Herpes Simplex Virus Type 1 Infection Stimulates p38/c-Jun
N-terminal Mitogen-activated Protein Kinase Pathways and Activates
Transcription Factor AP-1*
George
Zachos
§¶,
Barklie
Clements§, and
Joe
Conner
¶
From the
School of Biological and Biomedical
Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow,
G4 0BA and the § Institute of Virology, Institute of
Biomedical and Life Sciences, University of Glasgow, Church Street,
Glasgow G11 5JR, United Kingdom
 |
ABSTRACT |
Cells respond to environmental stress and
proinflammatory cytokines by stimulating the Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK) and the p38
mitogen-activated protein kinase cascades. Infection of eukaryotic
cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation
of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h
post-infection. By using a series of mutant viruses, we showed that the
virion transactivator protein VP16 stimulates p38/JNK, whereas no
immediate-early, early, or late viral expressed gene is involved. We
identified the stress-activated protein kinase kinase 1 as an upstream
activator of p38/JNK, and we demonstrated that activation of AP-1
binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in
the cellular levels of Jun protein. We suggest that activation of the
stress pathways by HSV-1 infection either represents a cascade
triggered by the virus to facilitate the lytic cycle or a defense
mechanism of the host cell against virus invasion.
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INTRODUCTION |
Eukaryotic cells respond to extracellular stimuli by recruiting
signal transduction pathways, many of which are mediated through activation of distinct mitogen-activated protein
(MAP)1 kinase cascades. The
best characterized of these are the extracellular signal regulated
kinase (ERK), the c-Jun N-terminal kinase/stress-activated protein
kinase (JNK/SAPK), and the p38 MAP kinase pathways (1, 2). Most
proliferative stimuli activate the ERK pathway, primarily through the
small GTP-binding protein Ras. Active Ras recruits to the plasma
membrane and activates the MAP kinase kinase kinase Raf-1. Active Raf-1
phosphorylates the MAP/ERK kinases (MEKs) 1 and 2, which in turn
stimulate the respective ERKs (3, 4). In a similar fashion, UV
irradiation, environmental stress, and proinflammatory cytokines
stimulate the JNK/SAPK and the p38 MAP kinase (p38 MAPK) cascades.
These kinases have been shown to phosphorylate a number of
transcription factors (5-7), including c-Jun (specifically phosphorylated by JNK/SAPK) and ATF-2 (phosphorylated by both JNK/SAPK
and p38 MAPK). Although there is coordinate regulation of JNK and p38,
they have distinct upstream activators as follows: p38 is activated by
MAP kinase kinases (MKKs) 3 and 6, whereas MKK 4 (SKK 1) and MKK 7 (SKK
4) activate both JNK and p38 (8, 9).
Two of the major classes of control elements that contribute to
transcriptional regulation of cellular genes by extracellular signals,
are the activator protein 1 (AP-1)-binding site, also known as the
phorbol 12-O-tetradecanoate-13-acetate response element (AP-1/TRE), and the activating transcription factor (ATF)-binding site,
also known as the cAMP response element (ATF/CRE) (10, 11). The
AP-1/TRE is recognized by a group of proteins encoded by the
c-jun and c-fos gene families. TRE-binding
proteins form homodimers or heterodimers (12, 13), which are induced by mitogenic stimuli, stress- and virus-induced alterations (14, 15).
Dimerization partners are affected by signal-regulated protein kinases
and alterations in the composition of the AP-1 complex result in
differential binding, growth, and oncogenic potential of AP-1 (12, 13,
16, 17). The ATF/CRE site is recognized by the ATF/CRE-binding (CREB)
proteins and is implicated in cAMP-, calcium-, and virus-induced
alterations (11). TRE- and CRE-binding proteins preferentially
recognize the corresponding sequences; however, cross-family
dimerization has been reported (18).
Herpes viruses are ubiquitous eukaryotic pathogens that
possess a common basic structure consisting of an icosahedral
nucleocapsid containing a linear, double-stranded DNA genome,
surrounded by a proteinaceous tegument and a membranous envelope.
Herpes simplex virus type 1 (HSV-1) is the best characterized member of
the family, and during the lytic cycle, viral gene expression can be
divided into three temporal stages, based upon the appearance of the
gene products (for review see Ref. 19). Immediate-early (IE) genes are
transcribed in the absence of de novo protein synthesis, and their products act to orchestrate the expression of the early and late
genes. Transcription of the five viral IE genes is initiated by the
virion tegument protein VP16 (Vmw65) through formation of a
multiprotein-DNA complex on viral promoters that includes the
pre-existing cellular proteins Oct-1 and host cell factor (HCF) (Ref.
20 and for review see Ref. 21).
Four of the five IE gene products (Vmw175, Vmw63, Vmw110, and Vmw68)
are phosphorylated nucleoproteins with regulatory activities that
prime the cell for efficient HSV-1 infection and control the expression
of viral early and late genes. Proteins Vmw175 and Vmw63 are absolutely
essential for lytic virus replication, whereas Vmw110 is not essential
but it is important for the efficient entry into the lytic phase of
infection and interacts with a number of cellular components (22-25).
