-infected cells. In contrast, both peptide-specific antibodies reacted with the 55-kDa protein as well as two
additional polypeptides of 58 and 47 kDa present in infected
cells (Fig. 6B, lane 2).
Identification of an EHV-1 gD neutralization epitope. gDspecific antibodies were tested for their ability to neutralize
virus by incubating each antibody with infectious EHV-1
and subsequently determining the capacity of the virus to
induce normal levels of viral TK in LM cells or to replicate
as judged by a plaque assay. LM cell monolayers were
infected with virus preincubated with dilutions of anti-19-
J. VIROL.
VOL. 66, 1992
mRNA AND EPITOPES OF EHV-1 gD
A
B
z
0
__
a*0
Ecom
O
I-
a.
a.
z
w
6457
l
O antl-19mor C' Inactivated
* anti-19mor untreated
* prelmmune C' Inactivated
401
0.
O prelmmun. untreated
20t
0
10
M.0.l
20
40
S0 160 320 640 1280
SERUM TITER
FIG. 7. Neutralization of EHV-1 infectivity by the anti-19-mer serum. (A) Virus neutralization as determined by inhibition of the induction
of viral TK activity. LM cell monolayers were infected at multiplicities of infection (M.O.I.; x axis) ranging from 0 (mock infected) to 10 PFU
per cell with virus preincubated with buffer (0) or the following sera diluted 1/160: preimmune serum (-), anti-EHV-1 virion serum with
demonstrated capacity to neutralize plaque formation (0) (61), and anti-19-mer serum (0). At 16 h after infection the monolayers were
harvested and cell lysates were assayed for virus-specific TK activity (DPM; y axis) by quantitation of the phosphorylation of
[1251]iododeoxycytidine (68). Each determination represents the average of three infected-cell cultures, each of which was assayed in
triplicate. (B) Virus neutralization as determined by reduction of EHV-1 plaque formation. Duplicate samples of preimmune serum or
anti-19-mer serum were either untreated or heated at 56°C for 1 h to inactive complement (C' inactivated). EHV-1 (3,000 PFU) was incubated
with twofold dilutions of either preimmune serum or anti-19-mer serum for 1 h at 37°C. The number of infectious virions remaining after
incubation was measured by plaque assays performed in triplicate. The data are presented as percent inhibition of plaque formation,
calculated relative to the number of plaques obtained with virus incubated with phosphate-buffered saline.
imately 50% of infectious virus was neutralized. Essentially
identical levels of neutralization were obtained with either
untreated or heated antisera, demonstrating that complement is not necessary for virus neutralization mediated by
the anti-19-mer serum. Similar assays performed with the
anti-20-mer serum revealed that it lacked neutralizing activity, confirming the results obtained by the TK assay method.
Overall, these results show that an EHV-1 neutralization
epitope maps at residues 4 through 22 of gD. A diagram
summarizing key elements of the gD gene and polypeptide is
shown in Fig. 8, and the domains of gD that are represented
by the 19-mer and 20-mer synthetic peptides are indicated.
