Journal of General Virology (1991), 72, 3077-3084. Printed in Great Britain
3077
The most abundant protein in bovine herpes 1 virions is a homologue of
herpes simplex virus type 1 UL47
Dale E. Carpenter and Vikram Misra*
Department o f Veterinary Microbiology, Western College o f Veterinary Medicine, University o f Saskatchewan,
Saskatoon, Saskatchewan, Canada S 7 N OWO
The bovine herpesvirus type 1 (BHV-1) protein VP8 is
present, in large amounts, in the tegument o f virions.
As a preliminary step towards determining the function
o f VP8 and the biological relevance for its abundant
presence, we describe the mapping of the location of its
gene and determination o f its nucleotide sequence. The
gene for VP8 was located between 0.088 and 0.108
map units on the BHV-1 genome and contained a 2226
bp reading frame encoding a 742 amino acid protein.
The protein, produced in vitro by transcribing and
translating the reading frame, was precipitated by
monoclonal antibodies and polyclonal serum directed
against VP8. T h e primary structure of VP8 showed
considerable homology with the product o f the U L 4 7
reading frame o f herpes simplex virus type 1.
Introduction
infected cells, has been shown to be located in the
tegument of virions (Marshall et al., 1986) and preliminary results (G. Weinmaster, unpublished results) suggest that it is a 7 phosphoprotein.
In this communication, as the first step towards
determining the function of VP8 and the biological
relevance for its abundant presence, we describe the
mapping of the location of its gene and the determination
of its nucleotide sequence. The amino acid sequence of
VP8 bears considerable resemblance to that of the herpes
simplex virus type 1 (HSV-1) gene UL47.
Virions of bovine herpesvirus type 1 (BHV-1) comprise
about 25 to 33 polypeptides (Pastoret et al., 1980; Misra
et al., 1981). Cells infected with the virus contain an
additional 15 polypeptides that are not represented in
purified virions (Misra et al., 1981). The nucleotide
sequences of the genes for some of these polypeptides
have been determined. These include BHV glycoprotein
B or gI (Misra et al., 1988; Whitbeck et al., 1988), BHVgC or glII (Fitzpatrick et al., 1989), BHV-gD or glV
(Tikoo et al., 1990) and thymidine kinase (TK) (Mittal &
Field, 1989; Smith et al., 1990). The location on the
genome of other viral genes, including those for the
D N A polymerase (Owen & Field, 1988) and the major
DNA-binding protein (Bandyopadhyay et al., 1990),
have also been mapped.
As with other herpesviruses the expression of BHV-1
genes is temporally regulated such that viral proteins can
be categorized as immediate early (0t), early (fl) or late ( J
depending upon the order of their synthesis in the
infected cell (Misra et al., 1981 ; Ludwig & Letchworth,
1987; Nelson et al., 1989). The ~, gene products can be
divided further into 71 and 72 subclasses. The ~,1 genes
are expressed earlier in infection than 72 genes and are
affected minimally if viral D N A replication is interrupted. The ~2 genes are expressed later in infection and
are not expressed if viral D N A synthesis is blocked.
Virions of BHV-1 contain an abundance of a protein
that has been designated VP8 (Misra et al., 1981), VP7
(Pastoret et al., 1980) or 107K (Marshall et al., 1986). The
protein, which is also the most abundant viral protein in
0001-0461 © 1991 SGM
Methods
Cells and virus. BHV-1 strain P8-2 was isolated at the University of
Wisconsin by J. R. Saunders. Details of virus propagation in MadinDarby bovine kidney (MDBK)cellshave been published (Misra et aL,
1981). To obtain radiolabelledinfectedcell proteins, cellsinfectedwith
BHV-I at a multiplicity of infection of 10 p.f.u, per cell were
maintained in either methionine-freemedium to which [35Slmethionine (100~tCi/ml) had been added or in phosphate-free medium
supplemented with 500 ~tCiof H332po4 per ml. Cells were harvested
20 h after infection.
Construction of a 2gtl l expression library. BHV-I genomicDNA (2.5
~tg) was partially digested with the restriction enzymes HaelII (2.5
units) and RsaI (7 units) for 8 min. The digest was electrophoresed
through a 0.8~ agarose gel and DNA fragments, 500 to 2000 bp in
length, were eluted from the gel. EcoRI linkers (Promega)were ligated
to the fragments,which werethen digestedwith EcoRI. The fragments
were clonedinto Protoclone2gt11 arms (Promega)and packaged using
the Packagene in vitro packaging system (Promega). The library was
grown on Escherichia coli host strain Y1090(r-)and expressionof the pgalactosidase-VP8 fusionprotein was inducedwith isopropylthio-fl-l>
3078
D. E. Carpenter and V. M i s r a
galactoside (Bethesda Research Laboratories). Plaques were transferred to nylon membranes by the method of Maniatis et aL (1982).
VP8polyclonal antisera. Antisera to VP8 were raised in rabbits. The
polypeptides of virions purified from infected ceils in eight t00 mm
diameter culture plates were separated by electrophoresis through a
preparative 7-5% (w/v) SDS-PAGE gel (Misra et al., 1981). The outer
lanes of the gel were stained with Coomassie blue to visualize the
proteins. The segment of the unstained gel, which corresponded to the
VP8 band, was excised and dialysed in water to remove SDS. The gel
slices were homogenized to a slurry with a Polytron homogenizer
(Brinkman Instruments). Freund's complete adjuvant (1 ml) and the
slurry (1 ml) were mixed and 0-4 ml inoculant was injected
intramuscularly and subcutaneously at several sites. At 2 week
intervals, 1 ml slurry and 1 ml Freund's incomplete adjuvant were
mixed and injected into the rabbits. Eight weeks after the initial
immunization, the rabbits were bled and serum was collected. The
rabbit serum was absorbed with ProtoBlot E. coli extract (Promega) to
remove cross-reacting antibodies. The resulting serum, diluted 1 : 500,
was used to detect VP8-expressing plaques from the 2gtl I library.
Bound antibody was visualized with horseradish peroxidase-labelled
anti-rabbit antibody (Bio-Rad).
Mapping of the VP8 gene on the BHV-1 viral genome. Viral genomic
DNA (2 rtg) was digested with the restriction enzymes EcoRI, HpaI,
HindlII and BgllI (5 units, 1 h at 37 °C) then electrophoresed through a
0.8% agarose gel. The DNA fragments were transferred to a nylon
membrane (Amersham) and hybridized to a radiolabelled DNA
fragment isolated from the 2gtl 1 clone that reacted with the anti-VP8
serum.
