Download Identification and characterization of the virion protein products of

Journal o f General Virology (1990), 71, 2953-2960.
2953
Printed in Great Britain
Identification and characterization of the virion protein products of herpes
simplex virus type 1 gene UL47
Gordon M c L e a n , 1 Frazer Rixon, 1 Nina Langeland, 2 Lars Haarr 2 and Howard Marsden 1.
1 Medical Research Council Virology Unit, Institute of Virology, University of Glasgow, Church Street,
Glasgow G l l 5JR, U.K. and 2 Department of Biochemistry, University of Bergen, Norway
We have identified two encoded proteins with an
antiserum raised against a synthetic oligopeptide
corresponding to amino acids 671 to 684 of the
predicted protein product of gene UL47 of herpes
simplex virus type 1 (HSV-1). They have apparent Mrs
of 82000 and 81000 and are both major virion
components located in the tegument. The 82/81K
proteins were first detected in infected cells in minor
amounts 6 h after infection at 37 °C but were later
(from 10 h until 24 h after infection) present in large
amounts. UL47 regulation was investigated using
phosphonoacetic acid (PAA), an inhibitor of DNA
synthesis: the amounts of the 82/81K protein synthesized were compared with those of 65KDBv, an early
gene product, and 21K/22K, a true late gene product.
The data showed that UL47 is regulated as a true late
gene.
Introduction
Viruses. HSV-1 strain 17 syn+ (Brown et al., 1973) was used in these
studies. The phosphonoacetic acid (PAA)-resistant mutant PAAr-1
was derived from HSV-1 17 syn + (Hay & Subak-Sharpe, 1976); the
mutation has been mapped within the DNA polymerase gene
(Crumpacker et al., 1980).
Over 30 proteins have been detected in virions of herpes
simplex virus type 1 (HSV-1) (Spear & Roizman, 1972;
Heine et al., 1974; Marsden et al., 1976; reviewed by
Dargan, 1986). However, the genes encoding only about
half the virion proteins have so far been identified.
Research towards identification of the genes encoding
others has recently been stimulated by the availability of
the complete sequence of the HSV-1 genome (McGeoch
et al., 1988). This sequence allows an approach whereby
antisera made against synthetic oligopeptides which
correspond to short amino acid sequences of predicted
gene products are used to detect previously unidentified
HSV proteins, or to assign encoding genes to already
recognized proteins. Using this approach our laboratory
has previously reported the identification of two virion
proteins: gG-1, an envelope glycoprotein encoded by
gene US4 (Frame et al., 1986a) and a 10K tegument
phosphoprotein encoded by gene US9 (Frame et al.,
1986b). We now report the identification of the products
of gene UL47 as major virion proteins of apparent Mrs
82000 and 81000 and show that the 82/81K proteins are
regulated as true late proteins.
Methods
Cells. BHK-21 C13 cells (Macpherson & Stoker, 1962) were used
throughout and were maintained in Eagle's medium supplemented
with 5 ~ tryptose phosphate and 10~ newborn calf serum (Gibco)
(ETCx0).
0000-9757 © 1990 SGM
Infection procedure. Cell monolayers at 75 ~ confluence were infected
for 1 h at a multiplicity of 20 p.f.u, in a volume of 0.5 ml of growth
medium per 50 mm dish, 1.5 ml per 90 mm Petri dish or 20 ml per roller
bottle. To inhibit viral DNA synthesis, cells were maintained from 1 h
before and then throughout infection in medium containing 300 ~g
PAA (Sigma) per ml.
Synthesis o f oligopeptides. Oligopeptides YGAAALRAHVSGRRA
and YLTPANLIRGDNA were synthesized by continuous flow Fmoc
chemistry (for reviews, see Atherton et al., 1979, Sheppard, 1983) using
an LKB Biolynx peptide synthesizer. Chemicals for these syntheses
were purchased from Biochrom with the exception of dimethylformamide (Rathburn Chemicals), trifluoroacetic acid (Aldrich Chemical
Company) and resin to which the first amino acid had been coupled via
an acid-labile bond (Peptide and Protein Research). The peptides were
synthesized both directly onto the resin and onto a branching lysine
core to generate a peptide with eight identical branches (Posnett et al.,
1988; Tam, 1988) using two columns of the synthesizer in series. The
lysine core was synthesized (onto an alanine residue coupled to the
resin) using Fmoc-Lys (Fmoc) pentafluorophenyl ester (purchased
from Peptide and Protein Research).