Vmw68 plays roles in the efficient late gene expression and
phosphorylation of the cellular RNA polymerase II (26, 27). The fifth
IE gene product (Vmw12) is a cytoplasmic protein postulated to assist
virus in avoiding immune detection (28). An additional HSV gene,
UL39, that encodes the large subunit (R1) of the viral
ribonucleotide reductase is expressed during IE times, but its function
at this stage of infection is not yet understood (29, 30). Expression
of early gene products occurs approximately 4-5 h postinfection, and
they are mostly virally encoded enzymes involved in DNA synthesis and
replication. Efficient expression of late genes that encode mostly
structural proteins commences approximately 6-7 h postinfection, and
their full expression requires viral DNA synthesis.
Recently, much attention has focused on the potential use of HSV-1 as a
gene delivery vector for the nervous system and other tissues (31).
Production of effective vectors requires a full understanding of the
biological mechanisms activated by viral infection and a detailed
analysis of the host cell response during the immediate-early and early
phases of viral invasion. Our study shows stimulation of the JNK/SAPK
and p38 MAPK stress pathways by viral protein VP16 during virus
immediate-early and early times, with a concomitant rise in AP-1
binding activity.
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EXPERIMENTAL PROCEDURES |
Cells and Viruses--
Baby hamster kidney (BHK) cells were
grown in BHK medium (Life Technologies, Inc.) supplemented with 10%
newborn calf serum. Chinese hamster ovary and HeLa cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum. Cells were grown as
monolayers, at 37 °C, in an atmosphere of 5% CO2.
Viruses dl403 (22) and D30EBA (32) that fail to express Vmw110 and
Vmw175, respectively, were kindly provided by Dr. R. Everett, Institute
of Virology, University of Glasgow, Glasgow, UK. Virus ICP6delta (33),
which fails to express the R1 subunit of the viral ribonucleotide
reductase, was a gift from Professor S. Weller, University of
Connecticut Health Center, Farmington, CT. The HSV-1
temperature-sensitive mutant tsk expresses an inactive form of Vmw175
at the non-permissive temperature of 38.5 °C (34). In the 27lacZ
virus, the gene encoding Vmw63 is inactivated by insertion of a
lacZ cassette (35). Virus in1814 expresses an inactive form
of virion protein VP16 which is defective in interacting with cellular
factors Oct-1 and HCF and was a gift from Dr C.M. Preston, Institute of
Virology, University of Glasgow, Glasgow, UK (36).
Proteins, Antibodies, and Plasmids--
Escherichia
coli for expression of a GST/Jun-(1-79) fusion protein and a
JNK/SAPK immunoprecipitating antiserum were provided by Dr. D. Gillespie, CRC Beatson Institute for Cancer Research, Glasgow, UK.
E. coli for expression of a GST/ATF-2-(19-96) fusion protein, purified GST/ATF-2 (19-96), purified recombinant
6-his-JNK/SAPK, and antibodies for the immunoprecipitation of SKK 1 and
SKK 4 (9) were obtained from Dr. S. Lawler, MRC Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, UK.
Antibodies for the detection of Vmw110 and Vmw175 in Western blotting
were obtained from Dr. R. Everett (37, 38). Antibodies specific for R1
and Vmw63 proteins were as described (39, 40). Polyclonal antibodies
against Jun, JunB, JunD, Fos, and ATF-2 were obtained from Santa Cruz
Biotechnology. A nonradioactive kit for detection of p38 MAPK activity
was used as directed by the manufacturer (New England Biolabs, Hitchin,
UK). Plasmids pMCI, expressing full-length VP16 and pMCI del, encoding
a truncated form lacking the transactivation domain, were kindly
provided by Dr. C. M. Preston.
Virus Infection and UV Irradiation of Cell
Monolayers--
Subconfluent cell monolayers were infected with either
wild-type HSV-1 (strain 17+) or mutant HSV-1 viruses at multiplicity of
infection of 10 pfu per cell, unless stated otherwise. Infected cells
were grown at 37 °C, 5% CO2, before harvesting. In
experiments with the tsk virus, infected cells were grown at 38.5 °C
(non-permissive temperature) or at 32 °C (permissive temperature)
before harvesting.
For UV irradiation of BHK cells, the medium was removed and the cell
monolayer was exposed to 80 J/m2 in a UV cross-linker. The
medium was replaced, and the cells were returned to the incubator for
30 min before harvesting.
Transfection of Cells--
BHK cells were grown in 30-mm dishes
and transiently transfected using LipofectAMINE (Life Technologies,
Inc.), as indicated by the manufacturer. Cells were harvested at
48 h post-transfection, and pull down kinase assays were peformed.
