Journal of General Virology (1992), 73, 303-311. Printedin Great Britain
303
The ULI3 virion protein of herpes simplex virus type I is phosphorylated
by a novel virus-induced protein kinase
Charles Cunningham, 1 Andrew J. Davison, t* Aidan Dolan, 1 Margaret C. Frame, ~
Duncan J. McGeoch, 1 David M. Meredith, 2 Helen W. M. Moss 1 and Anne C. Orr ~
1MRC Virology Unit, Institute of Virology, University of Glasgow, Church Street, Glasgow G l l 5JR and 2Department of
Microbiology, University of Leeds, Leeds LS2 9JT, U.K.
Herpes simplex virus type 1 (HSV-1) induces a protein
kinase (PK) activity in infected cell nuclei. In vitro, the
enzyme is able to phosphorylate exogenous casein
(albeit inefficiently) but not protamine, can use ATP or
G T P as a phosphate donor, is stimulated by high salt
concentrations and is insensitive to inhibition by
heparin. On the basis of these properties, the PK
appears to be distinct from previously described
cellular enzymes and from the cytoplasmic PK encoded
by the viral US3 gene. A major substrate of the enzyme
in vitro is a virus-induced protein with an Mr of 57000
(Vmw57). The gene encoding Vmw57 was mapped
using recombinants between HSV-1 and HSV-2 to a
region of the virus genome containing genes UL9 to
UL 15. Use of a monospecific rabbit antiserum showed
that Vmw57 is a virion structural protein encoded by
gene UL13. These results, in conjunction with previous
reports that the UL13 protein contains PK sequence
motifs, support the notions that the nuclear PK and
Vmw57 are identical, and that the observed reactivity is
due to autophosphorylation.
Introduction
peptide containing the eight amino acid residues at the
carboxy terminus of the US3 protein reacts specifically
with an HSV-1-induced protein with an Mr of 68 000. It
also recognizes a protein of identical Mr in an extensively
purified preparation of the HSV-1 PK which could be
phosphorylated by incubation with [~-32p]ATP. Purves
et al. (1987) have shown that an HSV-1 mutant in which
part of the US3 coding region has been deleted does not
induce the enzyme, in contrast to wild-type virus and a
derivative of the mutant in which US3 is reinserted. The
role of the US3 cytoplasmic PK in virus infection is
unknown, but it is not required for lytic growth of virus in
cell culture.
Smith & Smith (1989) and Chee et al. (1989) have
noted the presence in the HSV-1 genome of a second
gene encoding a protein with PK sequence motifs. This
gene, UL13, has counterparts not only in other alphaherpesviruses such as VZV (Davison & Scott, 1986;
McGeoch et al., 1988), but also in betaherpesviruses,
such as human cytomegalovirus (Chee et al., 1990) and
human herpesvirus 6 (Lawrence et al., 1990), and in the
gammaherpesvirus Epstein-Barr virus (Baer et al.,
1984). This is in contrast to the US3 PK, which has
counterparts only in the alphaherpesviruses. The protein
specified by UL13 has a predicted Mr of 57193
(McGeoch et al., 1986, 1988) and, like the US3 PK, has
sequence motifs indicating that it has the ability to
Protein kinase (PK) activity is associated with several
herpesviruses (Rubenstein et al., 1972; Randall et al.,
1972; Tan, 1975; Lemaster & Roizman, 1980; Blue &
Stobbs, 1981; Fliigel & Darai, 1982; Michelson et al.,
1984; Katan et al., 1985, 1986; Stevely et al., 1985;
Montalvo & Grose, 1986; Purves et al., 1986). The most
extensively characterized examples are cytoplasmic PKs
induced after infection of cells by the alphaherpesviruses
herpes simplex virus type 1 (HSV-1) and pseudorabies
virus (reviewed by Leader & Katan, 1988). The HSV-1
PK can utilize ATP but not GTP to phosphorylate serine
or threonine residues in basic substrates such as
protamine, but not acidic substrates such as casein. It is
active in high salt concentrations and appears to be
insensitive to effectors. The denatured subunit detected
in autophosphorylation experiments has an Mr of 68 000.
D N A sequence data have provided additional strong
evidence that alphaherpesviruses encode distinct PKs.