S~ nuclease mapping of the start of transcription. A variation of the
procedure of Berk & Sharp (1977) was used to map the start of
transcription of the VP8 gene. The VP8 gene was mapped, by Sl
nuclease digestion, in two segments. The 3-0 kb EcoRl G fragment
(Fig. 2) was cloned into the EcoRI site of the multiple cloning site of
pEMBL18. The orientation of the insertion was determined from the
location of an internal HindlII site. A HindlII digest would excise a 2-0
kb fragment if the insert was cloned in one direction or a 1.0 kb
fragment if it was cloned in the other direction. A clone that released a
2.0 kb fragment upon HindlII digestion was digested with ApaI and
HindlIl. A 1300 bp ApaI-EcoRI fragment of BHV-1 DNA, with the
entire multiple c!oning site adjacent to the EcoRI end, was eluted from
the gel and was labelled at the 5' ends with [y-32p]ATP using alkaline
phosphatase (Promega). The fragment was then digested with EcoRI to
remove the labelled multiple cloning site. The result was a 1300 bp
EcoRI-ApaI fragment labelled on one strand at the ApaI site. A similar
strategy was used to label the 1700 bp EcoRI-HindIII portion of the 3-7
kb HindlIl M fragment. The entire 3-7 kb HindlII M fragment was
cloned into the HindlIP site of the multiple cloning site. An internal
EcoRI restriction site was used to determine the orientation. The 1700
bp EcoRI-HindlII portion with the entire multiple cloning site at the
HindlII end was released with an EcoRI digest. This was eluted from a
gel, labelled and the multiple cloning site was removed with a HindIII
digest. This resulted in a 1700 bp EcoRI-HindlII fragment labelled 5"
on one strand at the EcoRI site. Labelled DNA was hybridized to total
infected cell RNA at a ratio of 1:5. Total infected cell RNA was
isolated from MDBK cells, by the method of Cathala et al. (1983), 20 h
after BHV-I infection. Products of S~ digestion were electrophoresed
on a urea-containing 6% (w/v) acrylamide gel. A ~2Pqabelled BstEII
digest of 2 DNA was denatured and electrophoresed to determine
the size of the fragments protected from digestion with $1 nuclease.
In vitro transcription and translation. The coding sequence for the
VP8 gene was cloned between the AccI and HindllI sites of pGEM-3Z
(Promega) in a two step cloning reaction. First the HindlIl M fragment
was cloned into the HindlII site of pGEM-3Z. An EcoRI digest was
used to determine the fragment orientation. A clone that would release
a 1.7 kb fragment upon EcoRI digestion was isolated. This clone was
digested with Accl which released a 600 bp fragment and a 1100 bp
fragment from the vector which still contains 2000 bp of the VP8 gene.
The 1100 bp fragment and the pGEM-partial VP8 fragment were
eluted from a gel and ligated. Regeneration of the VP8 gene coding
sequence in the pGEM vector was screened by EcoRI digestion. RNA
was transcribed from the T7 promoter using the Riboprobe system
(Promega). During transcription the transcripts were capped with the
cap analogue pGppp (Pharmacia). The capped RNA was translated in
a rabbit reticulocyte lysate containing PSlmethionine (Pelham &
Jackson, 1976). The reticulocyte lysate was a gift from A. J. Pawson
(Univeristy of Toronto).
Immunoprecipitation. Immunoprecipitations were carried out using
the method of Misra et al. (1982) using monclonal antibodies (MAbs) or
polyclonal antisera developed in our laboratory.
Sequencing. Sequenase and 7-deaza-dGTP reagents (United States
Biochemicals) were used to determine the nucleotide sequence of the
VP8 gene. Nested deletions of the DNA or subclones, constructed
using convenient restriction sites, were used to sequence both strands of
the entire VP8 coding region. Regions downstream from the VP8
coding region were sequenced on one strand only.
Protein sequence alignment. VP8 and UL47 protein sequences were
aligned using the CLUSTAL computer program of Higgins & Sharp
(1988).
Results
V P 8 is a y2 phosphoprotein
T h e p r o t e i n w e h a v e d e s i g n a t e d V P 8 ( M i s r a et al., 1981)
is m a d e in t h e l a t e r s t a g e s o f i n f e c t i o n a n d is t h e m o s t
a b u n d a n t o f t h e v i r i o n p r o t e i n s o f B H V - 1 ( M i s r a et al.,
1981; P a s t o r e t et al., 1980; M a r s h a l l et al., 1986). V P 8
h a s also b e e n s h o w n to r e s i d e in t h e v i r a l t e g u m e n t
( M a r s h a l l et al., 1986) a n d G . W e i n m a s t e r ( u n p u b l i s h e d
results) d e m o n s t r a t e d t h a t B H V - l - i n f e c t e d cells c o n t a i n
a phosphoprotein with an electrophoretic mobility
s i m i l a r to t h a t o f V P 8 .
T o c o n f i r m t h a t V P 8 is a 7 class p h o s p h o p r o t e i n ,
B H V - 1 i n f e c t e d cells w e r e l a b e l l e d w i t h e i t h e r [35S]methi o n i n e o r H 3 3 2 p o 4 , in t h e p r e s e n c e o r a b s e n c e o f 250 ~tg
phosphonoformic acid (PFA) per ml medium. VP8 and
B H V - g B , a f l class p r o t e i n ( N e l s o n et al., 1989), w e r e t h e n
i m m u n o p r e c i p i t a t e d f r o m t h e cell lysates. M o n o c l o n a l
a n t i b o d i e s w e r e u s e d for i m m u n o p r e c i p i t a t i o n .
F i g . 1 s h o w s t h a t a l t h o u g h 35S-labelled i n t a c t B H V - g B
( 1 3 0 K ) a n d its c l e a v a g e p r o d u c t s ( 7 0 K a n d 5 0 K ) w e r e
immunoprecipitated from PFA-treated and untreated
cells, V P 8 a n d 6 0 K p r o t e o l y t i c d e g r a d a t i o n p r o d u c t w e r e
i m m u n o p r e c i p i t a t e d o n l y f r o m cells in w h i c h v i r a l D N A
s y n t h e s i s h a d n o t b e e n i n t e r r u p t e d by P F A . V P 8 ,
i m m u n o p r e c i p i t a t e d f r o m u n t r e a t e d i n f e c t e d cells, w a s
l a b e l l e d w i t h 32p. T h e s e results c o n f i r m t h a t V P 8 is a y2
class p h o s p h o p r o t e i n .
BHV-1 homologue of HSV-1 UL47
(b)
(a)
Mock BHV-I BHV-1
VP8
VP8 BHV-gB
Infected
MAb against
Treatment
PFA
PFA
fragment was sequenced using a primer homologous to
the coding sequences of fl-galactosidase in 2gtll to
determine in which reading frame this fragment was
being expressed.