Preparation of Cell Extracts--
For immunocomplex and pull
down kinase assays, harvested cells from a 60-mm plate were incubated
on ice, for 30 min, in 250 µl of lysis buffer J (25 mM
HEPES, pH 8.0, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 20 mM
-glycerophosphate, 0.1 mM
NaVO3, 25 mM NaF, 1% Nonidet P-40). Cell
debris was removed by centrifugation at 13,000 × g.
For Western blotting, cells were lysed in buffer W (20 mM
HEPES, pH 7.6, 0.4 M KCl, 5 mM EDTA, 1 mM DTT, 1 mM NaVO3, 5 mM NaF, 10% glycerol, 0.4% Triton X-100, 1 µg/ml
okadaic acid) and treated as described above.
For DNA binding assays, nuclear extracts were prepared as described by
Dignam et al. (41).
Pull Down Kinase Assays--
Induction of GST fusion protein
expression in E. coli was achieved by treating cultures with
150 µg/ml isopropyl-1-thio-
-D-galactopyranoside for
3 h. Harvested bacteria were resuspended in NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM
EDTA, 0.1% Nonidet P-40) and lysed by sonication, and cell debris was
removed by centrifugation at 5000 × g. Bacterial
extracts were incubated at 25 °C for 30 min, with
glutathione-agarose (Sigma) previously swollen and washed extensively
in NETN buffer. The glutathione-agarose with bound GST fusion protein
was washed 4 times with 10 volumes of NETN buffer, and the gel was
resuspended in an equal volume of NETN buffer to give a 50% slurry.
Kinase activity in the cell extracts was analyzed by using the
glutathione-agarose with bound GST/Jun or GST/ATF-2 to pull down the
associating kinases and then determining incorporation of
32PO4 into the fusion proteins. Briefly, 60 µl of the 50% matrix slurry were added to 250 µl of cell extract
and incubated overnight, at 4 °C, with constant agitation. The
matrix was washed 4 times in wash buffer comprising 20 mM
HEPES, pH 7.6, 50 mM NaCl, 2.5 mM
MgCl2, 0.1 mM EDTA, 0.05% Triton X-100 and
resuspended in 30 µl of kinase assay buffer (20 mM HEPES,
pH 7.6, 20 mM MgCl2, 20 mM
-glycerophosphate, 0.1 mM NaVO3, 2 mM DTT, 0.1 µg/ml okadaic acid, 0.125 mM
[
-32P]ATP). The phosphorylation reaction was allowed
to proceed for 30 min, at 25 °C, and samples were electrophoresed by
SDS-polyacrylamide gel electrophoresis. 32PO4
incorporation was observed by autoradiography and quantified by
scintillation counting.
Immunocomplex Kinase Assays--
10 µl of a JNK/SAPK-specific
antiserum were added to 250 µl of cell extract, and the volume was
adjusted to 500 µl with buffer P (20 mM Tris, pH 8.0, 40 mM sodium pyrophosphate, 50 mM NaF, 0.1 mM NaVO3, 10 mM EDTA, 1% Triton
X-100, and 0.5% sodium deoxycholate). Samples were incubated overnight
at 4 °C, with constant agitation. 60 µl of a 50% slurry of
protein A-Sepharose (Sigma), previously swollen and extensively washed
in buffer P, were used to bind the immunocomplexed JNK/SAPK. 20 µl of
GST/Jun bound to glutathione-agarose were added to the matrix and
resuspended in 40 µl of kinase assay buffer, at 25 °C, for 30 min.
Samples were electrophoresed by SDS-polyacrylamide gel electrophoresis,
and 32PO4 incorporation was observed by
autoradiography and quantitated by scintillation counting.
Measurement of SKK 1 and SKK 4 Activity--
SKK 1 and SKK 4 activities were measured in cell extracts as described previously (9).
Briefly, SKK 1 or SKK 4 were immunoprecipitated with 10 µg of the
relevant antibody coupled to protein G-Sepharose 4B (Sigma), washed
extensively with 0.5 M NaCl, and incubated with 1 µl of
20 µM 6-his-SAPK/JNK and 2.5 µl of 40 mM
magnesium acetate and 0.4 mM unlabeled ATP for 30 min, at
25 °C. Activated JNK/SAPK was then assayed in 40 µl of a solution
containing 31.25 mM Tris, pH 7.4, 0.125 mM
EDTA, 1.25 mM NaVO3, 1 mM DTT, 12.5 mM magnesium acetate, 0.125 mM
[
-32P]ATP, and 0.25 mg/ml purified GST/ATF-2 as
substrate. Samples were incubated at 25 °C, for 30 min,
electrophoresed, and visualized by autoradiography.
Western Blotting--
Antibodies against Jun, JunB, JunD, Fos,
and ATF-2 were used at dilution of 1:300. Antibodies for R1 and Vmw63
were used at dilutions of 1:1000 and 1:100, respectively. Mouse
monoclonal antibodies for detection of Vmw110 and Vmw175 were used at
dilutions of 1:5000.
