From the Institut für Medizinische
Strahlenkunde und Zellforschung, Julius-Maximilians
Universität, D-97078 Würzburg, Germany,
Institut
für Mikrobiologie und Molekularbiologie, Justus-Liebig
Universität, D-35392 Giessen, Germany, and
Institut für Pharmakologie,
Medizinische Hochschule, D-30625 Hannover, Germany
Received for publication, October 31, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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Influenza A virus infection of cells results in
the induction of a variety of antiviral cytokines, including those that
are regulated by transcription factors of the activating protein-1 (AP-1) family. Here we show that influenza virus infection induces AP-1-dependent gene expression in productively infected
cells but not in cells that do not support viral replication. Among the
AP-1 factors identified to bind to their cognate DNA element during
viral infections of Madin-Darby canine kidney and U937 cells are those
that are regulated via phosphorylation by JNKs. Accordingly, we
observed that induction of AP-1-dependent gene expression
correlates with a strong activation of JNK in permissive cells, which
appears to be caused by viral RNA accumulation during replication.
Blockade of JNK signaling at several levels of the cascade by transient
expression of dominant negative kinase mutants and inhibitory proteins
resulted in inhibition of virus-induced JNK activation, reduced AP-1
activity, and impaired transactivation of the IFN- Influenza A virus is the prototype of the Orthomyxoviridae that
are characterized by a segmented negative strand RNA genome. The
function of the 10 different virus-encoded proteins has been well
elucidated (reviewed in Ref. 1). Transcription and amplification of the
viral genome is catalyzed by the RNA-dependent RNA
polymerase (RDRP)1 complex,
consisting of three proteins, PB2, PB1, and PA, which are associated
with the ribonucleoprotein complex. Genomic influenza virus RNAs carry
conserved nucleotide sequences at their 5'- and 3'-ends, which are in
part complementary and serve as promoters for synthesis of both
complementary RNA intermediates and novel viral RNA (vRNA) (1).
Because of the relatively small coding capacity of the viral genome, it
is not surprising that influenza A viruses extensively manipulate and
exploit host cell functions to support viral replication. On the other
hand, the host cell has developed defense mechanisms against viral
infections, which include an effective expression of antiviral
cytokines that may require activation of cellular signaling pathways.
Although only little is known about the intracellular signaling
cascades that are activated by influenza virus, there are numerous
reports on downstream target genes, such as interleukins (IL), tumor
necrosis factor- The best studied cell systems with respect to influenza virus-induced
cellular gene expression are macrophages and monocytes. These cells are
susceptible to influenza virus infection and express a set of cytokines
such as tumor necrosis factor- Several influenza virus-induced genes are dependent on activation of
the AP-1 family of transcription factors, including the IFN- JNKs, also known as stress-activated protein kinases (SAPKs) belong to
the family of mitogen-activated protein kinases (MAPKs) that are
regulated by dual phosphorylation of threonine and tyrosine residues in
a TXY motif (reviewed in Refs. 13 and 14). This activation
is mediated by dual specificity kinases termed MAPK kinases (MKKs). For
the JNK subgroup of MAPKs, at least two different MKKs have been
identified as specific activators, MKK4/SEK (for SAPK/ERK kinase) (15) and MKK7 (16,
17). It was recently shown that MKK4 and MKK7 act synergistically in
JNK activation, whereby MKK4 had a preference for the tyrosine residue
and MKK7 for the threonine residue within the TXY motif
(18). This suggests that the full activation of JNK may require
phosphorylation by two different MKKs. Since MKK4 or MKK7 are
preferentially activated by different upstream activators (19-21),
there is the potential for integrating the effects of different
extracellular signals. Specificity within the different signaling
cascades is accomplished by scaffold proteins, such as the
JNK-interacting protein-1 (JIP-1), which has been shown to form a
complex with JNK, MKK7, and the MKK7 activator mixed lineage kinase
(22).
In this report, we show that influenza virus infection of cells induces
JNK activation and AP-1-dependent gene expression in a
MKK4/SEK- and MKK7-dependent manner. This activation
selectively occurs in cells permissive for virus replication and
appears to be caused by accumulation of virus-specific RNA. Our
findings link influenza virus replication to the activation of an
important signaling pathway in the cell, which seems to be required for the antiviral response to infection.