Several motifs characteristic of PKs have been identified
in the predicted translation products of HSV-1 gene US3
and its counterpart, gene 66, in varicella-zoster virus
(VZV) (McGeoch & Davison, 1986). Two reports have
demonstrated unequivocally that the HSV-1 cytoplasmic
PK is the product of US3. Frame et al. (1987) have shown
that a polyclonal rabbit antiserum directed against a
0001-0598 © 1992SGM
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304
C. Cunningham and others
phosphorylate serine and threonine, rather than tyrosine,
residues.
In this paper, we report the properties of a novel
HSV-1 PK, and show that a major substrate of the
enzyme is a virion structural protein encoded by UL13.
We discuss the possibility that the latter protein is
identical to the PK.
Methods
Preparation of nuclear and virion extracts. Baby hamster kidney
(BHK) C 13 cells were either mock-infected or infected (usually for 8 h)
with HSV-1 strain 17 (Brown et al., 1973), HSV-2 strain HG52
(Timbury, 1971) or HSV- 1/HSV-2 intertypic recombinants (Marsden et
al., 1978; Chartrand et al., 1981) at a multiplicity of 20 in Eagle's
medium containing 2% (v/v) foetal calf serum (FCS).
Proteins were extracted from nuclei using salt washes and
ammonium sulphate precipitation as described by Piette et al. (1985).
Precipitated proteins were resuspended in 0.4 ml PK assay buffer
(50 mM-Tris-HCl pH 8.0, 50 raM-magnesium acetate, 0.5 M-NaC1,
0.1% v/v NP40, 1 mM-DTT) and stored at - 7 0 °C.
Virion extracts were prepared as described by Whittaker & Meredith
(1990). HSV-1 virions were prepared by sucrose density centrifugation,
and their purity was ascertained by SDS-PAGE. Virions were treated
with ether, extracted with 1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-l-propane sulphonate for 5 min at 0 °C, centrifuged at
12500 g for 10 rain through a 35% (w/v) sucrose cushion and the
supernatant was retained.
PK assay. Nuclear extract (5 to 25 gl containing approximately 0.1 to
0,5 gg protein) was incubated for 20 min at 37 °C with 50 ~tl PK assay
buffer containing 5 gCi [?-3:p]ATP or [y-32p]GTP (Amersham;
PB10218 or PB10244) in a total volume of 100 lal. NaC1 was included at
a final concentration of 1 M in earlier experiments, and in later
experiments at 1.5 M. Casein, protamine or heparin were added in
certain experiments. PK activity was measured by monitoring
radioisotope incorporation. Aliquots (5 gl) of reaction mixtures were
dried on filter paper discs (Whatman grade 1), washed extensively in
10% (w/v) TCA saturated with tetrasodium pyrophosphate, rinsed
with ethanol and dried. Precipitated radioactivity was determined by
scintillation counting. Aliquots of reaction mixtures were also analysed
by S D ~ P A G E .
3zp labelling of nuclear proteins in vivo. BHK cells were infected at a
multiplicity of 20 with HSV-1 and incubated from 2 to 12 h after
infection with 100 gCi/ml [32p]orthophosphate (Amersham; PBS11) in
phosphate-free Eagle's medium containing 2% FCS. Nuclear proteins
were then prepared as described above.
DNA-eellulose chromatography. Double-stranded DNA-cellulose
(Sigma) was equilibrated in 0.1 M-Tris-HC1 pH 7-5, 50mM-NaC1,
1 mM-2-mercaptoethanol, 1 mM-PMSF. Infected cell nuclear extract
was dialysed against this buffer and applied to a DNA-cellulose
column. The column was washed extensively, and bound proteins were
eluted using a 0.1 to 1.0 r,l linear NaC1 gradient.
Translation o f UL13 in vitro. Two expression plasmids containing
UL13 were prepared.
(i) A derivative of the cloning vector pTZ18U (Pharmacia) lacking
the HindlII site was prepared by digesting with HindIII, treating with
T4 DNA potymerase in the presence of the four dNTPs and ligating.
The 10.8 kbp KpnI f fragment of HSV-1 DNA, which contains the
entire coding regions of UL8 to UL13, was isolated from a plasmid
(Davison & Rixon, 1984) and ligated into the KpnI site of the modified
pTZ18U vector. Resulting plasmids with KpnI f in the orientation
appropriate for expression of UL13 were characterized by D N A
sequencing. One clone was selected for use and designated
pTZUL13(Kpn).
(ii) A 2.2 kbp XhoI partial digestion fragment, which contained only
UL13, was isolated from the KpnI f plasmid and ligated into the Sail
site of pTZ19U (Pharmacia). One clone, containing the insert in the
appropriate orientation for expression of UL13, was characterized by
DNA sequencing and designated pTZUL13(Xho).