Mock BHV-1 BHV-1
VP8 VP8 BHV-gB
PFA
PFA
PFA
3079
PFA
- - 200K
Mapping the location of VP8 on the BHV-1 genome
--92K
--69K
The 600 bp D N A fragment from the expression library
was used to probe fragments in EcoRI, HpaI, BglII and
HindIII digests of the BHV-1 genome. The location of
the sites for these enzymes onthe P8-2 strain of the BHV1 genome have been mapped (M. A. N. Beckie,
unpublished results). The probe hybridized to the 3 kb
EcoRI G fragment (0-096 to 0.119 map units), the 15 kb
HpaI D fragment (0.032 to 0-143 map units), the 3.7 kb
HindIII M fragment (0.083 to 0-111 map units) and the
12-5 kb BglII E fragment (0.026 to 0.119 map units).
These fragments all map at the left-hand end of the
BHV-1 genome (Fig. 2).
--46K
Fig. 1. VP8 is a ~,2 phosphoprotein. VP8 and BHV-gB, a fl protein,
were immunoprecipitated from lysates of (a) 35S- or (b) 32p-labelled
mock-infected or BHV-l-infected cells. Cells were either treated with
PFA (250 pg/ml), to interrupt viral DNA synthesis, or were left
untreated. The position of Mr markers, electrophoresed in the same gel
are indicated.
Screening the 2gt11 library
A BHV-1 expression library was constructed to isolate a
D N A probe for the VP8 gene. To determine which
phage in the expression library contained a portion of the
VP8 gene, polyclonal antisera to the protein were raised
in rabbits. Serum from rabbits, immunized three times
with VP8 protein, bound to the VP8 protein in Western
blot analysis. The VP8 antiserum detected a plaque from
the 2gtl 1 BHV-1 genomic library. This clone contained a
600 bp fragment of BHV-1 DNA. The 5' end of this
0
0'l
0.2
i
I
I
0"3
$1 nuclease mapping the VP8 transcriptional start site
The transcriptional start site of the VP8 gene was located
on the 3.7 kb HindlII M fragment by $1 nuclease
mapping. The HindlII M fragment was digested into a
1300 bp ApaI-EcoRI fragment and a ! 700 bp EcoRI-
0-4
0.5
0.6
0.7
0.8
0.9
1-0
I
I
I
I
I
I
I
IRs
UL
E
G
3.0
D
B
A
15.0
D
.|}~.~:}~:-~.~.~:i.:'i:i.-.~.~.~.~:~:i-~:~
4.3
15-0
N
I I
2.5
J
8-8
M
i:~*.:-':.~
3-7
E
1
10-0
18.0
G
13.7
A
I
L
C
I
18.3
O
I
I
7.5
15-8
G
F
K
II
C
H
D
I
I
I
8-2
A
Hindlll
ll.5
F
K
I
14-8
H
I
8-2
D
I
lJ
E
I I!~::::::~
Hpal
~45.0
B
20.0
EcoRI
A
I
11.7
I
8.3
1
9.0
I
I
16.0
F
1
24-0
I
11.5
C
1
Genome (132 kb)
F
.'l
23-5
E
B
I
C
I
~48.5
G
[
TRs
I
I
13.0
Us
Map units
13.0
B
I
BgllI
2-2
12.5
8-4
9.5 1.4
17.5
7-5
16.0
~38"0
18.5
1-3
Fig. 2. Mapping the location of the VP8 gene on the BHV-1 genome. The BHV-1 genome is depicted showing the location of the unique
long (UL), unique short (Us), internal repeating sequence (IRs) and terminal repeating sequence (TRs). Map units are indicated on the
top bar. The lower bars depict restriction maps of the genome of the P8-2 strain of BHV-1. The fragments are lettered above the
restriction maps in decreasing size with A indicating the largest fragment, B indicating the second largest fragment, etc. Sizes of the
restriction fragments in kb are indicated below the fragments. The HindlII restriction map has an additional set of fragments as there
are HindllI sites within the Us region. These fragments appear as the Us region can be present in two orientations. The restriction
fragments that hybridized to the 600 bp VP8 probe in Southern blot analysis are indicated as shaded boxes.
I
I
I
I
1
3080
D. E. Carpenter and V. Misra
(a)
(b)
1
2
3
4
5
5
1
2
3
4
Original probe
isolated from
expression library
(c)
VP8 transcription
start site
I
5
i
II
Accl
I
Accl
EcoRt
Hindlll
1700 bp
Apal
1300 bp
HindllI
700 bp
Fig. 3. Mapping the transcriptional start site of the VP8 gene. $1 nuclease protection analysis was performed on two different pieces of
the HindlII M fragment. (a) Result of $1 mapping the 1300 bp ApaI-EcoRI fragment. Radiolabelled 2BstEII size markers (lanes 5) were
electrophoresed to determine the size of protected fragments and the sizes are indicated in the centre. Lanes 1, labelled DNA (5' endlabelled at the ApaI site); lanes 2, D N A / R N A hybrid (labelled DNA hybridized to the RNA); lanes 3, Sl-treated (labelled DNA
hybridized to RNA and treated with S~ nuclease); lanes 4, second Sl-treated sample incubated for twice the time period. RNAprotected bands are indicated by << in lanes 3 and 4. (b) Result of $1 mapping the 1700 bp EcoRI-HindllI fragment. The lane numbers
are the same as for (a) except that the 1700 bp EcoRI-HindlII fragment, 5' end-labelled at the EcoRl site, was used. RNA-protected
bands are again indicated by <<. (c) Summary of results. The solid black bar represents the HindlII M fragment. Relevant restriction
sites are indicated and the sizes of restriction fragments are given below. The small dark shaded box represents the portion of the
HindlII fragment originally isolated from the Jlgtl 1 library and used as a probe for the VP8 gene. The large lightly shaded boxes
represent the DNA fragments protected from S~ degradation when the 1300 bp ApaI-EcoRI and 1700 bp EcoRI-HindlII portions of the
HindllI M fragment were subjected to $I nuclease protection analysis. The major transcriptional start site is indicated.
HindlII fragment (Fig. 3c). R N A from BHV-l-infected
cells protected the entire 1300 bp ApaI-EcoRI portion of
the 3-7 kb HindlII fragment from $1 digestion (Fig. 3a)
suggesting that the fragment was included, in its entirety,
within a transcript. The size of the 1700 bp EcoRIHindlII subfragment, labelled with 32p at the EcoRI site,
was reduced in size by 600 bp by $1 nuclease, indicating
that the beginning of the main transcriptional unit in this
fragment was about 1100 bp from the EcoRI site (Fig.
3 b). A minor transcription start site mapped about 650
bp from the EcoRI site.