DNA Binding Assays--
The AP-1 probe, encompassing binding
sites for the AP-1 complex, and the CRE probe, encompassing a binding
site for the ATF/CREB transcription factor, were as described
previously (18). Radioactive end labeling was performed using
T4 polynucleotide kinase and [
-32P]ATP
(42).
DNA-binding reactions were performed in a final volume of 20 µl, in
binding buffer consisting of 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 5%
glycerol, and 50 mg/ml poly(dI-dC). The appropriate oligonucleotide
probe (0.5 ng, 50,000 cpm/ng) was incubated with 10-20 µg of nuclear
extract for 12 h, at 4 °C, and after electrophoresis on 4%
polyacrylamide gel, binding was visualized by autoradiography. For
electrophoretic mobility shift assays, 0.5 µg of antibody (Santa Cruz
Biotechnology) was added to the reaction subsequent to addition of
radiolabeled probes.
 |
RESULTS |
Infection with Wild-type HSV-1 Stimulates JNK/SAPK--
BHK cells
were infected with wild-type HSV-1 virus, and pull down kinase assays
using GST/Jun fusion protein as substrate were performed, at various
time points, to measure JNK/SAPK activity (Fig.
1A, lanes 7-12). Untreated
cells harvested at the corresponding time points were used as control
(Fig. 1A, lanes 1-6). Band quantification identified a
marginal increase in the phosphorylation of GST/Jun by 3 h
postinfection (compare lanes 7 and 8 to
lanes 1 and 2), a 2-fold increase, indicating
activation of the JNK/SAPK kinases, at 6 h post-infection (compare
lanes 9 and 10 to lanes 3 and
4), and activation reached a maximum of 4-fold by 9 h
(compare lanes 11 and 12 to lanes 5 and 6). No further increase in stimulation of JNK/SAPK
kinases was detected at 12, 16, and 24 h postinfection (data not
shown). A 4-fold increase in JNK/SAPK activity in control UV-irradiated
cells, compared with untreated cells, was also found (lanes
15 and 16, and 13 and 14,
respectively). Conditioned media transferred from infected to
uninfected cells failed to activate JNK/SAPK, ruling out any autocrine
mechanism (data not shown).

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Fig. 1.
Wild-type HSV-1 infection stimulates JNK/SAPK
and activation coincides with the production of viral IE proteins.
A, pull down kinase assay for JNK/SAPK activity. Lanes
1-6, untreated cells, harvested at the same time points as the
virus-infected cells. Lanes 7 and 8, cells
infected with wild-type HSV-1, harvested at 3 h postinfection;
lanes 9 and 10, infected cells, harvested at
6 h postinfection; lanes 11 and 12, infected
cells, harvested at 9 h postinfection. Lanes 13 and
14, untreated cells (un); lanes 15 and
16, UV-irradiated cells. B, immunocomplex kinase
assay for JNK/SAPK activity. Lane 1, untreated cells;
lane 2, UV-irradiated cells; lanes 3 and
4, cells infected with wild-type HSV-1, harvested at 9 h postinfection. C, Western blot for the kinetics of
production of viral IE proteins. Lane 1, untreated cells;
lane 2, cells infected with wild-type HSV-1, harvested at
3 h postinfection; lane 3, infected cells, harvested at
6 h postinfection; lane 4, infected cells, harvested at
9 h postinfection. Proteins detected are indicated by
arrows.
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Similar increase in JNK/SAPK activity was observed in Chinese hamster
ovary and in HeLa cells infected with HSV-1 (data not shown).
Immunocomplex kinase assays using GST/Jun as substrate confirmed
stimulation of JNK/SAPK (Fig. 1B). We observed a 3-4-fold stimulation of JNK/SAPK in cells infected with wild-type HSV-1, harvested at 9 h postinfection (lanes 3 and
4), and in control UV-treated cells (lane 2),
compared with untreated cells (lane 1).
Western blotting experiments for Vmw63, Vmw110, Vmw175, and R1
immediate-early expressed proteins were also performed (Fig. 1C). By using extracts from untreated cells (lane
1) and from cells infected with HSV-1, harvested at 3, 6, and
9 h postinfection (lanes 2-4, respectively), we
observed that the kinetics of the JNK/SAPK activation apparently
coincided with the accumulation of virus IE proteins between 3 and 9 hours postinfection, thus raising the question whether a viral IE gene
is implicated in this phenomenon.
Wild-type HSV-1 Infection Stimulates p38 MAPK--
The effects of
wild-type HSV-1 infection of BHK cells on p38 MAPK activity were
determined by immunocomplex assays, using ATF-2 as a substrate (Fig.