The initiation codon for UL13 in pTZUL13(Kpn) and
pTZUL13(Xho) is situated 127 bp and 56 bp downstream, respectively,
from the initial KpnI and Sail cloning sites. In addition,
pTZUL13(Xho) contains an in-frame initiation codon in the multiple
cloning site, 69 bp upstream from that predicted to be the normal
translation initiation site for UL13.
pTZUL13(Kpn) was linearized with HindlII or NcoI prior to
transcription in vitro. HindlII cleaves in codons 155 to 156 and 172 to
173 of the UL13 coding sequence, and NcoI cuts downstream from
UL13. pTZUL13(Xho) was linearized with XhoI or EcoRI. XhoI
cleaves in codons 148 to 149 of the UL13 coding region, and EcoRI cuts
downstream from UL13.
In vitro transcription reactions were carried out for 2 h at 37 °C in
100 gl volumes containing 2 ~tg linearized plasmid, 40 mM-Tris-HC1
pH 7-5, 6 mM-MgC12, 2 mM-spermidine, 10 mM-NaCI, 2.5 mM of each
NTP (Promega), 100 units (U) RNasin (Boehringer Mannheim), 2 gCi
[3H]UTP (Amersham; TRK412) and 400 U T7 RNA polymerase
(Bethesda Research Laboratories). The yield of RNA was estimated by
monitoring radioisotopic incorporation. Synthesized R N A was extracted using 1:1 (v/v) phenol:chloroform, ethanol-precipitated,
dissolved in water at 2 mg/ml and stored at - 20 °C.
In vitro translation reactions were carried out using a New England
Nuclear reticulocyte lysate kit according to the manufacturer's
instructions. Synthesized proteins were treated with 50 pg/ml RNase A
and subjected to SDS-PAGE.
Preparation of polyclonal antisera against the UL13 protein. Amino
acid residues 149 to 207 of the UL 13 protein were expressed as a fusion
protein at the carboxy terminus ofEscherichia eoli/~-galactosidase. This
region contains PK motifs I and II (Smith & Smith, 1989). A 1.7 kbp
XhoI fragment from KpnI f containing the carboxy-terminal portion
(codons 149 to 518) of UL 13 was inserted in the appropriate orientation
into the Sail site of the fusion protein expression vector pUR288
(Riither & M/iller-Hill, 1983). It was found that the fusion protein
specified by this plasmid was degraded rapidly. An alternative
construction was made by digesting the plasmid with XbaI and SstII,
thus removing codons 208 to 518, treating with T4 DNA polymerase in
the presence of the four dNTPs and ligating. A clone containing the
appropriate plasmid was selected. The fusion protein generated after
induction with 1 mM-IPTG was insoluble, and therefore could not be
purified by affinity chromatography. Instead, extracts of induced
bacteria were prepared by lysozyme treatment and sonication, and
insoluble material containing the fusion protein was pelleted by
centrifugation. Proteins were separated by SDS-PAGE, and the fusion
protein was visualized by treatment of the gel with 1 M-KC1, excised
and electroeluted in 25 mM-Tris base, 192 mM-glycine, 0"1% (w/v) SDS.
The extracted fusion protein was lyophilized, washed with acetone,
resuspended in PBS and stored at - 7 0 °C.
Two sandylop rabbits were immunized with purified fusion protein
by Serotec Ltd., Oxford, U.K. as follows. Initially, preimmune sera
were obtained and each rabbit was inoculated subcutaneously with
40 gg protein in Freund's complete adjuvant. On day 28, sera were
prepared and each rabbit was boosted with 120 gg protein in Freund's
incomplete adjuvant. Further sera were obtained on days 35 and 49,
and the rabbits were boosted with 40 gg protein each on days 43 and 63.
Final sera were prepared on day 70.
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HSV-1 UL13 protein
Immune precipitation reactions. Immune precipitation reactions were
carried out essentially as described by Zweig et al. (1980). In some
experiments, antisera were preincubated with exogenous proteins for
2 h at 4 °C. Precipitated proteins were subjected to S D S - P A G E .
Gel electrophoresis o f proteins. S D S - P A G E was conducted using 10 %
or 12 ~ (w/v) polyacrylamide gels cross-linked with 1:37.5 (w/w) N,N'-
tetramethylene-bisacrylamide or 1:57 (w/w) N,N'-diallyltartardiamide. Gels from immune precipitation experiments were fixed in 10%
(v/v) acetic acid, 50% (v/v) methanol, treated with En3Hance (New
England Nuclear), dried and autoradiographed using Kodak XS-6 film
with a Dupont Cronex Lightning Plus intensifying screen at - 70 °C.