Following synthesis, the peptides were cleaved from the resin and
side-chain-protecting groups removed with 9 5 ~ trifluoroacetic acid
and 4 ~ (w/v) phenol in H20 using standard protocols. The Mrs of the
single peptides and the polylysine core were determined by mass
spectrometry (M-Scan Ltd) which gave values identical to those
expected. The amino acid composition of the branched peptide was
determined by amino acid analysis (Cambridge Research Biochemicals) and was in agreement with expectations. The peptides were
chosen in part for ease of synthesis and also because they correspond to
sequences near the termini of proteins, a location which is favourable to
the generation of protein-reactive sera (Palfreyman et al., 1984).
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2954
G. McLean and others
Antisera. All antisera were raised in rabbits. Antiserum 94497 was
raised against YGAAALRAHVSGRRA, which corresponds to amino
acids 671 to 684 of the 693 amino acid predicted UL47 gene product
(McGeoch et al., 1988) with an additional amino-terminal tyrosine (for
protein coupling purposes not relevant to this manuscript), presented as
an eight-branched peptide. The branched peptide was injected
intramuscularly, 100 ~tg per immunization per rabbit without an
additional hapten, in Freund's complete adjuvant on day 0 and then in
Freund's incomplete adjuvant on days 10, 30 and 40. Animals were bled
on day 50. Antiserum 18826, prepared against a synthetic oligopeptide
corresponding to amino acids 476 to 487 of the 65K DNA-binding
protein encoded by UL42, has previously been described (Parris et al.,
1988), as has antiserum 14327, which was prepared against a synthetic
oligopeptide corresponding to amino acids 151 to 161 of the predicted
US11 gene product (MacLean et al., 1987).
Radioisotopic labelling o f cells. Infected and mock-infected cells were
labelled with [35S]methionine (50 [aCi/ml) (Amersham) immediately
after adsorption in Eagle's medium containing 20% of the normal
concentration of methionine and supplemented with 2% foetal calf
serum (Emet/5C2). Approximately 24 h later the cells were washed
twice with phosphate-buffered saline and suspended in denaturing
buffer (0.05 M-Tris-HCI pH 6.8, 2 % SDS, 5 % 2-mercaptoethanol, 10 %
glycerol; enough bromophenol blue was added to visualize the dye
front) at a concentration of 2.5 × 108 cell equivalents/ml. This
suspension was heated at 70 °C for 5 min, stored at - 70 °C and boiled
immediately before electrophoresis.
Purification ofvirions. BHK cells in 80 oz plastic roller bottles were
infected at 37 °C with 40 ml ETC1o containing 1 p.f.u. HSV-1 strain
17+/500 cells. After 16 h the infection medium was removed and
replaced with 40 ml of Emet/sC2. After a further 8 h, [3sS]methionine
was added (25 ~tCi/ml) and incubation was continued for 2 days. The
cells were then shaken into the culture medium and pelleted by low
speed centrifugation (100O g for 10 min at 4 °C). After supernatant
clarification by a second low speed centrifugation, the supernatant
virions were pelleted (9000 g for 2 h at 4 °C). The virus pellet was
resuspended and further purified by one of two methods. In the first,
the resuspended pellet was centrifuged through a Ficoll 400 gradient,
the virion band was then collected and pelleted by centrifugation at
20000 g for 1 h (J. F. Szilfi.gyi & C. Cunningham, personal
communication). In the second, the resuspended pellet was purified by
centrifugation through 54% Percoll [in 0.025 M-sucrose, 0.01% bovine
serum albumin (BSA) and 0.5 mM-Tris-HCl pH 7.5] as described by
Marsden et al. (1987).