2A). Immunocomplexed p38 MAPK
activity was determined in samples harvested at 2, 4, 6, 8, and 10 h postinfection (Fig. 2A, lanes 2-6, respectively) and in
untreated cells (lane 1); Western blots were probed with an
antibody specific for the phosphorylated form of ATF-2 and demonstrated
an increase in phosphorylation by 6 h, with a maximum
activity detected at 10 h. ATF-2 phosphorylation by p38 MAPK was
also stimulated in UV-irradiated cells (lanes 7 and
8).

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Fig. 2.
Infection with wild-type HSV-1 stimulates p38
MAPK. A, immunocomplex assay for p38 MAPK activity.
Lane 1, untreated cells (un); lanes
2-6, cells infected with wild-type HSV-1, harvested at 2, 4, 6, 8, and 10 h postinfection, respectively; lanes 7 and
8, UV-irradiated cells. B, pull down kinase assay
for p38 MAPK and JNK/SAPK activity. Lanes 1 and
2, UV-irradiated cells; lanes 3 and 4, untreated cells; lanes 5 and 6, cells infected
with wild-type HSV-1, harvested at 3 h postinfection, lanes
7 and 8, infected cells, harvested at 6 h
postinfection; lanes 9 and 10, infected cells,
harvested at 9 h postinfection.
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Additionally, a GST/ATF-2 fusion protein was used as a substrate to
detect ATF-2 phosphorylation by both p38 MAPK and JNK/SAPK pathways
(Fig. 2B). Compared with untreated cells (lanes 3 and 4), a marginal increase in ATF-2 phosphorylation was
detected by 3 h postinfection (lanes 5 and
6), a 3-fold increase was observed by 6 h (lanes
7 and 8), with a further increase to 5-fold by 9 h
(lanes 9 and 10). A 5-fold activation was also
observed in control UV-irradiated cells (lanes 1 and
2). These results indicate that both p38 MAPK and JNK/SAPK
are stimulated by wild-type HSV-1 infection, and we considered the
possibility that a viral protein was implicated in this phenomenon.
Virion Protein VP16 Triggers Activation of JNK/SAPK during HSV
Infection--
We examined whether some component of the virion
tegument is responsible for activating JNK/SAPK. Virus in1814 has an
intact capsid structure and expresses an inactive form of virion
protein VP16. It possesses an intact transactivation domain but has a 12-base pair insertion in the VP16 coding sequences, disrupting the
domain required for interaction with Oct-1 and HCF. This virus expresses low levels of IE proteins after infection of cells at a
multiplicity of 10 pfu per cell; however, when used at a multiplicity of 100 pfu/cell, the virus replicates normally, and the kinetics and
levels of the IE genes expression correspond to those detected after infection with wild-type HSV-1 at a multiplicity of 10 pfu/cell (36).
GST/Jun pull down kinase assays were performed after cells were
infected with in1814 virus at a multiplicity of 100 pfu/cell (Fig.
3A). No activation of JNK/SAPK
was detected at 6 (lanes 3 and 4) and at 9 h
postinfection (lanes 5 and 6); activities were
similar to those of untreated cells (lanes 1 and
2). Similar results were obtained after infecting cells with
in1814 virus at 10 pfu/cell (data not shown). Importantly, during
infection with the in1814 revertant virus at 10 pfu/cell (Fig.
3B), activation of JNK/SAPK at 6 (lanes 3 and
4) and 9 h postinfection (lanes 5 and
6), compared with the untreated cells (lanes 1 and 2), was restored.

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Fig. 3.
Virion protein VP16 activates JNK/SAPK.
Pull down kinase assays for JNK/SAPK activity were performed. A,
lanes 1 and 2, untreated cells (un);
lanes 3 and 4, cells infected with virus in1814
(lacking functional VP16) harvested at 6 h postinfection;
lanes 5 and 6, infected cells, harvested at
9 h postinfection. B, lanes 1 and 2, untreated cells; lanes 3 and 4, cells infected
with in1814 revertant virus (wild-type) harvested at 6 h
postinfection; lanes 5 and 6, infected cells,
harvested at 9 h postinfection. C, lanes 1 and
2, untreated cells; lanes 3-8, cells transiently
transfected with different amounts of pMCI plasmid (expressing
wild-type VP-16) as indicated; lanes 9 and 10, UV-irradiated cells. D, lanes 1 and 2, untreated cells; lanes 3 and 4, cells transiently
transfected with 6 µg of pMCI del plasmid (expressing VP-16 which
lacks the transactivation domain).
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Expression of the VP16 protein in transient transfection assays in a
virus-free environment using different amounts of the pMCI plasmid
(Fig. 3C, lanes 3-8) was sufficient for activating JNK/SAPK, compared with cells transfected with the empty vector (lanes 1 and 2). Induction of JNK/SAPK by UV
irradiation is shown in lanes 9 and 10.
Furthermore, expression of a truncated VP16 protein lacking the
transactivation domain but still capable of interacting with the
cellular factors retained the ability to activate JNK (Fig.
3D, lanes 3 and 4, compared with
lanes 1 and 2). Thus, VP16 activates JNK/SAPK
through the domain required for interaction with cellular factors.