Viruses, Cells, and Viral Infections--
Avian influenza virus
A/Bratislava/79 (H7N7) (FPV), human influenza virus A/Asia/57 (H2N2)
(Asia), and the reassortant virus WSN-HK (H1N2), which contains seven
genes of human influenza A/WSN/33 (H1N1) and the neuraminidase gene
from human influenza virus A/HK/8/68 (H3N2), were used for infection of
different cell lines. Madin-Darby canine kidney (MDCK) cells were grown
in minimal essential medium; the human cervical carcinoma cell
line HeLa and human embryonic kidney HEK293 cells were grown in
Dulbecco's modified Eagle's medium; U937 promonocytic cells and A3.01
human T lymphoma cells were grown in RPMI 1640 medium. All growth media
contained 10% heat-inactivated fetal calf serum and antibiotics. Cells
were washed with phosphate-buffered saline and infected with various viruses at the indicated multiplicity of infection (MOI) in
phosphate-buffered saline/BA (phosphate-buffered saline containing
0.2% bovine serum albumin, 1 mM MgCl2, 0.9 mM CaCl2, 100 units/ml penicillin, 0.1 mg/ml
streptomycin) for 30 min at 37 °C. The inoculum was aspirated, and
cells were incubated with RPMI 1640/BA (U937 and A3.01 cells), minimal
essential medium/BA (MDCK), or Dulbecco's modified Eagle's medium/BA (HEK293 and HeLa cells) (medium containing 0.2% bovine serum
albumin and antibiotics). The amount of infectious virus grown in MDCK
cells was determined by plaque assays.
DNA Constructs and Cloning--
Expression vectors for
glutathione S-transferase (GST) fusion proteins
pEBG-SAPK Transient Transfections and Reporter Gene
Assays--
Transfection of HEK293 cells was performed by a calcium
phosphate coprecipitation method according to a modified Stratagene transfection protocol. A3.01 and U937 cells were transfected with DMRIE-C (Life Technologies), and MDCK and HeLa cells were transfected with FUGENE (Roche Molecular Biochemicals) or LipofectAMINE 2000 (Life
Technologies) according to the manufacturer's instructions. Unless
otherwise indicated, cells were harvested 24 h after transfection into 100 µl of lysis buffer (50 mM Na-MES, pH 7.8, 50 mM Tris-HCl, pH 7.8, 10 mM dithiothreitol, 2%
Triton X-100). The crude cell lysates were cleared by centrifugation,
and 50 µl of precleared cell extracts were added to 50 µl of
luciferase assay buffer (125 mM Na-MES, pH 7.8, 125 mM Tris-HCl, pH 7.8, 25 mM magnesium acetate, 2 mg/ml ATP). Immediately after injection of 50 µl of 1 mM
D-luciferin (AppliChem) into each sample, the luminescence
was measured for 5 s in a luminometer (Berthold). As a
transfection control, Electrophoretic Mobility Shift Assay (EMSA)--
Oligonucleotide
probes derived from the IL-8 promoter AP-1 site as described (28) were
labeled in a reaction mixture containing 200 ng of double-stranded DNA
probe, [ Immune Complex Kinase Assays and Western Blots--
Cells were
lysed in Triton lysis buffer (20 mM Tris/HCl, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM
EDTA, 50 mM sodium glycerophosphate, 20 mM
sodium pyrophosphate, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM sodium vanadate, 5 mM benzamidine) on ice
for 10-20 min. Cell lysates were then centrifuged, and supernatants
were incubated with a specific antiserum and protein A-agarose (Roche
Molecular Biochemicals) for 2 h at 4 °C to precipitate the
endogenous kinase. Endogenous JNK1 was precipitated with an anti-JNK1
antiserum (Santa Cruz Biotechnology). Transfected HA- or GST-tagged
SAPK Influenza Virus Induces AP-1-dependent Gene Expression
in Productively Infected Cells--
To selectively analyze the
regulation of virus-induced AP-1-dependent gene expression
without the involvement of other regulatory transcription factors, we
used a promoter reporter gene construct containing five copies of an
AP-1-binding DNA element in front of a luciferase reporter gene (26).
Productive infection of MDCK cells with influenza A virus resulted in a
virus titer-dependent transactivation of the AP-1 dependent
promoter as early as 4 h postinfection (p.i.) (Fig.