ATG
( a)
CAAT TATA
-352
-267
-
-44
KpnS
(b)
§I
|
Previously, DNA sequence analysis of the Us of EHV-1
KyA identified the EHV-1 gD and US9 genes and revealed a
deletion of 3,859 bp of unique sequences between these
genes (19, 20). In the present study, the gD mRNA was
identified and characterized and the gD gene product was
shown to be present in virions and to harbor a continuous
neutralization epitope within residues 4 to 22. Northern blot
analyses revealed three mRNAs (5.5, 3.8, and 1.7 kb) that
overlap the gD coding sequences and are transcribed in the
same direction as the gD ORF. The 3.8-kb mRNA was
shown to be a member of the late (beta-gamma) kinetic class
and was assigned as the gD mRNA on the basis of several
lines of evidence. (i) The 3.8-kb mRNA overlaps the gD
coding sequences, and the 5' terminus of this transcript is in
close proximity to the gD ORF. The 5' terminus of the 3.8-kb
mRNA was localized 68 nucleotides upstream of the second
in-frame methionine of the gD ORF, such that translation
would yield a 392-amino-acid polypeptide that possesses a
signal sequence at the amino terminus (19). (ii) Potential
cis-acting transcriptional elements upstream of the site of
transcription initiation of the 3.8-kb mRNA include a
(C)
NH2
v
c
BamHI
392
aa
F20-MER|
19-MER
l-NEUTRALIZATOIN
Im
DISCUSSION
aDORF
EPITOPE
*ccc
1
cc
i
COOH
CC
FIG. 8. Overview of the EHV-1 gD gene and polypeptide. (a)
Expanded map representing 5'-flanking sequences of the gD gene
showing potential cis-acting regulatory elements. The nucleotide
positions of cis-acting elements relative to the transcription initiation site (+1; denoted with an arrow) of the 3.8-kb gD mRNA are
indicated. Encircled elements indicate motifs predicted to promote
expression of the gD gene. Initiation of translation at position +68
would yield a 392-amino-acid (aa) polypeptide, and cleavage of the
signal sequence would result in a 366-amino-acid gD protein. (b)
Position of the gD ORF within the 2,229-bp KpnI-BamHI clone
pSZ-4 (19). (c) Schematic representation of the gD polypeptide
showing the following features: (1) 26-amino-acid signal sequence
(striped box), (2) 10 cysteine (C) residues (an asterisk denotes
cysteines conserved among gD homologs), (3) four potential
N-linked glycosylation sites (balloons with strings), and (4) a 17amino-acid transmembrane domain (open box). gD-specific antibodies were obtained by using as immunogens synthetic peptides
(designated 19-mer and 20-mer) which represent two domains (solid
boxes) of the gD polypeptide. gD-specific antibodies to the 19-mer
peptide inhibit virus infectivity in the absence of complement,
demonstrating that residues 4 through 22 possess a neutralization
epitope.
Downloaded from http://jvi.asm.org/ on December 29, 2014 by guest
5
6458
J. VIROL.
FLOWERS AND O'CALLAGHAN
transcription initiation site.
The 5' termini of the 3.8-kb gD mRNA and the 5.5-kb
mRNA lie within unique sequences, but their 3' ends lie
within the TR in a coterminal arrangement and map 18
nucleotides downstream of a consensus polyadenylation
signal. This polyadenylation signal appears to be used in
both inverted repeats since the 3' ends of the 2.3-kb mRNA
of the EHV-1 US2 gene and the 1.2-kb mRNA of the diploid
IR6 gene map to the same location in the TR (3). Taken
together, these studies reveal that two families of 3'-coterminal mRNAs map within the IRs of the KyA strain of
EHV-1. In the IR, the 2.3- and 1.2-kb mRNAs share 3'
termini, whereas in the TR the 5.5-, 3.8-, and 1.2-kb mRNAs
are 3' coterminal.
The 3.8-kb mRNA possesses a relatively long 3' untranslated region (2,250 nucleotides), which may be a consequence of the deletion of 3,859 bp of Us sequences downstream of the gD ORF (20). This deletion within the KyA
strain genome removed sequences encoding gI, gE, and a
unique 10-kDa ORF, as well as potential polyadenylation
signals located downstream of the gI and gE genes. DNA
sequence analysis of the US9 gene suggested that this
deletion also removed promoter elements necessary for US9
transcription. This observation was bolstered by the finding
that only the 5.5- and 3.8-kb mRNAs hybridize to probes
that contain US9 sequences. The absence of expression of
the US9 gene would not be predicted to affect EHV-1
replication in vitro since the US9 genes of HSV-1 and
pseudorabies virus are nonessential (35, 49, 51). Direct proof
that the deletion removed essential US9 promoter elements
requires identification of the US9 transcription start site in
EHV-1 strains that possess an intact Us.