Mr standards were included in all gels.
80
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Results
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Time post-infection (h)
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Induction of a nuclear PK by HSV-1
Endogenous protein phosphorylation was studied in
nuclear extracts of mock- or HSV-l-infected cells.
Nuclear PK activity was measured at various times after
infection by monitoring incorporation of 32p from
[7-32p]ATP into TCA-precipitable material (Fig. l a).
The level of activity in mock-infected cells was indistinguishable from the background, but enzyme activity in
HSV-l-infected cells increased until about 5 h after
infection and then remained approximately constant.
SDS-PAGE of the samples showed that nuclear extracts
of HSV-l-infected cells were able to phosphorylate a
major endogenous protein species with an apparent Mr
of 57 000 (Vmw57; Fig. 1b, lanes 5 to 8). (The estimated
Mr depended on the cross-linker used in polyacrylamide
gels; when N,N'-diallyltartardiamide was used instead of
N,N'-methylene-bisacrylamide it was 51000.) In contrast, very little PK activity was detected in nuclear
extracts prepared from mock-infected cells (Fig. lb, lanes
1 to 4). When cells were infected with HSV-1 and
labelled with 32p in vivo, a minor phosphoprotein with
the same Mr as Vmw57 was detected in nuclear extracts
(Fig. lc).
Biochemical properties of the nuclear PK
The PK which phosphorylates Vmw57 was characterized with respect to salt dependence, substrate specificity, phosphate donor and inhibition by heparin.
Phosphorylation was detected in the presence of NaC1 at
concentrations ranging from 0.1 to 2.5 M, but was optimal
at 1.5 M (Fig. 2a, lanes 8 to 14). A concentration of 1.5 M
was used in subsequent assays. A cellular PK able to
phosphorylate exogenous casein (Mr 23600) in the
presence of 1.5 M-NaC1 was also detected (Fig. 2b, lane
3). This activity was rather variable in different
experiments, and was optimal at 0.2 M-NaC1 (data not
shown). The substantial enhancement of casein kinase
activity in infected cells indicates that the virus-induced
(b)
1 2 3 4
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(c)
2 3 4
155K122K-
~ ; ~ ~-~--Vmw57
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Fig. 1. Induction of a nuclear P K by HSV-1. (a) Time course o f
induction of P K activity in a nuclear extract of HSV-l-infected cells.
P K activity in the presence of 1 M-NaCI was monitored by TCA
precipitation. The horizontal dashed line indicates the level of activity
in mock-infected cells. (b) Autoradiograph of samples monitored in (a)
subjected to SDS-PAGE. Lanes 1 to 4 and 5 to 8 show samples taken at
1, 2, 3 and 12 h after mock or HSV-l-infection, respectively. Mrs of
marker proteins in HSV-l-infected cells are shown to the left. (c)
Autoradiograph comparing Vmw57 labelled in vitro with phosphoproteins labelled in vivo. Lanes 1 and 2 show nuclear extracts of mock- and
HSV-l-infected cells labelled in vitro. Lanes 3 and 4 show nuclear
extracts of mock- and HSV-l-infected ceils, respectively, which had
been labelled in vivo with [32P]orthophosphate" MrS of marker proteins
are shown to the left.
PK is able to phosphorylate casein (Fig. 2b, lane 4).
Phosphorylation of Vmw57 was also stimulated moderately by casein and markedly by protamine (Fig. 2b, lanes
4 and 6). Protamine, which has an Me of less than 10000,
was shown not to be phosphorylated using polyacrylamide gels (data not shown) of higher concentration. The
HSV-l-induced PK was able to use ATP or GTP as a
phosphate donor (Fig. 2b, lanes 2 and 8), and was
insensitive to inhibition by heparin at concentrations up
to 25 p.g/ml (Fig. 2c).