NP40 extraction o f HSV-1 virions. Extraction was as described by
Frame et al. (1986b) with some modifications. Briefly, NP40 (Pierce
Chemical or BDH Chemicals) was diluted to various concentrations in
20 mM-Tris-HCl, 5 mM-NaC1 pH 7.5. Unlabelled or [35S]methioninelabelled HSV-1 virions were incubated in the different concentrations
of NP40 for 30 rain at room temperature then centrifuged at 40000
r.p.m, for 30 min at 4 °C in a TLA100.2 rotor using a Beckman TL-100
tabletop centrifuge. The supernatant was removed and mixed with 0-5
volumes of 3 × denaturing buffer (see above); the pellet was suspended
in denaturing buffer. These suspensions were heated to 100 °C for 5
min immediately before electrophoresis. For those experiments
involving iodination of virion proteins the centrifugation step was
omitted.
Iodination o f virion proteins. Virion proteins were iodinated essentially
as described by Markwell & Fox (1978). Briefly, 2 mg lodo-gen (Pierce)
(1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril) was dissolved in 2 ml
chloroform immediately before use, 25 gtl was transferred to an
Eppendorf tube, dried and stored under nitrogen. 125Iodine (200 laCi)
(Amersham; IMS. 30) was added, followed by 30 ~tl of Percoll-purified
virions which had been diluted in phosphate buffer (pH 7-4) to a final
buffer concentration of 10 mM. Incubation was performed for 10 min at
between 0 and 4 °C and the reaction stopped by transferring the
solution to another tube without Iodo-gen.
Gel electrophoresis and Western blotting. SDS-PAGE of proteins was
conducted either with 5 to 12.5~ gradient gels cross-linked with 5 ~
(wt/wt) N,N'-methylene-bisacrylamide (BIS) or 9% gels cross-linked
with 2-5% (wt/wt) N,N'-diallyltartardiamide (DATD) using the buffer
system of Laemmli (1970). Western blots were performed essentially as
described (Towbin et al., 1979) with some modifications (Frame et al.,
1986b).
Antisera 14327, 18826 and 94497 were diluted 50-, 100- and 100-fold,
respectively, for the experiments described here. Blotted proteins were
incubated with antisera and bound antibody was visualized with 1251.
labelled Protein A. Where indicated, the antiserum was incubated with
peptide for 1 h before exposure to the blotted protein. The apparent
Mrs of reactive polypeptides were determined by alignment of the 35S
and 1251 autoradiographic images (Haarr et al., 1985).
Silver staining. Following electrophoresis, gels were fixed for 30 min
in 30% ethanol/10% acetic acid. Gels were then removed from the fix
solution and incubated for a further 30 rain in a solution containing
30 % ethanol, 0.5 M-acetic acid, 0.5 % gluteraldehyde and 0-2 % sodium
thiosulphate. The gels were then rinsed thoroughly three times in water
for 10 min and soaked in 0.1% silver nitrate/0.02 % formaldehyde for 15
to 30 min. Gels were then developed in a solution containing 2.5%
sodium bicarbonate/0-01% formaldehyde pH 11-8 for between 5 and 15
min until all bands were visible. Development was stopped by addition
of 0.05 M-EDTA for 5 min followed by a further wash in deionized
water. All solutions for silver staining were prepared using high-purity
filtered HPLC grade water.
Results
Fig. 1 shows the position of the open reading frame of
gene UL47 on the prototype arrangement of the HSV-1
genome (McGeoch et al., 1988), together with the
position and sequence of the peptide against which
antiserum 94497 was raised. The reactivity on Western
blots of this antiserum with proteins from HSV-1infected cells is shown in Fig. 2. The immune serum
specifically detects a strong polypeptide band of apparent Mr 82K in 5 to 12.5 % gels which is not detected by the
pre-immune serum (Fig. 2) and which is not present in
mock-infected cells (Fig. 3, lane 1).
We next investigated the regulation of expression of
this 82K protein. Cells were harvested at various times
after infection and polypeptides were separated by SDSPAGE, transferred to a nitrocellulose membrane and
probed with antiserum 94497. The protein was just
detectable at 6 and 8 h after infection, the amount was
strongly up-regulated by 10 h and found in largest
amounts at 12 h, whereas later the amount of protein
present in the cells declined slightly (Fig. 3). This
dramatic up-regulation was reproducibly observed and
suggests that gene UL47 is regulated as a late gene.
Johnson et al. (1986) suggested an operational definition of true late genes as those whose expression is most
severely reduced, compared to all other groups of genes,
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H S V - 1 U L 4 7 gene products
I
I
I
I
I
I
I
I
I
I
I
0.0
0.1
0-2
0.3
0.4
0.5
0.6
0.7
0-8
0.9
1-0
TRE
U~.