Immunoprecipitation
To confirm that the transcription start site, identified by
the $1 protection experiments, was that of VP8, a 3.1 kb
AccI-HindlII piece of D N A from the HindlII M
fragment was cloned between the AccI and HindIII sites
of pGEM-3Z. The AccI site in the HindlII M fragment
was located immediately upstream from the putative
start site of the VP8 transcript (Fig. 3c). In vitro
transcription, from the T7 promoter of pGEM-3Z,
resulted in a single 3.1 kb R N A transcript. In vitro
translation of this R N A produced a 90K protein which
could be immunoprecipitated from the reticulocyte
lysate by the anti-VP8 sera used initially to screen the
2gtl 1 BHV-1 genomic library but not with monoclonal
antibodies to BHV-gB (Fig. 4). Monoclonal antibodies to
VP8 also immunoprecipitated the in vitro translated
protein (data not shown). The VP8 antisera and
monoclonal antibodies to VP8 and BHV-gB immunoprecipitated the appropriate proteins from lysates of BHV-1
BHV-1 homologue of HSV-1 UL47
kDa
1
2
3
4
5
6
7
200K - -
M
D
A
A
GTCTACACGGGATTTAGTTTTCGCGCCCGCGGCTTTCTAGGCCCCCLIAGACGCCATGGACGCCGCTAGGGA
12
24
36
48
60
G
R
P
E
R
R
P
R
R
S
G
T
Y
R
T
H
P
F
Q
R
P
S
A
R
D
72
R
TGGGCGGCCTGAGCGCCGCCCGCGCCGCTCCGG~CGTACCGCACGCACCCGTTCCAGCGCCCCTCTGCCCG
84
92.5K--
3081
R
S
L
P
D
F
L
96
D
A
L
R
R
P
P
D
A
108
A
D
A
E
D
T
S
E
120
A
A
E
R
P
E
N
V
Y
R
132
V
R
R
P
Y
I
D
G
144
R
GCGGAGCCTGCTGGACGCCCTGCGCGCTGCnGACGCCGAGGCCGCGGAGCGCCCGCGGGTCCGGCGCCCGCG
156
Q
168
E
180
192
D
D
204
216
D
GCCTGACTTCCAGCGGCCCCCGGACGAGGACACCAGTGAGGACGAG~CGTGTATGATTACATCGACGGCGA
228
68K--
240
252
264
276
288
S S D S A D D Y D S D Y F T A N R G P N H G A G
TAGCAGCGACAGCGCCGACGACTATGATAGCGATTATTTTACTGCT~CCGCGGCCCC~TCACGGCGCCGG
300
312
324
336
348
D
A
M
D
T
D
A
P
P
E
R
A
P
E
G
G
A
P
Q
D
Y
L
T
360
A
CGATGCTATGGACACAGACGCACCACCCGAGCGCGCCCCGG~GGGGGTGCCCCGC~GACTACTTGACGGC
372
384
396
408
420
432
HLRAIEAc~GPESAPHRSLLERTART
CCACCTGCGCGCCATCGAGGCC
444
V
Y
~
H
E
F
P
CCCGAGTCAGCGCCCCACCGGAGCCTGCTGGAGCGCACGGCCCGGAC
456
468
480
492
P
R
D
L
S
A
G
S
R
A
P
A
Q
R
A
R
504
R
CGTGTATGCGCACGAGTTTCCCCCGCGCGATTTGAGTGCGGGCTCCAGGGCGCCGGCACAGCGCGCGCGGCG
516
46K--
528
540
552
564
576
S L R G F p R G G G G G Q E P G P D D E G D D A
GAGCCTCCGCGGCTTCCCGCGTGGCGGCGGGGGCGGCCAGG~CCCGGGCCAGACGACG~GGCGACGACGC
588
600
612
624
636
A
D
L
R
E
D
L
V
P
D
E
A
Y
A
H
L
E
R
D
E
R
L
S
648
E
CGCAGACCTGCGCGAGGACCTTGTGCCAGATGAGGCCTACGCGCACCTAGAGCGCGACGAGCGGCTGTCGGA
660
Fig. 4. Immunoprecipitation of the VP8 protein from in vitro
translation reaction and infected cell lysate. Lanes: 1, reticulocyte
lysate with no exogenous RNA added; 2, in vitro translation of in vitro
transcribed RNA containing the VP8 coding region; 3, immunoprecipitate of in vitro translated VP8 using the polyclonal VP8 antisera
originally used to screen the 2gtl 1 library; 4, immunoprecipitate of in
vitro translation using gB MAb; 5, immunoprecipitate of BHV-1infected cell lysate with VP8 MAb; 6, immunoprecipitate of BHV-1infected cell lysate with polyclonal VP8 antisera; 7, immunoprecipitate
of BHV-l-infected cell lysate with gB MAb. Mr markers are indicated.
672
684
696
708
720
G P P L L N M E A A A A A A G E R S V V E E L F
AGGGCCCCCGCTCCTC~CATGGAGGCGGCCGCTGCGGCTGCGGGG~AGAGGAGCGTGGTGGAGGAGCTGTT
732
744
756
768
780
782
T Y A P A Q P Q V E V p L P R I L E G R V R P S
TACGTACGCCCCTGCCCAGCCTCAGGTAGAGGTGCCGCTGCCCAGGATTTTGGAGGGCCGGGTGCGGCCCAG
804
816
828
840
852
864
A
F
F
R
R
A
A
Q
M
P
L
D
M
A
S
D
A
L
C
R
P
H
G
L
T
P
P
N
D
I
T
T
W
Q
R
V
V
R
T
V
D
P
E
CGCCTTCTTCGCGCAGATGCCGCTGGACGCGCTGTGCCGCACGCCGCCC~CGATCAGCGCGTGGTGCGCGA
876
W
888
T
900
912
L
S
924
936
E
GCGGCGCGCTTGGGACATGGCCGGTACGCCGCATGGGCTCCT~T~CCACGTGGAGCACGGTGGACCCGGA
948
960
972
984
996
1008
F S I G G M Y V G A P E G T R P R L V W R R A M
ATTCTCGATCGGCGGCATGTACGTGGGCGCCCCTGAGGGCACCCGGCCCCGGCTAGTGTGGCGGCGCGCGAT
1020
1032
1044
1056
1068
1080
K Q A M A L Q Y R L G V G G L C R A V D G A R M
G~GCAGGCCATGGCGCTGCAGTACCGGCTGGGGGTGGGGGGCCTGTGCCGAGCAGTAGACGGCGCACG~T
1092
1104
1116
1128
1140
1152
P P T E A L L F L A A R A A A R S A Q L P F F V
GCCGCCCACTGAGGCGCTGCTCTTTTTGGCGGCACGCGCTGCTGCGCGTAGTGCGC~CTGCCATTTTTTGT
1164
1176
1188
1200
1212
1224
A
A
G
A
R
G
R
R
R
A
A
P
A
R
G
G
G
W
A
A
G
S
H
A
CGCGGCCGGGGCGCGCGGGCGGCGCCGCGCGGCGCCTGCCCGCGGCGGCGGTTGGGCTGCTGGCAGCCACGC
1236
1248
1260
1272
2284
V H A T G R V P H A T L F R G S M G S L I Y W H
AGTTCACGCCACCGGACGCGTCCCCCACGCGACGCTCTTTCGCGGCTC~TGGGCTCCCTGATTTACTGGCA
1308
1320
1332
1344
1356
infected cells. The in vitro translation of VP8 also yielded
an approx 50K protein. This protein was translated from
RNA endogenous to the lysate and appeared even if no
external RNA was added to the lysate.