Viral IE, Early, or Late Genes Do Not Trigger Stimulation of
JNK/SAPK--
The primary function of VP16 is to activate
transcription of the IE genes. We therefore investigated the possible
contribution of IE genes in stimulating JNK. BHK cells were infected
with mutant viruses ICP6delta, 27lacZ, dI403, and E30DBA that fail to
express functional forms of R1, Vmw63, Vmw110, and Vmw175 proteins,
respectively. Pull down kinase assays in extracts of cells harvested
9 h postinfection were performed using GST/Jun as substrate. All
mutant viruses stimulated JNK/SAPK to approximately the same levels as
those obtained with extracts from cells infected with wild-type virus (data not shown). In addition, similar kinetics of JNK/SAPK stimulation were observed with the different mutant viruses and wild-type HSV-1
(data not shown). Overall, our results indicate that immediate-early proteins Vmw63, Vmw110, Vmw175, and R1 are not directly responsible for
the induction of JNK/SAPK activity.
The role of IE gene products in JNK/SAPK activation was further studied
in cells infected with the temperature-sensitive mutant tsk. The tsk
virus has a mutation in the gene encoding Vmw175 such that the protein
fails to transactivate early and late gene expression at the
non-permissive temperature of 38.5 °C and IE gene products
accumulate (34). At the permissive temperature of 32 °C the tsk
virus replicates as wild type. GST/Jun pull down kinase assays were
performed using cellular extracts after infection with tsk. Identical
patterns of JNK/SAPK activation were observed at both the permissive
and non-permissive temperatures (data not shown). Thus, an accumulation
of IE gene products at 38.5 °C did not augment the induction of
JNK/SAPK activity, suggesting that none of these proteins is the direct
cause of this phenomenon.
Furthermore, viruses failing to produce functional Vmw175 or Vmw63 also
fail to express efficiently early and late genes (34, 35, data not
shown). We can therefore exclude early and late viral proteins from
causing activation of JNK/SAPK.
The Upstream Activator of p38/JNK, SKK1, Is Stimulated during HSV-1
Infection--
The activities of SKK 1 and SKK 4, which are upstream
activators of p38/JNK, were assayed after infection with wild-type
HSV-1 virus (Fig. 4). Levels of GST/ATF-2
phosphorylation were stimulated by SKK 1 immunoprecipitated from
extracts prepared at 5 (Fig. 4A, lanes 1 and
2) and 8 h postinfection (lanes 3 and
4) and by exposure to UV light (lanes 7 and
8), compared with untreated cells (lanes 5 and
6). SKK 4 activity was increased in samples extracted after
UV irradiation (Fig. 4B, lanes 7 and
8); however, the levels of GST/ATF-2 phosphorylation were
similar in infected (lanes 1-4) compared with untreated
cells (lanes 5 and 6). These results implicate
SKK 1, but not SKK 4, in stimulating p38/JNK during HSV-1
infection.

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Fig. 4.
SKK 1 but not SKK 4 is stimulated during
HSV-1 infection. A, pull down kinase assay for SKK 1 activity. Lanes 1 and 2, cells infected with
wild-type HSV-1, harvested at 5 h postinfection; lanes
3 and 4, cells harvested at 8 h postinfection;
lanes 5 and 6, untreated cells (un);
lanes 7 and 8, UV irradiated cells. B,
pull down kinase assay for SKK 4 activity. Lanes 1 and
2, cells infected with wild-type HSV-1, harvested at 5 h postinfection; lanes 3 and 4, infected cells
harvested at 8 h postinfection; lanes 5 and
6, untreated cells; lanes 7 and 8, UV
irradiated cells.
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Elevated AP-1 Binding Proceeds Stimulation of the Stress Pathways
during HSV-1 Infection--
Members of the Jun/Fos and ATF/CREB
families of transcription factors are common targets for activation by
JNK/SAPK and p38 MAPK pathways. BHK cells infected with wild-type HSV-1
virus were examined for AP-1 and CREB binding activity, by means of gel
retardation assays, at various time points of postinfection (Fig.
5). Increased DNA binding by the AP-1
transcription factor to its consensus oligonucleotide was observed at 6 (lane 5) and 9 h (lane 6) postinfection and
reached a maximum by 11 h postinfection (lane 7),
compared with untreated cells (lane 3). No alterations in
the DNA binding activity of the ATF/CREB family of transcription
factors to the consensus CRE probe was observed (lanes
10-13). UV irradiation induced DNA binding of both AP-1 and CREB
factors (lanes 1 and 2 and 8 and
9, respectively).

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Fig. 5.