1
2
2955
3
IRL IRs U~ TRs
UL47
U"3
6 9 ~ ~ ~ _ _ ~
/
~
•UL47
f'-'l
] NH2
ARRGSVHARLAAAG(Y)
684
671
Fig. 1. Schematic presentation of the predicted protein product ofgene
ULA7 of HSV-1 strain 17 with the location and sequence of the peptide
against which the antiserum 94497 was raised. The HSV-1 genome is
shown with the unique long (UL) and unique short (Us) regions, the
terminal repeats (TRL and TRs), internal repeats (IRL and IRs) and the
location ofgene UL47 (McGeoch
1988). The fractional length of
the genome is shown at the top of the figure. The numbers at the end of
the open reading frame and peptide refer to amino acid numbers in the
predicted sequence. The amino-terminal tyrosine (Y) residue in
parentheses is not part of the predicted amino acid sequence of UL47
(see text).
etal.,
under conditions of severely inhibited virus DNA
replication. To determine whether gene UL47 was
regulated as a true late gene according to this definition,
the expression of the 82K protein was examined in the
presence of 300 ~tg/ml PAA. At this concentration, virus
DNA replication is reduced to undetectable levels (< 5~o
of the no drug control) in cells infected with HSV-1 strain
17 (Johnson et al., 1986). The PAA-resistant mutant,
PAAr-1, (Hay & Subak-Sharpe, 1976), which induces
wild-type levels of virus DNA synthesis in the presence
of 300 p.g/ml PAA, was included in the experiment as a
control. For comparison, the behaviour of two other
proteins was also examined: 21K, a true late protein
(Johnson et al., 1986) and 65KDBP, an early protein
(Schenk & Ludwig, 1988; Goodrich et al., t989) whose
synthesis is reduced three- to four-fold in the presence of
PAA (Goodrich et al., 1989). The reactivities on Western
blots of the rabbit antisera 18826 and 14327 when used to
detect expression of 65KDBp and 21K respectively have
previously been described (Parris et al., 1988; MacLean
et al., 1987). Antiserum 18826 reacts with only a single
protein, 65KDBp (Parris et al., 1988), whereas antiserum
14327 reacts with predominantly 21K and proteins of
apparent MT22K, 17-5K, 14K and 11K, which have been
shown to be related to 21K (MacLean et al., 1987).
The results of this experiment are shown in Fig. 4 in
which infected cells were harvested at 0, 6 h, 12 h and 24
h after adsorption. Polypeptide 82K is first detected at 12
h after adsorption both in wild-type virus-infected cells
Fig. 2. Western blotting with the 94497 serum showing specific
recognition of an 82K polypeptide band in HSV-l-infected cells. Lane
1 shows 3sS-labelled HSV-l-infected cell proteins transferred to
nitrocellulose. Lanes 2 and 3 show the polypeptides on the nitrocellulose that were detected by the pre-immune or immune 94497 sera,
respectively. In this and subsequent figures, unless stated otherwise,
polypeptides were separated on 5 to 12-5~ SDS-polyacrylamide gels
and the alignment of the 3sS-labelled and 125I-labelled lanes was as
(1985).
described by Haarr
et aL
in the absence of PAA and in PAA-resistant virusinfected cells in the presence of PAA; in cells infected
with wild-type virus in the presence of P A A no synthesis
of 82K could be detected at any time after infection (a).
This finding is exactly what would be expected for true
late proteins and essentially parallels the result with 21K
(b), previously found to be a true late protein (Johnson et
al., 1986). In contrast, 65KDBp is clearly detectable by 6 h
post-infection and is only slightly inhibited by PAA in
the wild-type virus-infected cells (c). The sensitivity of
detection of 82K by antiserum 94497 was examined by
Western blotting twofold serial dilutions of HSV-1infected cells. The serum could easily detect 0.5~ (i.e.
1:256 dilution) of the level of protein induced in cells
infected with wild-type virus in the absence of PAA (d).
These experiments show that in the absence of viral
DNA synthesis the expression of gene UL47 is reduced
to less than 0.5~ of normal. We conclude from these
experiments that UL47, the gene encoding 82K, is
regulated as a true late gene.