1A). The early onset of
luciferase expression suggests that the effects are directly induced by
the virus and not by an autocrine event. Essentially the same results were obtained in the promonocytic U937 cell line, which is also permissive for influenza virus replication (Fig. 1B). In
cells that only allow an abortive virus replication, such as T
lymphocytes (A3.01) (29) or HeLa cells (30), the
AP-1-dependent promoter activity remained completely silent
(Fig. 1B), indicating that productive replication is
required to induce AP-1-dependent transcription.
AP-1 Factors Are Constitutively Bound to Their Cognate DNA Element
in MDCK and U937 Cells--
A first regulatory step in AP-1 activation
is transcriptional up-regulation of different AP-1 components upon cell
stimulation (10). To analyze whether influenza A virus infection
results in an up-regulation of AP-1 factors, EMSAs from nuclear lysates of infected and noninfected MDCK cells were performed. Although there
was a slight up-regulation of AP-1 factors 1-2 h after infection (Fig.
2A), the major portion of AP-1
factors was already bound to DNA in uninfected cells. Competition with
the nonlabeled AP-1 DNA probe showed that the protein-DNA complexes
observed were AP-1-specific (Fig. 2A). To analyze which AP-1
factors are constituents of the complex, supershift assays using
specific antibodies against certain AP-1 proteins were performed. The
shifted protein-DNA complexes in the presence of the antibodies
indicated that c-Jun, c-Fos, and JunD were components of the
AP-1 complexes. Further, with regard to these three factors, the
composition of the AP-1 complexes did not differ 4 or 8 h
postinfection (p.i.) (Fig. 2A). The same results
were obtained with U937 cells, albeit in these cells more AP-1 specific
complexes were observed (Fig. 2B). Furthermore, ATF-2 was
identified as another AP-1 binding factor (Fig. 2C), while
transcription factors JunB, Fra-1, and CREB did not bind to the
AP-1-specific DNA probes in both cell lines (Fig. 2C and data not shown). Thus, c-Jun, c-Fos, JunD, and ATF-2 were identified as
AP-1 binding factors in uninfected and infected cells and may contribute to the observed induction of AP-1-dependent
transcription.
ATF-2 Is Phosphorylated in Response to Virus Infection--
The
transcriptional activity of AP-1 complexes is potentiated upon
phosphorylation of AP-1 factors (10). One of these factors is ATF-2,
which plays a critical role in the virus-induced expression of the
antiviral cytokine IFN- Productive Infection of Cells with Different Influenza A Viruses
Results in a Strong Activation of JNK--
Since the data shown in
Fig. 3B suggest that JNK causes virus-induced
phosphorylation of ATF-2, we analyzed whether infection with different
influenza A viruses results in JNK activation. Cells were infected and
JNK activity was assessed in immune complex kinase assays at different
time points postinfection. A strong activation of JNK was observed upon
productive infection of MDCK, U937, or HEK293 cells. Activation
kinetics did not differ between the virus isolates but were dependent
on the cell line chosen. For example, infection of MDCK cells with the
influenza A virus strain Asia (Fig.
4A), the FPV strain (Fig.
4B) or the reassortant virus WSN-HK (data not shown)
resulted in an onset of JNK activation about 2-4 h postinfection,
whereas the same virus isolates induced JNK activity only after 8 h postinfection in HEK293 cells. In U937 cells, JNK activation was
observed as early as 1-2 h after infection with FPV. The dependence of
virus-induced JNK activation on the cell type was most obvious in
nonpermissive HeLa cells, where JNK was not activated at all (Fig.
4E). The same results were observed in Asia-infected A3.01 T
lymphocytes (data not shown). This indicates that the potential of
influenza A viruses to replicate in a certain cell type correlates with
both the ability to induce JNK activity (Fig. 4) and
AP-1-dependent gene expression (Fig. 1). In support of this
assumption, we observed that inhibition of virus replication by the
anti- influenza virus drug amantadine (35) results in a 50% decrease
in JNK activity 8 h postinfection (Fig.
5A) concomitant with a roughly
50% reduction in virus titers analyzed in parallel.
Virus-induced JNK Activation Is Mediated by MKK4/SEK and
MKK7--
We next analyzed which known JNK activator(s)
(MKK4/SEK, MKK7, or both) is activated in response to virus infection.
Cells were transfected with vectors expressing tagged versions of
either dominant negative or wild-type forms of SEK/MKK4 (Fig.