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C. Cunningham and others
306
(a) 1
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(b) 1
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Fig. 2. Biochemical properties of the nuclear PK. (a) Effect of NaC1. Nuclear extracts of mock- (lanes 1 to 7) or HSV-l-infected (lanes 8
to 14) cells were labelled in vitro in the presence of 0.1, 0.2, 0-5, 1.0, 1-5, 2-0 or 2.5 M-NaC1, respectively. Samples were subjected to S D S P A G E and the gel was autoradiographed. (b) Effect of exogenous substrates. Nuclear extracts of mock- (lanes 1, 3, 5 and 7) or HSV-1infected (lanes 2, 4, 6 and 8) cells were labelled in vitro in the presence of[?-32p]ATP (lanes 1 and 2), [V-32p]ATP plus 1 ~tg casein (lanes 3
and 4), [?-32p]ATP plus 1 ktg protamine (lanes 5 and 6) or [),-32p]GTP (lanes 7 and 8). All reactions included 1.5 M-NaCI. Samples were
subjected to S D S - P A G E and the gel was autoradiographed. The position of casein is indicated by a vertical line. (c) Effect of heparin.
P K activity in the presence of 1-5 M-NaC1 was monitored by T C A precipitation.
Fractionation of the nuclear PK on DNA-cellulose
The nuclear location of the PK suggested a possible
interaction with DNA, so nuclear extracts of HSV-1infected cells were fractionated further by chromatography on DNA-cellulose. Fractions were eluted with a
0-1 to 1.0 M-NaCI gradient and assayed for PK activity in
the presence of 1.5 M-NaC1. The fraction that eluted at
approximately 0.5 M-NaC1 gave maximal incorporation
of 3zp into TCA-precipitable material (Fig. 3a). The
activity in this fraction was assayed at NaC1 concentrations ranging from 0-3 to 2.0 M in the presence of
exogenous casein, and phosphorylated proteins were
examined by SDS-PAGE (Fig. 3b). The eluted PK
promoted phosphorylation of Vmw57 and casein to a
similar degree at each NaCI concentration. The optimal
NaC1 concentration was similar to that observed
previously in unfractionated nuclear extracts. Coomassie
blue staining indicated that the elution profile of total
Vmw57 was identical to that of Vmw57 which could be
phosphorylated, and that several proteins were present
in the peak fraction (data not shown). Despite the last
point, the results imply that Vmw57 and the virusinduced PK co-elute from DNA-cellulose. No activity
capable of phosphorylating Vmw57 or casein was eluted
when nuclear extracts from mock-infected cells were
subjected to DNA-cellulose chromatography (data not
shown).
Viral origin of Vmw57
Nuclear extracts were prepared from HSV-2-infected
cells and assayed for activity in the presence of 1.5 M-
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Fig. 3. Fractionation of the nuclear P K on DNA-cellulose. (a)
Fractions were eluted from DNA-ceUulose using a 0.1 to 1.0 M-NaCI
gradient, and P K activity in the presence of 1.5 M-NaC1 was monitored
by T C A precipitation. (b) Autoradiograph showing proteins phosphorylated in vitro by incubation of the peak fraction from (a) in the
presence of 5 p.g casein and 0.3, 1.0, 1.5 or 2.0 M-NaCI (lanes 1 to 4,
respectively).
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HSV-1 UL13 protein
(a)
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Characterization of a rabbit antiserum against the HSV-1
UL13 protein
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the region of the HSV-1 D N A molecule encoding
Vmw57. The genome structures of the recombinants are
shown in Fig. 4 (a). Nuclear extracts of cells infected with
each recombinant were prepared, and the Mrs of major
phosphorylated species were compared with those of
cells infected with HSV-1 or HSV-2 (Fig. 4b). This
analysis revealed that Vmw57 maps to a region between
the HSV-1 BamHI c-a (defined in R12-4) and BgllI o-p
sites (defined in RE6), which contains genes UL9 to
U L I 5 (McGeoch et al., 1988).
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Fig. 4. Viral origin of Vmw57. (a) Genome structures of intertypic
recombinants between HSV-1 and HSV-2that were used to locate the
region of the genome encoding Vmw57. The HSV genome structure is
shown at the top, with horizontal lines and open rectangles denoting
unique and inverted repeat regions, respectively. Recombinant
structures are shown as solid lines superimposed on dashed lines
indicating HSV-I (upper) and HSV-2 (lower) sequences. Crossover
limits are shown as solid vertical lines. The scale shows the fractional
genome length. (b) Autoradiograph showing electrophoretic mobilities
of HSV-1 Vmw57 (lanes 1, 4, 7, 10 and 13), its counterpart in HSV-2
(lanes 3, 6, 9, 12 and 15) and the corresponding proteins specified by
intertypic recombinants Bx6 (17-1) (lane 2), Bxl (24) (lane 5), RE6 (lane
8), Bx5 (7-2) (lane 11) and R12-4 (lane 14). Nuclear extracts of infected
cells were phosphorylated in vitro in the presence of 1-5 M-NaC1and
subjected to SDS-PAGE. Only those portions of autoradiographs
representing the major phosphoprotein (Vmw57) are shown.