To test whether the protein product of gene UL47 is a
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G. M c L e a n and others
2956
MI
0
1 2
3 4 5 6
8
10 12 18
24
(a)
-UL47
1
2
3
4
5 6
8 9 10
7
11 12
-81/82K
UL47-
(b)
1
2
3
4
5
6
8
7
9
10
11
12
-21K
(c)
1
2 3 4
5 6
7 8
9 10 11 12
UL42Fig. 3. The 82K product of UL47 is expressed late in HSV-l-infected
cells. BHK cells were mock-infected (MI) or infected with HSV-1
strain 17, harvested at the times shown (in h), separated by SDS-PAGE
and probed with serum 94497. Proteins from mock-infected cells were
included for comparison. An autoradiographic image of the 12sIProtein A used to detect bound antibody is shown.
structural c o m p o n e n t o f virus particles, virions were
purified from the supernatant o f [35S]methioninelabelled infected cells (Rixon et al., 1988). Fig. 5 (a) is an
electron m i c r o g r a p h o f such a preparation and shows it
to be substantially free o f c o n t a m i n a t i n g cellular debris.
Virion proteins were subjected to gel electrophoresis,
transferred to a nitrocellulose m e m b r a n e and p r o b e d
with serum 94497. Both 5 to 12-5~ gradient gels crosslinked with BIS (Fig. 5, lanes 1 to 6) and 6 ~ gels crosslinked with D A T D (lanes 7 to 9) were used because in
earlier experiments (Marsden et al., 1976) a polypeptide
doublet o f a p p a r e n t Mrs o f 82K and 81K had been
observed in purified virions. The 6 ~ gels cross-linked
with D A T D were found to resolve this doublet more
clearly. Fig. 5(b) (lanes 1 and 7) show the methioninelabelled virion polypeptides which were transferred to
the membrane. The profile is similar to that reported
earlier (Heine et al., 1974; Marsden et al., 1976; D a r g a n ,
1986; Rixon et al., 1988) in that the major proteins seen
include the major capsid protein (Vmw155), gB, Vmw82,
the 65K trans-inducing factor (Vmw65 or 65KTw), gD,
Vmw51 ( V P I 9 C ) and Vmw37. It lacks the large
tegument protein Vmw273 because the latter is too large
to be electroeluted from the gel and transferred to the
nitrocellulose m e m b r a n e (Rixon et al., 1988). Serum
94497 reacts specifically with V m w 8 2 (lane 2) and
Vmw82/81 (lane 8) and this reaction is blocked by the
peptide against which the antiserum was raised (lanes 3
to 5 and lane 9) but not by an unrelated peptide (lane 6).
Lane 7 shows virion proteins blotted from a 6 ~ gel crosslinked with D A T D in which Vmw82/81 has been
resolved as a doublet. T w o separate exposures o f a strip
-65Kr~Bp
'
(d)
1 2
3
.....
. ':i;;
4 5 6
:
7
~;~
8
~ =~i~:
~
9 10
Fig. 4. The 82K product of gene UL47 is regulated as a true late
protein. Proteins were extracted from BHK cells infected with HSV-1
strain 17 in the absence (lanes 1 to 4, a, b and c) or presence (lanes 5 to 8,
a, b and c) of 300 Ixg/mlPAA. A third set of cells were infected with the
PAA-resistant mutant PAAr-1 in the presence of 300 ~tg/mlPAA (lanes
9 to 12, a, b and c). Cells were harvested at 0 (lanes 1, 5 and 9, a, b and c),
6 (lanes 2, 6 and 10, a, b and c), 12 (lanes 3, 7 and 11, a, b and c) and 24 h
(lanes 4, 8 and 12, a, b and c) after infection and polypeptides were
separated by SDS-PAGE. Three identical gels were run, polypeptides
were transferred to nitrocellulose membranes and probed with serum
94497 to detect the product ofgene UL47 (a), serum 14327to detect the
product of gene US11 (b) and serum 18826to detect the product of gene
UL42 (c). (d) Immunoblot with serum 94497 using doubling dilutions of
proteins extracted from BHK cells infected with HSV-1 strain 17 in the
absence of PAA and harvested 18 h after infection. The autoradiographs have been trimmed to show only the relevant region. Lanes 1 to
10, dilutions of 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024
and 1:2028 respectively.
probed with serum 94497 show that both proteins are
recognized by the serum (lanes 8 and 9) and that the
reactions are blocked by the peptide against which the
antiserum was raised (lane 10). This experiment demonstrates that both proteins are encoded by gene U L 4 7 and
identifies the U L 4 7 gene products as the previously
recognized Vmw82/81 o f HSV-1 virions.