E
L
R
E
L
Y
A
Y
L
L
G
P
V
M
L
T
A
V
L
A
L
R
L
F
V
A
P
T
A
A
P
A
L
C
S
E
A
P
A
E
R
A
F
R
E
V
A
R
Y
A
G
Y
T
A
N
R
W
L
Y
A
K
I
P
A
G
L
Q
R
Y
A
A
G
A
E
R
L
S
1296
1368
A
CGAGCTGCGCGTGATGCTGACTGCGGTGCCGGCCCTGTGCGCGCGCTACGCGGGCGCCGGGCTGCAGTCGGC
1380
L
1392
H
1404
1416
G
E
1428
L
1440
$
CGAGCTGTACCTGCTGGCGCTACGGCACTCAGAGGCGCCCGGCTACACGGCAAATGAGCGCTACGCGCTCTC
1452
T
1464
L
1476
1488
V
L
1500
H
1512
L
GGCGTACCTGACGCTGTTTGTAGCGCTCGCGGAGCGGGCCGTGCGCTGGCTGTATCTAGCGGGCGCGCACCT
1524
H
1536
A
1548
1560
R
Y
1572
P
1584
L
GCTCGGGCCGCACCCCACAGCGGCGGCCTTCCGCG~GTGCGCGCC~GATCCCGTACGAGCGGCTGCCGCT
1596
1608
1620
1632
1644
1656
Sequencing
G S A T L H D A E V E T V D S A T F Q E A L A F
AGGCAGCGCGACGCTGCACGACGCCG~GTGGAGACGGTGGACTCGGCCACCTTCCAGGAGGCCCTGGI~
1668
1680
1692
1704
1716
After confirming that the DNA from the HindlII M
fragment contained the gene for VP8, the nucleotide
sequence of both strands of the fragment from the AccI
site to 2500 bp downstream was determined (Fig. 5). The
fragment contained a 2226 bp open reading frame which
would encode a protein 742 amino acids in length. This
coding sequence was located between 0-088 and 0.108
map units on the BHV-1 genome.
TAGCGCGCTGGCACATGTTTACGGGGAGGCCTACGTAGCGGTGCGGACCGCGACGACGCTGCTGATGGCCGA
S
A
L
A
H
V
Y
G
1740
Y
A
A
H
E
A
Y
V
A
1752
A
E
R
R
D
V
R
T
A
1764
V
R
E
M
T
T
L
L
M
1776
T
A
A
F
L
G
A
E
1788
V
G
L
I
1800
A
GTACGCGGCCCACGCTGAGCGCCGGGACGTGCGCGAGATGACAGCGGCCTTCCTGGGCGTGGGGCTGATCGC
1812
1824
1836
1848
1860
1872
Q R L M G S L E P A A E L R S R R S G V R G P A
GCAGCGGCTGATGGGCAGCCTCG~CCTGCTGCTG~CTGCGTAGCCGGCGCAGCGGTGTACGGGGGGCGGC
1884
1896
1908
1920
1932
C
P
T
p
V
S
V
R
E
G
T
V
E
F
W
L
A
R
Y
S
A
R
D
G
L
L
A
D
A
M
R
E
L
A
L
P
L
L
R
p
V
1944
R
GTGTCCGACGGTGCGCGAGGGCACGCTCGCGCGGTACAGCCTGCTAGCGGACGCGGCACTGCCGCTGGTGCG
1956
L
1968
E
1980
1992
V
R
2004
V
2016
A
CCCGGTGTCCCTGGTGGAGTTCTGGGAGGCCCGCGACGGCGTCATGCGCGAGCTGCGGCTGCGGCCCGTGGC
2028
2040
2052
2064
2076
S P P L A G K R R V M E L Y L S L D S I E A L V
GAGCCCGCCCCTGGCCGGC~GCGGCGGGTCATGGAGCTGTACCTCTCGCTGGACAGCATAGAGGCGCT~
2100
2112
2124
2136
2148
G
R
E
P
H
L
P
L
G
S
R
T
G
D
G
P
V
L
G
P
L
V
D
I
A
E
A
L
A
D
2088
2
H
CGGCCGCGAGCCGCTAGGTTCGCGGCCGGTGCTTGGGCCGCTCGTGGACATCGCGGAGGCGCTGGCGGACCA
2172
Protein sequence alignment
A comparison of the VP8 amino acid sequence with the
sequence of HSV-1 (McGeoch et al., 1988) revealed
considerable homology with the protein encoded by the
open reading frame UL47. A more detailed alignment,
using the CLUSTAL program (Higgins & Sharp, 1988)
Fig. 5. Nucleotide and amino acid sequences of the coding region for
the VP8 gene. The sequence for the segment of the D N A containing a
large open reading frame, from which in vitro transcription/translation
produced the VP8 protein, is listed above. The AccI restriction site used
V
2184
R
2196
G
P
R
L
G
2208
G
R
2220
2232
*
CCCGCACCTCGTCACGGGCGATGGGCGGGGCCCGCGCCTGGGCGGCCGCTAGCGGCAGGCCCGCGCTTACCG
2244
2256
~
2280
2292
2304
CGGCGGCTGGGACCGCCCACATCAGCCACCACGCCCGCGGCGGGCG~GGCGGGAGCATACGGATATATCGGC
2316
2328
2340
2362
-- 2 3 6 4
2376
AGTGCG~CTTACGGTTCGCGGTGA~GGGCGGCGCCGCTTTACGCACCGGCCGGCGGAGAGCCCCGGAGTA
2388
2400
2412
2424
2436
2448
GCTCGCCCGCCCGTCTCTTCCCCACGGACGACGGCTGCGAGCGTCTACT
2460
2472
2484
2496
to clone the fragment into pGEM-3Z is indicated. The ApaI restriction
site located within the fragment isolated as a probe is also indicated
between 2260 bp and 2270 bp. The putative promoter consensus
sequences (i.e. TATA box and SP1 sites) for a gene that shares a 3'
terminus with the VP8 gene are indicated between 2340 and 2376 bp
(underlined). The region > 60 bp downstream from the VP8 coding
sequence was sequenced on one strand only.