5B) or MKK7 (Fig. 5C). Cells were infected for
8 h, and transfected MKKs were specifically precipitated from cell
lysates with the respective anti-tag antibody. Activities of SEK/MKK4
or MKK7 were then assessed in immune complex kinase assays using
recombinant GST-tagged SAPK Virus-induced Activation of AP-1-dependent Gene
Expression Is Mediated by the JNK Signaling Pathway--
The
observation that influenza virus infection causes activation of
JNK/SAPK via MKK4 and MKK7 suggests that this pathway mediates
AP-1-dependent gene expression. For a final proof, we measured virus-induced AP-1 dependent gene expression in the presence of transdominant interfering mutants of the JNK signaling cascade. Expression of dominant negative JNK/SAPK or a dominant negative form of
c-Jun (TAM67) almost abolished luciferase expression induced by viral
infection, suggesting that JNK/SAPK and its target transcription factor
c-Jun are essential mediators of this process (Fig.
6A). As observed in the kinase
assays (Fig. 5G), expression of MKK7wt and active MKK7(S3E)
enhances the effect of virus infection on AP-1-dependent
gene expression (Fig. 6B), whereas expression of the
inhibitory mutant SEK(K>R) or JIP-1 results in an inhibition, both in
U937 and MDCK cells (Fig. 6C). Thus, influenza virus-induced AP-1 dependent gene expression is regulated by MKK4/SEK and
MKK7-mediated activation of JNK/SAPK and c-Jun.
Accumulation of RNA Synthesized by the Viral Polymerase Complex
Results in Activation of JNK--
A remaining question is which viral
components are responsible for activation of JNK in the host cell.
Recently, we identified influenza A virus hemagglutinin, nucleoprotein,
and matrix protein as viral transactivators toward the transcription
factor NF- The JNK Signaling Pathway Is Critical for Virus-induced Activation
of the IFN- The elucidation of intracellular signaling pathways that are
activated by influenza A virus infection is important for the understanding of both viral replication strategies and host defense mechanisms. Here we show that productive infection of cells results in
a strong activation of the JNK subgroup of MAPK signaling pathways. Further, expression of dominant interfering mutants revealed that the
JNK signaling pathway is a major player in transduction of virus-induced signals leading to AP-1-dependent expression
of IFN- AP-1 family transcription factors are regulated both on a
transcriptional and posttranslational level (10). Our results demonstrate that transcriptional up-regulation of AP-1 factors only
plays a minor role for influenza virus-induced
AP-1-dependent gene expression in MDCK and U937 cells. In
both uninfected and infected cells, the major portion of AP-1
components were constitutively bound to AP-1 sites, and the composition
of the complexes was not significantly changed during infection (Fig.
2). This is consistent with the appearance of constitutively expressed
JunD and ATF-2 in the complex. The weak up-regulation 1-2 h after
infection (Fig. 2) may be attributed to c-Jun and c-Fos that were also
identified in the complexes. It is still an open question which
virus-induced signaling events are responsible for the observed
up-regulation of these factors. Recently, it was shown that
pharmacological inhibition of the mitogenic Raf/MEK/ERK signaling
cascade at the level of MEK results in a block of
12-O-tetradecanoylphorbol-13-acetate-induced c-Jun and c-Fos
mRNA synthesis (38). Using the same inhibitor, U0126, in EMSA
experiments, we could not interfere with influenza virus-induced
up-regulation of AP-1
factors,2 although we
observed an activation of the Raf/MEK/ERK cascade early after infection
(39). Given that a variety of signaling pathways are known to be
involved in regulation of AP-1 factor expression (40), inhibition of
MEK by U0126 may not be sufficient to result in detectable differences
in the EMSA.