Immunoblotting experiments employing gD-specific antibodies revealed that the EHV-1 gD gene product is present
in purified virions as a 55-kDa species. The precursorproduct relationships of the 58- and 47-kDa polypeptides,
detected in infected-cell extracts, to the mature 55-kDa
protein, are unknown. Recently, Whittaker et al. (65) re-
ported that EHV-1 glycoprotein 17/18, originally described
by O'Callaghan and Randall (44), is gD and is present in
EHV-1 virions of the AB1 strain as a 60-kDa glycoprotein.
Monoclonal antibodies generated to virion gD were shown to
possess neutralizing activity. However, the locations of the
reactive epitopes were not identified, and it is not known
whether any of these epitopes correspond to the neutralization epitope mapped in this report. Studies with glycanases
and inhibitors of glycosylation showed that the glycosylation
of gD involved mainly N-linked oligosaccharides (65). Love
et al. (36) used antibodies raised to an EHV-1 gD fusion
product expressed in Escherichia coli to identify the gD gene
product in virions and infected cells.
The 19-mer and 20-mer peptides represent EHV-1 gD
continuous antigenic sites that align very closely with continuous epitopes of HSV-1 gD recognized by monoclonal
antibodies assigned to groups VII and II, respectively (39).
The EHV-1 anti-19-mer antibody recognizes residues 4
through 22 of EHV-1 gD, whereas the HSV-1 group VII
epitopes lie within residues 1 through 23 of HSV-1 gD.
Similarly, the EHV-1 anti-20-mer antibody binds to residues
267 through 285 of EHV-1 gD, whereas HSV-1 group II
monoclonal antibodies react with amino acids located within
residues 264 through 287 of HSV-1 gD. Homology between
these continuous antigenic domains of EHV-1 gD and
HSV-1 gD is limited, and neither the anti-19-mer nor the
anti-20-mer antibody reacts with HSV-1 gD in immunoblotting experiments (data not shown). Group VII monoclonal
antibodies neutralize HSV-1 infectivity, reduce plaque size,
and inhibit syncytium formation (38). Immunization of animals with peptides containing the antigenic targets of group
VII monoclonal antibodies elicits virus-neutralizing antibodies (7) and protects mice from lethal HSV-1 challenge (13).
The EHV-1 anti-19-mer serum was shown to neutralize virus
in cell culture; however, it remains to be determined whether
immunization with the 19-mer peptide will afford protection
to a lethal EHV-1 challenge, for example, in the hamsterEHV-1 model.
The analysis of gD-negative mutants of HSV-1, pseudorabies virus, and bovine herpesvirus type 1 has demonstrated
that the gD class of glycoproteins is essential for virus
penetration into target cells (17, 18, 31, 33, 40, 47, 53).
Complement-independent virus neutralization exhibited by
the anti-19-mer serum implicates a similar role for EHV-1 gD
in virus entry. Indeed, the recent findings of Whittaker et al.
(65) demonstrate that monoclonal antibodies to gD prevent
EHV-1 penetration into rabbit kidney cells. The colocalization of two continuous antigenic domains within the EHV-1
and HSV-1 gD polypeptides, as well as the conservation of
cysteine residues (19) known to be requisite for proper
conformation of HSV-1 gD (67), suggests similarities in
structure and function for these two viral glycoproteins.
ACKNOWLEDGMENTS
We are grateful to Scarlett P. Flowers for assistance with the
RNA-mapping studies and to Suzanne Zavecz and Bridget Higgenbotham for technical support. We thank Michael Wolcott for assistance in the generation of gD-specific antibodies.
This investigation was supported by Public Health Service research grant AI 22001 from the National Institutes of Health, a
Grayson-Jockey Club Research Foundation Inc. research grant, and
grant 89-37266-4735 LTom the U.S. Department of Agriculture Animal Molecular Biology Program.
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CCAAT box and a TATA box, and their spatial arrangement
to the transcription start site of the gD mRNA is in accordance with most eukaryotic promoters. (iii) Recently, sequences upstream of the gD mRNA transcription start site
were shown to contain a functional promoter that requires
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species were not detected with probes that overlap the gD
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