3082
D. E. Carpenter and V. Misra
VP8
M DAARDGRPERRPRRS GTYRTH p FQRpSARRS LLDALRAA D A E A A E R P R V R R PRPDFQRP
UL47
M-SARE* .**.
-PAGRRRRAST
*. * . * * . . *
.................................
RPRA .... S
***,
Mapunits
0
0.1
I
VP8
UL47
PDEDTSEDENVYDY
I DGDSS DSADDYDS DYFTANRGPNHGAGDAMDTDAPPERAPEGGAP
PVADEPAGDGV-GFMG
. . . . . . . . . y I/~AV F R G D D D S E L E A L E E M A G D E p p V R R R R E G P R
,.,
...... , ....
, .. , . . . . . .
, ..,. ,.** * . i..
VP8
UL47
QDy LTAH LRAI EA LPESA PHRS LLERTARTVYAHEF
PPRDLSAGSRA
PAQRARRS LRG FP
AR .... RRRASEAPP--TSHR
.... RASR ........ QRPGPDAARSQSVRGR
..... LD
** ** *
,,**
*..*
.*
.... *...
*.*
VP8
UL47
RGGGGGQEPGPDDEGDDAADLREDLVPDEAYAHLER-DDDEVPRGPPQARQGGY
....... LGPVDARAI
.....
* ...*.
* * .* *
VP8
UL47
RS V~E E LFT yA pAQ PQVEVPLPR I LEGRVRPS A F FAQM PLDALCRT PPN DQRWRE
RRAW
EAVGPEDGGGAR5
PPKVEV ..... LEG RVPG pE LRAA F PLDR L- -A PQVAVWDESVR
S AL
.*
*
. *...*.***
***** ....
*..***
*
.* . . . .
*.*
%/P8
UL47
DblAGTPHG LLI TTWSTVD P E FS - - - I GGMYVGAPEGTR
PR LVWRRAMKQAMA
LQ -YR LGV
AL-GHPAGF
.... y pCPDSAFGLSRVGVMHFAS
PD- -NPAVFFRQTLQQGEALAWY
ITGD
.. * * *.
..
*,.*.
.* * . . . * .
.* . .* . . . . *. * * . *
*
VP8
UL47
GG LCRAV DGARM P PTEA LLF LAA ................
RAAARSAQ LPF FVAAGARGRR
G i L D L T D R R T K T S A A Q A M S F L A O A V V R L A I N G W V C G T R L H A E A R G S D L D D R A A E LP-RQ F A
* *
.......
*. * * *
.*,**..,*
,*.
*
VP8
UL47
RAA PARGGGWAAG S HAVHATGRV ..... PHAT LFRGSMGS LI YWHELRVMLTAVPALCAR
S LTALRPVG -AA; PLLSAGG LVS pQSG PDAAVFRS S LGS LLYWPGVRALLDRDCRVAAR
• .. *
* **..
, *.* *
*.*..**.*.***.**...*..*.
VP8
UL47
HSV-1
DERLSEG pPLLNMEAAAAAAGE
LGRVGGSRVAPS
PLFLEELQYEE
DDYP
*.*
..*...*
.* . . . . . .
(~)
YAG--AGLQSAE
Ly LLALRHS EAPGYTANERYALSAY
LTLFVALAERAVRWLyLAGAHLL
YAG RMTY LAT GAL- LARFN PDAVRCV LTREAA F LGRV LDV LAVMAE QTVQW LS VVVGARL
GPHPTAAAFREVRAKI
PYERLPLGSATLHDAEVETVDSATFQEALAFSALAHVYGEAYVA
HPHVHH PAFADVARE ELF RALP LGS PAWGAEH
EALG DTAARRLLAN
SG LNAVLGAAV¥
**
. * * .*
.
.
***** .... ** * .......
** *,*. * *.*
VD8
UL47
VRTATT LLMAEYA .... AHAERRDVREMTAA
F LGVG L IAQR LMG- - -S LEPAAE LRS RRS
LHTA LATVT LKYARACG DAHRRR D DAAATRA I LAAG LV LQRLLG FA DTVVACVTLAAF
DG
..**
. .
.**
***
. * *.*..**
***.*
.....
* ,
VP8
GVRG PAC PTVREGT LARY S LLADAA L PLVR PVS LV E FWEAR DGVMRE LR LRpVAS P PLAG
GFTAFEVGTY- -TPLRYACVLRATQPLYARTTPAKFWADVRAAAEHVDLRPASSApRAP
*
*.
*
*
**
.
*. * *
.....
** . . . . . .
***..*.*
*
A
*
VP8
~3L47
KRRVME Ly LS LDS I EALVGRE PLGS RPVLG P LVD I AE ALADHPHLVTG
DG ..........
VSGTADPAFLLKDLEPF
P- PAPVS GGSV LG PRVRW
DIMS Q FRKLLMG DEGAAA LRAHVS
. . . .
*...* ....
*...- . * * * *
* ........
*. * * .
VP8
UL47
--RGPRLGGR--GRRATGLGGPPRp
*.. ***,
Fig. 6. Comparison of the amino acid sequence of BHV-1 VP8 and
HSV-1 UL47 as determined by the CLUSTAL sequence alignment
computer program. An asterisk below amino acids indicates identical
residues, and a point below corresponding amino acids indicates
conservative substitutions.
showed that regions of homology were located throughout the two molecules (Fig. 6). The CLUSTAL alignment program's method for deciding conservative
replacements is based on the log odds matrix of Dayhoff
(1978). This relies on the frequency at which various
amino acids are replaced by each other.
Discussion
Several lines of evidence suggest that the BHV-1 protein
VP8 is a homologue of the product of the HSV-1 gene
UL47. First, the genomic map location of VP8 is similar
to UL47 with respect to other shared homologues, (e.g.
gB, gC, DNA polymerase, TK and capsid protein) of the
viruses (Fig. 7). Varicella-zoster virus (VZV) also has a
gene arrangement collinear to those of BHV-1 and HSV1, with the open reading frame 11 mapping in the same
relative location as VP8 and UL47 (Wirth et al., 1989;
McGeoch et al., 1988; Davison & Scott, 1986).