To investigate where in the virion the 82/81K protein
is located, purified virions were treated with various
concentrations o f N P 4 0 and solubilized proteins were
separated from larger virion structures by centrifugation.
Proteins in both the pellet (P) and the supernatant (S)
were resolved by S D S - P A G E using a 5 ~ to 1 2 . 5 ~ gel
and proteins were visualized by silver staining (Fig. 6).
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HSV-1 UL47 gene products
1
2
3
4
5
6
7
2957
8
Vmw155gBVmw82/81Vmw65gD-
-UL47
Vmw37-
•2• ••
(b)
1 2
3 4 5 6
7
8910
Vmw155-~i17
~
Vmwl
Vmw82Vmw65gDVmw51"
:2; :
: :
,
Fig. 6. Localization of Vmw82/81 within the virion by treatment of
virions with NP40. Following treatment of virions with various
concentrations of NP40 (lanes 1 and 2, 0.00~; lanes 3 and 4, 0.01~;
lanes 5 and 6, 0.10~; lanes 7 and 8, 1.00~), solubilized proteins were
identified as those remaining in the supernatant following ultracentrifugation as described. Proteins in the pellet (odd-numbered lanes) and
supernatant (even-numbered lanes) were separated by SDS-PAGE
and silver-stained.
Vmw37-
al., 1988) were solubilized, as was Vmw82/81.
Fig. 5. The 82/81K polypeptide products of gene UL47 are constituents of the HSV-1 virion. (a) Electron micrography of the virion
preparation used for the experiment shown in (b). Virions were
prepared as described in the text. Virion sample (2 p.l) was applied to a
parlodion-coated copper grid, allowed to adsorb for 5 min, blotted dry
and stained with 3~ phosphotungstic acid pH 7-0. (b) HSV-1 virion
proteins, labelled with [35S]methionine,were separated by SDS-PAGE
using either 5 to 12-5% gels cross-linked with BIS (lanes 1 to 6) or 6~
gels cross-linked with DATD (lanes 7 to 9) and then transferred to
nitrocellulose membrane strips. Lanes 1 and 7 show autoradiographic
images of 35S-labelled virion proteins. Lanes 2 to 6, 8, 9 and 10 were
probed with serum 94497 in the absence of polypeptide (lanes 2, 8 and
9) or in the presence of 1 l.tg/ml(lane 3), 10 lag/ml (lane 4) and 100 lag/ml
(lanes 5 and 10) of branched peptide YGAAALRAHVSGRRA
against which the antiserum was raised or 100 ~tg/ml of the unrelated
branched peptide YLTPANLIRGDNA (lane 6). Bound antibody was
visualized with 125I-ProteinA (lanes 2 to 6, 8, 9 and 10). Lanes 8 and 9
show two different exposures from the same nitrocellulose strip. The
major virion proteins transferred to nitrocellulose are indicated on the
left.
T h e data show that in the absence of N P 4 0 or with 0-01
N P 4 0 the virions a p p e a r to be intact because no proteins
are found in the supernatant. With 0.1 ~ and 1 ~ N P 4 0
both gB (an envelope glycoprotein) and V m w 6 5 (a
tegument protein; R o i z m a n & Furlong, 1974; Preston et
In
contrast, concentrations of N P 4 0 as high as 1 ~o did not
solubilize Vmw155 (the major capsid protein), showing
that the capsids remain intact. These data suggest that
Vmw82/81 is located in the virion tegument or envelope.
To determine in which o f these two structures
Vmw82/81 is located, virion proteins were iodinated
before or after disruption with 1 X NP40. In this
experiment, NP40-solubilized proteins were not separated from the remainder of the virion prior to iodination.
The first condition (without NP40) should label only
protein exposed on the surface of virions whereas the
latter condition (with N P 4 0 ) should label additionally
some internal proteins.
Iodinated proteins were analysed by S D S - P A G E (Fig.