NaC1. The observation that the major phosphorylated
species had a slightly greater Mr than HSV-1 Vmw57
(Fig. 4b, compare lanes 1 and 3) indicates that the
protein is virus-encoded. A series of HSV-1/HSV-2
intertypic recombinants was analysed in order to identify
O f the primary translation products of genes UL9 to
UL15, that of UL13 is nearest in Mr to Vmw57. To
ascertain whether UL13 encodes Vmw57, antisera were
raised in two rabbits which had been inoculated with a
fusion protein of/~-galactosidase linked to amino acid
residues 149 to 207 of the predicted UL13 protein. As a
substrate for testing the antisera, UL13 was transcribed
and translated in vitro. When p T Z U L 1 3 ( K p n ) was cut
downstream from UL13 using NcoI, a UL13-specific
protein with an apparent Mr of 57 000 was produced (Fig.
5 a, lane 1). When pTZUL13(Xho) was cut downstream
from UL13 using EcoRI, two UL13-specific proteins
were detected. One corresponded in size to that produced
by pTZUL13(Kpn) and comigrated with Vmw57 (Fig.
5 a, lane 2; compare Fig. 5 c, lanes 1 and 2). The other had
a greater Mr and was presumably the result of translation
initiation at the upstream in-frame A T G codon present
in the multiple cloning site. When pTZUL13(Xho) was
cut within UL13 using XhoI, neither UL13-specific
protein was produced; instead, truncated proteins with
MrS of approximately 18 000 were noted (Fig. 5 a, lane 3).
Antiserum obtained from one rabbit 49 days after the
initial inoculation precipitated the UL13-specific proteins, whereas preimmune serum did not (Fig. 5b, lanes 1
to 3, 5 and 7; Fig. 5 c, lanes 3 and 4); preincubation with
purified UL13 fusion protein inhibited antibody reactivity (Fig. 5c, lane 5). These results indicate that one
rabbit produced antibodies which reacted specifically
with the UL13 protein. Antibodies against the UL13
protein were not detected in sera from the other rabbit. It
should be noted that the heavy chains of I g G migrated
just above the UL13 proteins and distorted this region in
the gels, causing the two ULl3-specific proteins to
migrate as a single band at a greater rate than in samples
not subjected to immune precipitation (compare Fig. 5 b,
lanes 2 and 6).
Identification o f the UL13 protein as Vmw57
Nuclear extracts of HSV-l-infected cells were phosphorylated in vitro and immune-precipitated using
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308
C. C u n n i n g h a m a n d o t h e r s
(a)
1
2
3
4
(b)
1
2
3
4
5
~---UL13
6
7
8
(c)
9
1
2
3
4
5
Vmw57---~
-UL13
"~---UL13
Fig. 5. Characterization of a rabbit antiserum against the HSV-I ULI3 protein. (a) Autoradiograph showing in vitro translation
products of pTZUL13(Kpn) cut with NcoI (lane 1), pTZUL13(Xho)cut with EcoRI (lane 2) or Xhol (lane 3), and endogenous mRNAs
in the translation kit (lane 4). (b) Autoradiograph showing the resultsof immune precipitation of in vitro translation productsof ULI 3. In
vitro translation products ofpTZUL 13(Kpn) cut with NcoI, pTZUL13(Xho)cut with EcoRI and endogenousmRNAs in the translation
kit are shown in lanes 4, 6 and 8, respectively.Proteins precipitated from these samplesby preimmune serum are shown in lanes 5, 7 and
9, respectively,and those precipitated by 49 day immune serum are shown in lanes 1, 2 and 3, respectively.(c) Autoradiograph showing
inhibition of immune precipitation of in vitro translation products of UL13. Lane 1 shows phosphoproteins resulting from in vitro
labelling of a nuclear extract of HSV-l-infected cells. Lane 2 showsin vitro translation products of pTZUL13(Xho)cut with EcoRI, and
lanes 3 to 5 show proteins precipitated from this sample using preimmune serum, 49 day immune serum and 49 day serum which had
been preincubated with 2 ~tg UL13 fusion protein, respectively.
reactive antisera. Results of the more important of a
range of experiments are described below. It should be
noted that the pattern of labelling varied between
different nuclear extracts. In some, Vmw57 was the
major phosphorylated protein detected, but in others a
major phosphoprotein with an Mr of 38000 was also
detected (e.g. Fig. 5c, lane 1). In addition, minor
phosphoproteins were visualized after over-exposure of
autoradiographs (Fig. 6a, lane 1). Similar attempts to
detect Vmw57 in [35S]methionine-labelled cells were not
successful (data not shown).