Wild-type HSV-1 infection induces AP-1 but
not CREB binding. Gel retardation assay for AP-1 and CREB binding
to the corresponding consensus oligonucleotides. Lanes 1-7,
AP-1 probe; lanes 8-13, CRE probe. Lanes 1 and
3 and 8 and 10, untreated cells
(un); lanes 2 and 9, UV-irradiated
cells; lanes 4 and 11, cells infected with
wild-type HSV-1, harvested at 3 h postinfection; lane
5, cells harvested at 6 h postinfection; lanes 6 and 12, cells harvested at 9 h postinfection;
lanes 7 and 13, cells harvested at 11 h
postinfection. Arrows indicate specific protein-DNA
complexes.
|
|
In order to identify the proteins that participate in the activated
AP-1 complex, we used antisera specific for the most abundant Jun/Fos
family members (Jun, JunB, JunD, and Fos) and for ATF-2, in
electrophoretic mobility shift assays, in untreated cells and cells
infected with HSV-1, at various times postinfection. In untreated cells
(Fig. 6, lanes 1-6), we
detected Jun (lane 2), JunB (lane 3), and JunD
(lane 4) as major components of the AP-1 complex. Neither
Fos (lane 5) nor ATF-2 (lane 6) was detected. Following induction of AP-1 binding activity at 6 h postinfection, the relative amounts of JunB and JunD were unchanged, whereas those of
Jun decreased (data not shown). At 11 h postinfection (Fig. 6,
lanes 7-12), the activated AP-1 complex was composed mainly
of JunB (lane 9) and JunD (lane 10). Jun became a
minor participant (lane 8), and Fos (lane 11) was
detected in very small amounts, only after overexposure of the film,
and ATF-2 was not detected at all (lane 12).

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|
Fig. 6.
Substitution of Jun by JunB and JunD in the
formation of the induced AP-1 complex. Electrophoretic mobility
shift assays using AP-1 probe were performed to determine composition
of AP-1 complexes. Lanes 1-6, untreated cells; lanes
7-12, cells infected with wild-type HSV-1, harvested at 11 h
postinfection. Antibodies used against components of the AP-1 complex
were as indicated. Arrow a, Jun·DNA complex;
arrow b, JunB·DNA complex; arrow
c, JunD·DNA complex.
|
|
Intracellular levels of Jun/Fos family members and of ATF-2 were
examined by Western blotting, at several times postinfection (Fig.
7). Levels of Jun protein decreased
between 6 and 11 h postinfection (lanes 1-5), whereas
JunB (lanes 6-9), JunD and ATF-2 (data not shown) levels
remained constant.

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|
Fig. 7.
Decrease in cellular levels of Jun during
HSV-1 infection. Western blots were performed to determine the
cellular amounts of AP-1 components during infection. Lanes
1-5, levels of Jun protein. Lane 1, untreated cells
(un); lanes 2-5, cells infected with wild-type
HSV-1, harvested at 3, 6, 9, and 11 h postinfection, respectively.
Lanes 6-9, levels of JunB protein. Lane
6, untreated cells; lanes 7-9, cells infected with
wild-type HSV-1, harvested at 3, 9, and 11 h postinfection,
respectively. Proteins are indicated by arrows.
|
|
Our results suggest that the AP-1 transcription factor, but not CREB,
is a downstream candidate for mediating signals from the activated
p38/SAPK pathways to the target genes. They also show increased
participation of JunB and JunD in the AP-1 complex during HSV
infection, with a simultaneous decrease in both relative contribution
in the complex and total cellular amounts of Jun protein.
 |
DISCUSSION |
Two independent yet parallel signaling cascades are responsible
for coordinating the cellular responses to environmental stress. JNK/SAPK and p38 MAPK play a central role in these pathways, and their
substrates include proteins of the Jun/Fos and ATF families, forming
the AP-1 transcription factor (1-7, 18).
In our study, we showed that HSV-1 infection of cells stimulated both
JNK/SAPK and p38 MAPK activity after 3 h of infection and that
activity levels increased up to a maximum of 4-fold between 3 and
9 h postinfection; these increased levels were maintained throughout the remainder of the virus replicative cycle.
By using a series of mutant viruses, we identified the virion
transactivator protein VP16 as the activator of the p38/JNK pathway and
mapped this activity to the domain that is responsible for interaction
with host cellular factors. We also showed that VP16 is both necessary
and sufficient for activating the stress pathways even in the absence
of any viral context. Preparations of UV-irradiated virus retained
their ability to stimulate JNK/SAPK, which is consistent with a role
for VP16 in activating JNK/SAPK (data not shown). No structural protein
of the capsid nor any viral IE, early, or late gene product was found
to be involved in triggering stimulation of p38/JNK. However, the fact
that the kinetics of the JNK/SAPK activation apparently coincided with the accumulation of IE proteins between 3 and 9 hours postinfection, may suggest a secondary, complementary role for some viral IE proteins.