7). The positions of gB, Vmw82/81, Vmw65 (transinducing factor) and g D were obtained by alignment
with [35S]methionine-labelled virion proteins (data not
shown). Both gB and g D were labelled in the presence
(lane 1) and absence (lane 2) of N P 4 0 as would be
expected of surface proteins, whereas Vmw82/81 and
Vmw65 (a tegument protein) were labelled only in the
presence o f NP40. The heavily labelled band just above
the 65K trans-inducing factor in lane 1 was also present
after iodination of the BSA-containing Percoll solution,
and most likely represents BSA. F r o m this experiment,
we concluded that Vmw82/81 is located in the virion
tegument.
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2958
G. M c L e a n and others
1
2
Vn
Fig. 7. Vmw82/81is locatedin the tegument.Percoll-purifiedvirions
wereincubatedat between0 and 4 °Ceitherin the absence(lane2)or in
the presence (lane 1) of 1~ NP40. All sampleswere iodinated;acidinsoluble radioactivity from ~25I varied from 20000 to 600000
c.p.m./~tl. Polypeptideswere then separated on SDS polyacrylamide
slab gels of 9~ acrylamidecross-linkedwith DATD.
Discussion
Significant differences exist in the published DNA
sequences obtained for gene UL47 from HSV-1 strain F
(McKnight et al., 1987) and HSV-1 strain 17 (McGeoch
et al., 1988). In particular, there is an extra G residue at
base 101 163 of strain 17 which is not present in the
sequence of strain F. As a consequence, the predicted
amino acid sequences differ from the aligned prolines at
position 650 of strain F and 651 of strain 17; the former
continues for a further 14 residues whereas the latter
continues for a further 42 residues. The sequence
YGAAALRAHVSGRRA, against which antiserum
94497 was raised, corresponds to amino acids 671 to 684
of the UL47 product predicted for strain 17 (McGeoch et
al., 1988), with an additional N-terminal tyrosine, but
does not exist in the UL47 product predicted for strain F
(McKnight et al., 1987). Our data showing that antiserum 94497 specifically identifies the products of gene
UL47 of HSV-1 strain 17 as abundant virion proteins
with apparent Mrs:of 82000 and 81000 confirms the
correctness of the UL47 DNA sequence obtained for
strain 17 but does not exclude the possibility that there is
a strain-specific sequence difference between strain 17
and strain F.
We have established that expression of the UL47 gene
is regulated in a true late manner. Our findings support earlier studies which reported a 4.7 kb true late
mRNA transcribed in a leftward direction from this
region of the genome (Hall et al., 1982). The quantitative
data reported here (Fig. 4) show that the UL47 protein
product levels in the absence of viral DNA synthesis are
less than 0-5~ of those in a normal infection.
Regulation of HSV-1 gene expression has been most
frequently examined at the mRNA level. Evidence exists
for nine true late mRNAs (Holland et al., 1980, I984;
Frink et al., Hall et al., 1982; Silver & Roizman, 1985;
Johnson et al., 1986). Comparison of the positions and
sizes of these transcripts with the DNA sequence
obtained by McGeoch et al. (1988) suggests that these
true late mRNAs may originate from genes UL22,
UL31, UL32, UL38, UL44, UL45, UL47, UL49 and
US11, but quantitative data for mRNA regulation has
been obtained only for UL49 (Silver & Roizman, 1985)
and US11 (Johnson et al., 1986). Quantitative data will
have to be obtained for the others to identify these genes
unambiguously as true late. At present, the designation
of HSV-1 UL38 as a true late gene seems doubtful.
Firstly, assembly of capsids is not dependent on virus
DNA replication (reviewed by Dargan, 1986) but is
dependent on the expression of HSV-1 UL38 (Pertuiset
et al., 1989), the product of which is an abundant capsid
protein (Rixon et al., 1990). Secondly, qualitative data
have been obtained by Yei et al. (1990) suggesting that
the HSV-1 gene is not regulated as a true late gene
whereas the HSV-2 gene is.
At the protein level, the only gene previously shown
quantitatively to be regulated in a true late manner is
US11 (Johnson et al., 1986). Its product is a 21K nonstructural protein (Rixon & McGeoch, 1984) which
localizes to the nucleoli of infected cells (MacLean et al.,
1987) and binds directly or indirectly to DNA, most
likely to the 'a' sequence (Dalziel & Marsden, 1984;
MacLean et al., 1987).