When sera obtained 28, 35, 49 and 70 days after the
initial immunization were assayed for their ability to
react with 32p_labelled Vmw57, antibodies were detected
in the earliest sample (data not shown). Preincubation of
antiserum obtained at 70 days with 2 ~tg or more of
purified UL13 fusion protein inhibited immune precipitation of 32p-labelled Vmw57 (Fig. 6a, lane 5; b, lanes 2
to 6). Preincubation with PBS (Fig. 6a, lane 4), pure flgalactosidase (Fig. 6a, lane 6) or a fusion protein of flgalactosidase linked to a portion of UL15 (Fig. 6a,
lane 7) or UL17 protein (Fig. 6b, lane 7) failed to inhibit
binding to Vmw57. The results of these experiments
show that an antiserum raised against the UL13 protein
reacted specifically with Vmw57, and demonstrate
unequivocally that UL13 encodes Vmw57.
I d e n t i f i c a t i o n o f V m w 5 7 as a virion p r o t e i n
S D S - P A G E of an extract of purified virions which had
been phosphorylated in vitro revealed a major phosphoprotein which comigrated with Vmw57 (Fig. 7, lanes 1
and 10). Like Vmw57, this protein was immuneprecipitated by the UL13 antiserum (Fig. 7, lanes 3 and
7), and binding was inhibited by preincubation of the
antiserum with the UL13 fusion protein but not with the
UL17 fusion protein (Fig. 7, lanes 4, 5, 8 and 9). These
results show that Vmw57 is a structural component of the
virion, and strongly suggest that a P K responsible for its
phosphorylation is also present in virus particles.
Discussion
HSV-1 induces a P K activity in the nuclei of infected
cells. The activity is similar to the cytoplasmic P K
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H S V - 1 U L 1 3 protein
(a)
1
2
3
4
5
6
309
7
1
2
3
4
5
6
7
8
9
10
~---Vmw57
nw57
(b)
1
2
3
4
5
6
Fig. 7. Identification of Vmw57 as a virion protein. Autoradiograph
showing proteins immune-precipitated from a nuclear extract of HSV1-infected cells (lanes 2 to 5) or a virion extract (lanes 6 to 9) which had
been phosphorylated in vitro. Lanes 1 and 10 show the labelled proteins
in the nuclear and virion extracts, respectively. Remaining lanes show
proteins precipitated by the preimmune serum (lanes 2 and 6), the 70
day immune serum (lane 3 and 7), and the 70 day immune serum which
had been preincubated with 4 lag UL13 (lanes 4 and 8) or UL17 fusion
protein (lanes 5 and 9).
7
q
Vmw57
Fig. 6. Identification of the UL13 protein as Vmw57. (a) Autoradiograph showing proteins immune-precipitated by the 70 day immune
serum from a nuclear extract of HSV-l-infected cells which had been
phosphorylated in vitro. Lane 1 shows the labelled nuclear extract;
lanes 2 and 3 show proteins precipitated after preincubation of
preimmune serum with 2 lag UL13 fusion protein or PBS, respectively;
lanes 4 to 7 show proteins precipitated after preincubation of the 70
day immune serum with PBS, 6 ~tg UL13 fusion protein, 6 p.g pure flgalactosidase or 6 p.g UL15 fusion protein, respectively. (b) Autoradiograph showing the effect of preincubating 70 day immune serum with
different amounts of the UL13 fusion protein on immune precipitation
of proteins from a nuclear extract of HSV-l-infected cells which had
been phosphorylated in vitro. Lane 1 shows the labelled nuclear extract;
lanes 2 to 6 show proteins precipitated after preincubation with 0.25,
0.5, 1, 2 or 4 lag UL13 fusion protein; lane 7 shows proteins precipitated
after preincubation with 4 lag UL17 fusion protein.