In an analysis of the viral features that give rise to the strong JNK
activation, we have now identified accumulation of virus-specific RNA
as a major trigger. This finding is consistent with two other reports
published while this manuscript was in preparation (41, 42). In these
studies, it was shown that treatment with the dsRNA analog poly(IC) as
well as infection with vesicular stomatitis virus (41) or
encephalomyocarditis virus (42) results in JNK activation. However,
poly(IC) might have its limitations to represent RNA of replicating
single-stranded RNA viruses. In our study, we have reconstituted the
influenza virus replicase complex to mimic ongoing viral genome
transcription and replication in the absence of infection. This is the
first demonstration that RNA accumulation within a virus-like setting
causes activation of the JNK signaling cascade. This finding further
indicates that viral RNA still has signaling capacity, although it is
packaged with NP to form RNP complexes. It will now be important to
identify the direct cellular signaling sensors for viral RNA. The only cellular signaling enzyme known so far to be directly regulated by
dsRNA is the 68-kDa double-stranded RNA-dependent
kinase (43), which inhibits protein synthesis by phosphorylation
of the Usually, 5-6 h after infection with influenza A virus, an efficient
host-cell protein synthesis shut off is initiated (1). The induction of
JNK activity 2-4 h postinfection and the amount of luciferase that is
accumulated as early as 4 h postinfection show that the viral
induction of both AP-1-dependent gene expression and
transactivation of the IFN- Another interesting observation is the inability of nonpermissive
cells, such as T lymphocytes or HeLa cells, to support virus-induced JNK1 and AP-1 activation. These cells are readily infected; however, they undergo an abortive infection at some point during the viral life
cycle. In HeLa cells, synthesis of viral proteins was found to be
normal, but there appears to be a defect during budding of novel virus
particles (46), presumably due to a misincorporation of the HA into the
membrane (47). In T lymphocytes, the point at which viral replication
is impaired is less well understood; however, there is also a
significant amount of viral proteins synthesized without formation of
new virus particles (29). It will now be of great interest to elucidate
why there is a lack of JNK activation despite the fact that
virus-specific RNA is still synthesized in these cells.
In summary, our results demonstrate for the first time that productive
influenza A virus infection results in the activation of a member of
the MAPK signaling cascades, which are important mediators of
intracellular signaling. Virus-induced activation of
AP-1-dependent gene expression occurs early during the
viral life cycle, is mediated by viral RNA-induced activation of the JNK signaling cascade, and appears to be part of the innate immune response to infection.
promoter. Virus
yields from transfected and infected cells in which JNK signaling was
inhibited were higher compared with the levels from control cells.
Therefore, we conclude that virus-induced activation of JNK and AP-1 is
part of the innate antiviral response of the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and interferons (IFN), which are immediately
transcribed in response to infection (for a review, see Ref. 2).
, IL-1, IL-6, and IFN-
/
upon
infection (3-5). Other virus-induced genes include monocyte
chemoattractant protein-1 in monocytes (6) or IL-8 in human lung
epithelial cells (7, 8) or rat kidney cells (9).
gene
that encodes for one of the most potent antiviral cytokines. This
transcription factor family consists of homodimers and heterodimers of
Jun (c-Jun, JunB, JunD) Fos (c-Fos, FosB, Fra-1, Fra-2), or the
activating transcription factor (ATF-2, ATF-3) proteins, which bind to
a common DNA site, the AP-1 site (reviewed in Ref. 10). The regulation
of AP-1 activity occurs at two major levels. First, the abundance of
the proteins is commonly regulated by controlling the transcription of
AP-1 factor genes (11). The regulation of transcription is best
understood for c-jun and c-fos genes that
are rapidly up-regulated upon cell stimulation (11). At a second level,
the activity of different AP-1 factors is posttranscriptionally modulated by phosphorylation events. This includes AP-1 factors c-Jun
and the constitutively expressed JunD and ATF-2, which are phosphorylated by kinases of the JNK family, resulting in an increased transactivation potential of the factors (reviewed in Refs. 10 and
12).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pEBG-SEK1 and pGEXKG-c-Jun-(1-135) were a kind gift of
J. Kyriakis and L. Zon (Charlestown, MA). Generation of
pEBG-SEK1(K>R) and pEBG-SAPK
(KK>RR) was described previously (23).
The cDNAs of a hemagglutinin epitope (HA)-tagged version of
SAPK
, the transactivation domain deletion mutant of c-Jun, TAM67,
and the JNK binding domain of JIP-1 (22) were cloned into the multiple
cloning site of pRSPA (24). Expression vectors pCS3 MKK7wt, MKK7(S3E),
and MKK7(K>M) were a kind gift of P. M. Holland (Fred
Hutchinson Cancer Research Center, Seattle, WA) and were described
earlier (25). The 5× AP-1 luciferase construct contains five copies of
a conserved 9-base pair AP-1 motif, also known as the TRE
(12-O-tetradecanoylphorbol-13-acetate-responsive element) (26), in front of a luciferase reporter gene. The
empty pHMG expression vector and pHMG expression plasmids for influenza virus NP, PB1, PB2, and PA were kindly provided by J. Pavlovic (Institute of Medical Virology, Zürich, Switzerland), and
constructs that allow RNA polymerase I-driven synthesis of influenza
vRNA-like templates carrying the antisense reading frame for a GFP
(pPol I GFP) or a luciferase (pPol I Luc) reporter gene were kindly provided by P. Palese (Department of Microbiology, Mount Sinai School
of Medicine, New York). The IFN-
promoter/enhanceosome luciferase construct was a generous gift of J. Hiscott (Lady
Davis Institute for Medical Research, McGill University, Montreal,
Canada) (27). The double-stranded RNA (dsRNA) analog poly(IC) and the p38-specific inhibitor SB202190 were purchased from Sigma or
Calbiochem, respectively.