We have now shown that the 82/81K products of
UL47 are abundant structural proteins and, because they
are readily released from virions by NP40, they must be
components of the tegument or envelope. Iodination in
the presence but not in the absence of NP40 indicates
that Vmw82/81 is located in the tegument. Examination
of the amino acid sequence predicted for UL47 reveals
no features characteristic of a membrane protein, which
would support this conclusion.
By examination of earlier studies (Roizman & Furlong, 1974; Heine et al., 1974; Honess & Roizman, 1973),
and comparison of the apparent MrS, relative intensity
and virion location of the proteins described there, we
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H S V - 1 UL47 gene products
speculate that Vmw82/81 corresponds to virion proteins
13 and 14. However, this speculation remains to be
experimentally tested.
The tegument is a poorly defined structure which lies
between the capsid and the envelope and constitutes
approximately 65 ~ of the virion by volume (Schrag et
al., t989). Many tegument proteins are poorly characterized but some appear to function at the early stages of
infection. Prominent among these is 65KTw, a protein of
apparent Mr 65 000, which stimulates transcription from
immediate early genes (Post et al., 1981; Batterson &
Roizman, 1983; Campbell et al., 1984). It has been
reported (McKnight et al., 1987) that the products of
genes UL46 and UL47 act to modulate (positively and
negatively, respectively) the activity of 65KT~F. It is
therefore of interest that the 82/81K products of gene
UL47 should be present in virions in amounts comparable to that of 65KTIF (Vmw65). 65KTIF is also of
importance as a structural component of the virion (this
might account for its abundance in virions) because a
temperature-sensitive mutation in the gene encoding this
protein confers decreased thermostability on virions
(Moss, 1989). Whether Vmw82/81 will also have an
equivalent structural role and whether it is essential for
virus growth and assembly remain to be determined.
It is of interest that two proteins originate from UL47.
In a previous analysis using two-dimensional (2D) gel
electrophoresis (Marsden et al., 1983) we identified two
polypeptides (spots 24 and 25) of apparent Mr 81000 and
one polypeptide (spot 402) of apparent Mr 82000. We
cannot unambiguously equate spots 24, 25 and 402 with
either of the 82K or 81K polypeptides but, because no
other spots having these apparent MrS were detected on
2D gels and because all three spots are labelled following
iodination of NP40-treated virions but not untreated
virions (data not shown), we think it likely that spot 402
corresponds to Vmw82 and spots 24 and 25 correspond to
Vmw81. Our earlier studies (Marsden et al., 1983)
demonstrated that spot 402 was a processed product
whereas spots 24 and 25 were synthesized in vitro. We
speculate that spots 24 and 25 both arise from UL47 by
initiation of translation at the first and second AUGs
(codons 1 and 34 respectively) of the mRNA. There is a
precedent for this suggestion from studies on the
translation of the HSV thymidine kinase, in which it was
shown that translation is initiated at the first three
AUGs of the mRNAs (Haarr et al., 1985).
Our identification of the UL47 gene product as an
82/81K virion protein and the availability of antiserum
directed against this protein should facilitate its purification and allow in vitro experiments to test directly its role
in modulating trans-activation by 65KTIF. It will also be
important to determine whether it has any role in virion
structure.
2959
We thank Professor J. H. Subak-Sharpe for his interest in this work
and for critically reading the manuscript. G. McL. was supported by an
MRC Studentship for training in research methods. This work was
supported in part by grants from the Norwegian Research Council for
Science and the Humanities and from the Norwegian Society for
Fighting Cancer.
Note in proof. HSV-1 mutants in which gene UL47 has been deleted
can grow in tissue culture [Barker, D. E. & Roizman, B. (1990). Virology
177, 684-691]. Misra and Carpenter (personal communication) have
identified the gene encoding VP8, a major 107K tegument protein of
bovine herpesvirus 1 [Marshall et al. (1986). Journal of Virology 57,
745-753] and have shown by sequencing the gene that VP8 is
homologous to UL47 of HSV-1. They have also shown that VP8 is a
phosphoprotein which is expressed late in cells infected with BHV-1.
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