e n c o d e d b y HSV-1 gene US3 c h a r a c t e r i z e d p r e v i o u s l y in
t h a t it has the unusual p r o p e r t y o f b e i n g s t i m u l a t e d in
vitro by high salt c o n c e n t r a t i o n s . H o w e v e r , the two
e n z y m e s differ in subcellular location, a n d in the a b i l i t y
o f the n u c l e a r P K to utilize G T P as a p h o s p h a t e d o n o r
a n d to p h o s p h o r y l a t e exogenous c a s e i n b u t not prota m i n e . T h e n u c l e a r P K has p r o p e r t i e s s o m e w h a t s i m i l a r
to those d e s c r i b e d for a P K isolated f r o m liver nuclei
( B a y d o u n et al., 1986; D e l p e c h et al., 1986), e x c e p t t h a t it
is significantly less sensitive to i n h i b i t i o n b y h e p a r i n
( B a y d o u n et al., 1986). Thus, the H S V - l - i n d u c e d n u c l e a r
P K c a n n o t be identified w i t h e i t h e r the US3 c y t o p l a s m i c
P K or cell n u c l e a r P K s c h a r a c t e r i z e d previously. It m a y
c o r r e s p o n d to the v i r i o n k i n a s e d e s c r i b e d by L e m a s t e r &
R o i z m a n (1980), b u t c o m p a r a t i v e studies to confirm this
h a v e n o t b e e n c a r r i e d out.
V m w 5 7 is a m a j o r s u b s t r a t e o f the n u c l e a r P K in vitro.
T h i s p r o t e i n was shown b y analysis o f i n t e r t y p i c
r e c o m b i n a n t s to be e n c o d e d b y one o f HSV-1 genes U L 9
to U L 1 5 , a n d a d d i t i o n a l e x p e r i m e n t s using a specific
a n t i s e r u m s h o w e d it to be e n c o d e d b y the U L 1 3 gene.
M o r e o v e r , V m w 5 7 was s h o w n to be p r e s e n t in virions. It
is r a t h e r m o r e difficult to d e t e r m i n e w h e t h e r the n u c l e a r
P K is viral or cellular in origin, since several P K
activities t h a t are p r o b a b l y cellular in o r i g i n h a v e b e e n
d e t e c t e d in HSV-1 virions (Stevely et al., 1985). O u r
analyses were c a r r i e d out using p a r t i a l l y purified
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310
C. Cunningham and others
e n z y m e , a n d we have by n o m e a n s ruled out the
possibility that the activity p e r t a i n s to a cellular e n z y m e
i n d u c e d by HSV-1 i n f e c t i o n rather t h a n to a virus gene
product. T h e r e are, however, three lines of c i r c u m s t a n tial e v i d e n c e s u p p o r t i n g the hypothesis t h a t V m w 5 7 is
the P K a n d that the observed labelling of V m w 5 7 in vitro
represents a u t o p h o s p h o r y l a t i o n . First, V m w 5 7 a n d the
P K f r a c t i o n a t e d together d u r i n g D N A - c e l l u l o s e chrom a t o g r a p h y . Second, virions a p p e a r to c o n t a i n V m w 5 7
a n d the P K because v i r i o n extracts were able to
phosphorylate e n d o g e n o u s Vmw57. T h i r d , the U L 1 3
p r o t e i n has b e e n predicted to f u n c t i o n as a s e r i n e t h r e o n i n e p r o t e i n kinase o n the basis of its p r i m a r y
a m i n o acid sequence (Smith & Smith, 1989; Chee et al.,
1989).
P r o o f that Vmw57 is the P K will i n v o l v e further
extensive studies. O n e a p p r o a c h is to u n d e r t a k e purification to h o m o g e n e i t y of the activity from HSV-1-infected
cells or a heterologous expression system. I n p r e l i m i n a r y
e x p e r i m e n t s we have b e e n unsuccessful in r e t a i n i n g the
P K activity from H S V - l - i n f e c t e d cells in purification
steps b e y o n d DNA--cellulose c h r o m a t o g r a p h y . Also, we
have b e e n u n a b l e to isolate a r e c o m b i n a n t v a c c i n i a virus
expressing the UL13 p r o t e i n by t r a n s c r i p t i o n from a
powerful late promoter. W e i n t e r p r e t this as i n d i c a t i n g
that high levels of V m w 5 7 are lethal to growth of
v a c c i n i a virus. A n a l t e r n a t i v e a p p r o a c h to the p r o b l e m
w h i c h has m e t with initial success involves isolating a n
HSV-1 m u t a n t with a lesion in UL13 (L. Coulter, H.
Moss & D. M c G e o c h , u n p u b l i s h e d data). Such a m u t a n t
is currently b e i n g used to characterize the P K .
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