Additional evidence is found in RNA transcript
mapping. UL47 is transcribed as a 4.7 kb ),2 transcript
0.2
0-3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
I
I
I
I
I
I
I
I
I
UL
~
VZV
UL47 gC
homologue
orfl 1
BHV-1
CV6~ gC
**
VP8
UL47
UL47
I
Us
D~NA gB TK Capsid
polymerase
UL
Us
CZS3"---"CS~
DNA gB Deoxy- Capsid
polymerase pyrimidine
kinase
UL
E
I
Us
L__
DNA gB TK Capsid
polymerase
Fig. 7. Collinear gene arrangement of herpesviruses. The genomes of
HSV-1, VZV and BHV-I are compared showing the unique long (UL),
unique short (Us) and repeating sequences (open boxes). Map units are
indicated on the bar above. Homologous genes and direction of
transcription are indicated by arrows. Information for VZV genes is
from Davison & Scott (1986), for HSV-1 McGeoch et al. (1988) and for
BHV-gC (glII) Fitzpatrick et al. (1989), DNA polymerase Owen &
Field (1988), gB Misra et al. (1988), TK Mittal & Field (1989) and
capsid Wirth et al. (1989).
which shares a 3' terminus with a 2.5 kb 71 transcript
(Hall et al., 1982). This is very similar to the transcript
map of the corresponding region of the BHV-1 genome.
Wirth et al 0989) showed that the 3-7 kb HindlII M
fragment hybridizes to a 4-5 kb ~2 and a 2-7 kb 71
transcript. In Northern blot analysis, we have found the
1300 bp ApaI-EcoRI fragment, containing the distal half
of the VP8 gene, hybridized to a 4.5 kb transcript (data
not shown) of which only the first 2700 bp would encode
the VP8 protein. Immediately downstream from the VP8
coding sequence there is a promoter consensus sequence
(Fig. 5) which may be the regulatory sequence for the
transcript that shares a 3' terminus with VP8.
Finally, we have sequenced the BHV-1 VP8 gene and
shown that the derived amino acid sequence has
considerable homology to the derived amino acid
sequence of HSV-1 UL47 (McGeoch et al., 1988). Blocks
of identical amino acids appear throughout the sequences; however, the carboxy half of the molecules
appear to have maintained greater conservation. The
proteins differ in size by 49 amino acids. This size
difference generally seems to be due to changes in the
amino terminal half of the proteins. VP8 also shares
considerable homology with VZV open reading frame
11. Once again there seems to be more conservation of
the carboxy half of the proteins with variability in the
amino half of the molecule.
In HSV-l-infected cells, viral gene expression is
initiated when the major structural tegument protein
I
B H V - 1 homologue o f H S V - 1 UL47
V m w 6 5 (~-trans-inducing factor or g-TIF) forms a
complex with cellular proteins which then bind to cisacting elements in the g gene promoters ( R o i z m a n et al.,
1988). A review of V m w 6 5 interaction with cellular
proteins is presented by Stern et al. (1989) and G o d i n g &
O ' H a r e (1989).
The gene products o f the HSV-1 o p e n reading frames
U L 4 7 and U L 4 6 were initially implicated as modulators
o f the ~-TIF trans-activation in transient expression
systems ( M c K n i g h t et al., 1987). A more recent paper
indicates that deletion o f UL46, within the context o f the
virus, has no a p p a r e n t effect on ~ - T I F trans-activation
but deletion of U L 4 7 has a significant effect ( Z h a n g et
al., 1991). It is not k n o w n by w h a t m e c h a n i s m the
product of U L 4 7 affects ~-TIF trans-activation. T h e
coding sequence for U L 4 7 is reportedly not required for
viral replication in cell culture (Barker & R o i z m a n , 1990;
Z h a n g et al., 1991).
Previous research demonstrates t h a t VP8 protein is
located in the tegument o f BHV-1. Bolton et al. (1983)
showed that if BHV-1 virions are disrupted with the
detergent NP40, approximately h a l f o f the VP8 protein
fractionates with the m e m b r a n e s and the other half
fractionates with the nucleocapsids. Marshall et al.
(1986) confirmed these results with similar experiments
but also iodinated the surface proteins o f BHV-1 virions.
Unlike the glycoproteins, VP8 is not iodinated on intact
infected cells but can be iodinated after viral disruption
with NP40.
Although there is little published data on BHV-1 gene
regulation suggesting that it is identical to HSV-1 gene
regulation, we find it useful to base hypotheses on
systems which, at least initially, a p p e a r to be similar. T h e
identification o f VP8 as the major t e g u m e n t protein o f
BHV-1 and demonstration that it is homologous to the
HSV-1 U L 4 7 gene product is consistent with the
identification o f the HSV-1 U L 4 7 gene product as a
major tegument protein o f H S V - l virions ( M c L e a n et al.,
1990). U p o n viral infection, UL47, a modulator o f g-TIF,
as well as the a - T I F is carried into the cell. U'L47 m a y act
by complexing with the g - T I F and cellular proteins or by
modifying g - T I F or cellular proteins, changing their
ability to trans-activate immediate early genes. It has
been k n o w n for some time that H S V - I carries a protein
kinase in its tegument (Lemaster & R o i z m a n , 1980). It is
possible U L 4 7 m a y have kinase activity that modulates
the activity o f the p h o s p h o p r o t e i n V m w 6 5 (~-TIF). W e
have recently shown t h a t BHV-1 carries protein kinase
activity in its tegument. Initial results suggest this kinase
activity m a y be attributed to the VP8 protein and we are
investigating this possibility futher.
We thank Anne Marie Adachi and Randy Nelson for technical
assistance in preparing the monoclonal antibodies against VP8 and
BHV-gB. We are also grateful to Howard Marsden for offering useful
3083
suggestions about the manuscript. The project was funded through a
contract with Agriculture Canada.
References
BANDYOPADHYAY,S. K., MITTAL, S. K. & FIELD, H. J. (1990).
Identification of the gene homologous to HSV-1 major DNA binding
protein in the BHV-1 genome. VeterinaryMicrobiology 22, 203-212.
BARKER,D. E. & ROIZMAN,B. (1990). Identification of three genes
nonessential for growth in cell culture-near the right terminus of the
unique sequences of long component of herpes simplex virus 1.
Virology 177, 684-691.
BERK, A. J, & SHARP, P. A. (1977). Sizing and mapping of early
adenovirus mRNAs by gel electrophoresis of S1 endonucleasedigested hybrids. Cell 12, 721-732.
BOLTON,D. C., ZEE, Y. C. & ARDANS,A. A. (1983). Identification of
envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis. Veterinary
Microbiology 8, 57-68.
CATRALA,G., SAVOUgET,J. F., MENDEZ,B., WEST,B. L., KARIN,M.,
MARTIAL,J. A. & BAXTER,J. D. (1983). Laboratory methods: a
method for isolation of intact, translationally active ribonucleic acid.
DNA 2, 329-335.
DAVlSON,A. J. & Sco'lq', J. E. (1986). The complete DNA sequence of
varicella-zoster virus. Journal of General Virology 67, 1759-1816.