VP16 regulates viral IE genes expression; however, unlike most
transcriptional activators, it is recruited to IE gene promoters by
association with the cellular proteins Oct-1 and HCF (20, 21). VP16 is
reported to mimic Luman protein in its interaction with HCF. Luman is a
human transcription factor of the CREB/ATF family that requires HCF to
activate transcription from CRE sites (43). HSV VP16 mimics this
interaction with HCF to monitor the physiological state of the host
cell (43), and this may result in the observed activation of the stress
pathway as a host cell response. Alternatively, stimulation of the
stress pathway may occur via interaction of VP16 with an unknown
cellular protein.
SKK 1 and SKK 4 are upstream activators of p38 and JNK. SKK 4 is known
to be activated by proinflammatory cytokines, whereas stressful stimuli
like UV irradiation and osmotic stress activate both SKK 1 and SKK 4 (9). Surprisingly, we identified SKK 1, but not SKK 4, as upstream
activator of p38/JNK, and these findings suggest that HSV-1 infection
activates stress pathways by a different mechanism than other stress stimuli.
Gel retardation assays demonstrated that induction of AP-1 binding
proceeded stimulation of p38/JNK. AP-1 activation was detected at
6 h and reached a maximum by 11 h postinfection. Activation of AP-1 might occur via the ras/ERK pathway (44), but
induction of ERKs during HSV-1 infection was not observed using kinase
assays in myelin basic protein polyacrylamide gels (data not shown), as
described by Shackelford (45). The in vivo targets of
virus-stimulated p38/JNK do not include ATF-2, since CREB binding
activity was not elevated and ATF-2 was not part of the activated AP-1
complex. Furthermore, induction of AP-1 binding coincided with an
increased participation of JunB and JunD in the AP-1 complex and with a simultaneous decrease both in the contribution of Jun to AP-1 and in
the total cellular levels of Jun protein. Thus, Jun does not appear to
be a target for stimulated p38/JNK.
Effective JNK substrates require a separate docking site and
specificity-conferring residues flanking the phospho-acceptor (16).
JunB has a functional JNK-docking site but lacks specificity-conferring residues, whereas JunD lacks a JNK-docking site, requiring
heterodimerization with docking competent partners in order to be
phosphorylated by JNK (16). Therefore, JunD may be a substrate for JNK
during activation by HSV-1 infection and JunB may serve as the JNK
docking partner. Significantly, progressive exclusion of Jun from the activated AP-1 complex could confer different attributes to AP-1 compared with the uninduced complex. Members of the Jun/Fos family differ in their characteristics (12, 17); Jun behaves as a positive
regulator of cell growth and may cause transformation when
overexpressed (12, 17, 46), whereas JunD antagonizes both of these
effects and is linked to AP-1-induced apoptosis (46, 47).
The biological role of p38/JNK and subsequent AP-1 activation by HSV-1
infection is unknown. Stimulation of p38/JNK and AP-1 activation may
represent a mechanism by which the virus manipulates cellular processes
to promote successful virus replication. There is evidence to suggest
that JNK/SAPK activation regulates the cell cycle (48, 49). In
addition, a subset of cellular genes transactivated by AP-1 may ensure
efficient viral gene expression and DNA replication and facilitate
virus growth. Alternatively, stimulation of stress-activated signaling
pathways could represent a spontaneous cellular defense mechanism to
viral invasion, with the aim of abrogating virus replication by
programmed cell death (50, 51).
The emergence of HSV-1 as a candidate for gene delivery (31) makes
further investigations regarding cellular stress response to viral
infection of great importance.
 |
ACKNOWLEDGEMENTS |
We are grateful for technical support
provided by Julia Dunlop (Institute of Virology, Glasgow, UK) and Rosie
Rankin (Glasgow Caledonian University, UK). We are indebted to Drs.
Dave Gillespie and Liz Black (CRC Beatson Laboratories, Glasgow, UK)
and to Dr. Sean Lawler (MRC Protein Phosphorylation Unit, Dundee, UK)
for reagents and helpful advice. We are also grateful for other
essential reagents provided by Drs. R. Everett, C. M. Preston, A. McLean (Institute of Virology, Glasgow, UK), and Prof. S. Weller
(University of Connecticut Health Center, Farmington, CT).
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Supported by Project Grant 050608 from The Wellcome Trust.
To whom correspondence should be addressed: School of
Biological and Biomedical Sciences, Glasgow Caledonian University,
Cowcaddens Rd., Glasgow, G4 0BA, UK. Tel.: 44 141 331 3219; Fax: 44 141 331 3208; E-mail: J.Conner{at}gcal.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
MAPK, MAP kinase;
MKK, MAPK kinase;
ERK, extracellular signal regulated kinase;
JNK/SAPK, c-Jun N-terminal
kinase/stress-activated protein kinase;
TRE, 2-O-tetradecanoate-13-acetate response element;
ATF, activating transcription factor;
CRE, cAMP response element;
CREB, CRE-binding proteins;
HSV-1, herpes simplex virus type 1;
IE, immediate-early;
BHK, baby hamster kidney;
GST, glutathione
S-transferase;
DTT, dithiothreitol;
pfu, plaque-forming
units;
HCF, host cell factor.
 |
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