-galactosidase activity of cotransfected Rous
sarcoma virus-
-galactosidase vector was analyzed, and luciferase
activities were normalized on the basis of protein content. Mean and
S.D. values of at least three independent experiments performed in
duplicate or triplicate are shown in Figs. 1, 6, and 8, A
and B.
-32P]dCTP, 1 mM dATP, 1 mM dGTP, 1 mM dTTP, 500 mM
Tris-HCl, pH 7.5, 100 mM MgCl2, and 2 units of
Klenow fragment. After a 30-min incubation at 37 °C,
oligonucleotides were separated on a G-25 Sephadex spin column (Roche
Molecular Biochemicals) and finally resuspended in TE (30,000 cpm/µl). For the typical binding reactions, 5 µg of nuclear lysates
were incubated on ice for 5 min in the absence or presence of
competitor DNA in an 18-µl reaction mixture containing 25 mM Tris, pH 7.5, 1 mM EDTA, 0.5 mM
1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, 100 mM KCl, 0.1% (v/v) Nonidet P-40, 1 µg (w/v) of bovine
serum albumin, 10% (v/v) glycerol, and 0.5 µg (w/v) of poly(dI-dC); 60,000 cpm of labeled oligonucleotide were added, and the mixture was
incubated for 15 min at 25 °C. The antisera for supershifts were all
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
samples were loaded on a 5% nondenaturing polyacrylamide gel
equilibrated with 0.5× Tris borate-EDTA buffer and electrophoresed for
2.5 h at 180 V. Gels were dried, and DNA-protein complexes were
visualized by autoradiography.
, GST-tagged SEK/MKK4, or Myc-tagged MKK7 were detected with
either a monoclonal antibody against the HA epitope (12CA5), an
antiserum against GST (23), or the monoclonal anti-Myc epitope antibody
(9E10). Immune complexes were used for in vitro kinase
assays as previously described (23). Briefly, immunoprecipitated kinases were washed twice, both in Triton lysis buffer supplemented with 500 mM NaCl and in kinase buffer (10 mM
MgCl2, 25 mM
glycerophosphate, 25 mM HEPES pH 7.5, 5 mM benzamidine, 0.5 mM dithiothreitol, and 1 mM sodium vanadate).
The assays were performed in kinase buffer supplemented with 5 µCi of
[
-32P]ATP, 0.1 mM ATP, and recombinant
GST-c-Jun-(1-135) or GST-SAPK
as substrates for JNK/SAPK or
MKK4 and -7, respectively, at 30 °C for 15 min. Proteins were
separated by SDS-polyacrylamide gel electrophoresis and blotted onto
polyvinylidene difluoride membranes. Phosphorylated substrates were
detected by a BAS 2000 Bio Imaging Analyzer (Fuji) and by
autoradiography. Equal loading of immunoprecipitated kinases was
analyzed by Western blotting using the appropriate anti-kinase
antiserum or anti-tag antibody in a standard enhanced chemiluminescence
reaction (Amersham Pharmacia Biotech). To detect activated ATF-2, an
anti-phospho-ATF-2 antiserum was used (New England Biolabs). Equal
ATF-2 loading in these assays was controlled with a pan-ATF-2 antiserum
(Santa Cruz Biotechnology).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Productive influenza A virus infection
results in transactivation of an AP-1 dependent promoter. MDCK
cells (A) or U937, A3.01, and HeLa cells (B) were
transfected with a 5× AP-1 luciferase construct and infected 24 h
later with the influenza strain FPV at the MOIs indicated. Cells were
harvested 4 or 8 h postinfection (p.i.), and luciferase
activities in the lysates were determined as described. Results are
given as -fold activation of relative luciferase activity compared with
the uninfected control. HeLa cells were also infected with the Asia
strain with essentially the same results (data not shown).