DAYIIOFF,M. (editor) (1978). Atlas of Protein Sequence and Structure,
vol. 5, supplement 3, pp. 345-358. Washington, D.C. : National
Biomedical Research Foundation.
FITZPATRICK,D., BABIUK,L. A. & ZAMB,T. J. (1989). Nucleotide
sequence of bovine herpesvirus type 1 glycoprotein glII, a structural
model as a new member of the immunoglobulin superfamily and
implications for the homologous glycoproteins of other herpes
viruses. Virology 173, 46-57.
GODING,C. R. & O'HARE, P. (1989). Herpes simplex virus Vmw65octamer binding protein interaction : a paradigm for combinatorial
control of transcription. Virology 173, 363-367.
HALL,L. M., DRAPER,K. G., FRINK,R. J., COSTA,R. H. & WAGNER,
E. K. (1982). Herpes simplex virus mRNA species maping in EcoRI
fragment I. Journal of Virology 43, 594--607.
HIGGINS,D. G. &. SHARP,P. M. (1988). CLUSTAL: a package for
performing multiple sequence alignments on a micro computer. Gene
73, 237-244.
LEMASTER,S. & ROIZMAN,B. (1980). Herpes simplex virus phosphoproteins. II. Characterization of the virion protein kinase and of the
polypeptides phosphorylated in the virion. Journal of Virology 35,
798-811.
LUDWIG,G. V. & LETCHWORTH,G. J., III (1987). Temporal control of
bovine herpesvirus 1 glycoprotein synthesis. Journal of Virology 61,
3292-3294.
MCGEOCH, D. J., DALRYMPLE,M. A., DAVISON,A. J., DOLAN,A.,
FRAME,M. C., MCNAB,D., PERRY,L. J., SCOTT,J. E. & TAYLOR,P.
(1988). The complete DNA sequence of the long unique region in the
genome of herpes simplex virus type I. Journalof General Virology69,
1531-1574.
MCKNIGHT, J. L. C., PELLETI',P. E., JENKINS,F. J. & ROIZMAN,B.
(1987). Characterization and nucleotide sequence of two herpes
simplex virus 1 genes whose products modulate ~-trans-inducing
factor-dependent activation of the ccgenes. Journal of Virology 61,
992-1001.
MCLEAN,G., RIXON,F., LANGELAND,N., HAAP,R, L. & MARSDEN,H.
(1990). Identification and characterization of the virion protein
products of herpes simplex virus type 1 gene UL47. Journal of
General Virology 71, 2953-2960.
MANIATIS, T., FRITSCH, E. F. & SAMBROOK, J. (1982). Molecular
Cloning: a Laboratory Manual. New York: Cold Spring Harbor
Laboratory.
MARSHALL,R. L., RODmGUEZ,L. L., & LETCHWOR'mG. J., III (1986).
Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus) by biochemical and immunological
methods. Journal of Virology 57, 745-753.
3084
D. E. Carpenter and V. Misra
MISRA, V., BLUMENTHAL,R. M. & BABIUK, L. A. (1981). Proteins
specified by bovine herpesvirus-1 (infectious bovine rhinotracheitis
virus). Journal of Virology 40, 367-378.
MISRA,V., GILCHRIST,J. E., WE1NMASTER,G., QUALTIERE,L., VANDEN
HURK, S. & BABIUK, L. A. (1982). A herpesvirus induced 'early'
glycoprotein: characterization and possible role in immune cytolysis.
Journal of Virology 43, 1046-1054.
MISRA, V., NELSON, R. & SMITH, M. (1988). Sequence of a bovine
heresvirus type-1 glycoprotein gene that is homologous to the herpes
simplex gene for the glycoprotein gB. Virology 166, 542-549.
MII"rAL, S. K. & FIELD, J. (1989). Analysis of the bovine herpesvirus
type 1 thymidine kinase (TK) gene from wild-type and TK-deficient
mutants. Journal of General Virology 70, 901-918.
NELSON, R., ADACHI, A.-M., CHISHOLM, H. & MISRA, V. (1989).
Temperature-sensitive mutants of bovine herpesvirus type 1:
mutants which make unaltered levels of 'early' glycoproteins but fail
to synthesize a 'late' glycoprotein. Journal of General Virology 70,
125-132.
OWEN, L. J. & FIELD, H. J. (1988). Genomic location and sequence
analysis of the putative bovine herpesvirus-1 polymerase gene.
Archives of Virology 98, 27-38.
PASTORET,P. P., BURTONBOY,G., AGUILAR-SETIEN,A., GODART, M.,
LAMY,M. E. & SCHOENAERS,F. (1980). Comparison between strains
of infectious bovine rhinotracheitis virus (bovid herpesvirus I) from
respiratory and genital origins, using polyacrylamide gel electrophoresis of structural proteins. Veterinary Microbiology 5, 187-192.
PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient mRNAdependent translation system from reticulocyte lysates. European
Journal of Biochemistry 67, 493-497.
ROIZMAN, B., KRISTIE, T., McKNIGHT, J. L. C., MICHAEL, N.,
MAVROMARA-NAZOS,P. & SPECTOR,D. (1988). The trans-activation
of herpes simplex virus gene expression: comparison of two factors
and their cis sites. Biochimie 70, 1031-1043.
SMITH, G. A., YOUNG, P. L. & MATTICK,J. S. (1990). The location and
nucleotide sequence of the thymidine kinase gene of bovine
herpesvirus type 1.2. Journal of General Virology 71, 2417-2424.
STERN, S., TANAKA,M. & HERR, W. (1989). The oct-1 homoeodomain
directs formation of a multiprotein-DNA complex with HSV
transactivator VP16. Nature, London 341, 624-630.
TIKOO, S. K., FITZPATRICK,D. R., BABIUK,L. A. & ZAMB,T. J. (1990).
Molecular cloning, sequencing and expression of functional bovine
herpesvirus I glycoprotein glV in transfected bovine cells. Journalof
Virology 64, 5132-5142.
WIRTH, U. V., GUNKEL, K., ENGELS, i . & SCHWYZER, M. (1989).
Spatial and temporal distribution of bovine herpesvirus 1 transcripts. Journal of Virology 63, 4882--4889.
WHITBECK,J. C., HELLO,L. J. & LAWRENCE,W. C. (1988). Comparison
of the bovine herpesvirus lgI gene and the herpes simplex virus type
1 gB gene, Journal of Virology 62, 3319-3327.
ZHANG, Y., SIRKO, D. A. & MCKNIGHT, J. L. (1991). Role of herpes
simplex virus type 1 UL46 and UL47 in alpha TIF-mediated
transcriptional induction: characterization of three viral deletion
mutants. Journal of Virology 65, 829-841.
(Received 28 June 1991; Accepted 5 August 1991)