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Fig. 2.
AP-1 factors are constitutively bound to AP-1
sites in MDCK and U937 cells during influenza A virus infection.
MDCK (A) or U937 cells (B and C) were
infected with FPV at a MOI of 5 for the times indicated, and nuclear
lysates were subjected to EMSA using a labeled AP-1 DNA probe. Where
indicated, binding reactions were performed in the presence of a 1:10
and 1:100 surplus of unlabeled oligonucleotides (competitor) or in the
presence of specific antibodies against c-Jun, c-Fos, JunD, ATF-2,
JunB, CREB-1, and Fra-1. As a control, nuclear lysate was replaced by
H2O (A, lane 16).
Roman numerals to the right of each
panel are indicative of the main AP-1 complex (I)
and the supershifted complexes induced after binding to the antibodies
(II-V). p.i., postinfection.
(31, 32). ATF-2 was indeed phosphorylated
upon infection with different influenza A virus strains in permissive
293 (Fig. 3A), COS7 (Fig.
3B), and U937 cells (data not shown). Two different MAPKs
were reported to phosphorylate ATF-2, p38 and JNK/SAPK (33, 34).
Although p38 is activated upon influenza virus infection (data not
shown), ATF-2 phosphorylation was not decreased or abolished in cells
treated with effective concentrations of the p38-specific inhibitor
SB202190 (Fig. 3B). This indicates that the JNK/SAPK
subgroup of MAPKs may be primarily responsible for the observed
phosphorylation event.
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Fig. 3.
ATF-2 phosphorylation upon influenza virus
infection occurs independently of p38 MAPK. 293 cells
(A) or COS7 cells (B) were infected with the
influenza strains FPV (A and B) or Asia
(B) at a MOI of 8 for the times indicated in the presence
(B) or absence (A and B) of the
p38-specific inhibitor SB202190 (20 µM). Cell lysates
were subjected to SDS-polyacrylamide gel electrophoresis, blotted onto
nitrocellulose membranes, and incubated with an antibody specific for
phosphorylated ATF-2. Equal loading of ATF-2 was confirmed using a
pan-ATF-2 antiserum. ATF-2 phosphorylation was also detected in lysates
of virus-infected U937 cells with an earlier onset consistent with the
kinetics of JNK activation in these cells (data not shown).
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Fig. 4.
Influenza A virus induces a strong activation
of JNK in productively infected cells. MDCK (A and
B), HEK293 (C and D), HeLa
(E), and U937 cells (F) were infected with Asia
(A, C, and E) or FPV (B,
D, and E) at a MOI of 5. Cells were harvested at
the indicated times postinfection, and JNK1 was immunoprecipitated from
cell lysates. Immune complexes were subjected to in vitro
kinase assays using purified GST-c-Jun-(1-135) as a substrate.
Proteins were separated by SDS-polyacrylamide gel electrophoresis, and
labeled substrate bands were detected by exposure to x-ray films. Equal
loading of JNK1 proteins was confirmed by Western blot.
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Fig. 5.
Virus-induced activation of JNK/SAPK is
mediated by MKK4/SEK and MKK7. A, MDCK cells were
infected with FPV at an MOI of 8 for 8 h in the presence or
absence of the anti-influenza viral compound amantadine (1 µM). The activity of endogenous JNK1 was determined as
described. B, C, and D, HEK293 cells
were transiently transfected with plasmids expressing GST-tagged wild
type (SEK wt) or dominant negative SEK/MKK4
(SEK(K>R)) (B and D) or
Myc-tagged wild type (MKK7 wt) or dominant negative MKK7
(MKK7(K>M)). 24 h after transfection, cells
were infected for 8 h, and activity of transfected MKKs in the
lysates was determined in immune complex kinase assays with GST-SAPK
(B and C) or in a coupled assay with GST-SAPK
and GST-c-Jun-(1-135) as substrates (D). E-G,
HA-tagged JNK/SAPK
was cotransfected with vectors expressing
MKK7(K>M), SEK(K>R), the JNK-binding domain of JIP-1, wild-type MKK7,
or active MKK7(S3E) as indicated in a 1:1 (E-G) or 1:2
ratio (F). 24 h after transfection, cells were infected
for 8 h, and cell lysates were used to immunoprecipitate
JNK/SAPK
with an anti-HA antiserum. JNK/SAPK
activity was
subsequently assayed in immune complex kinase assays as described.
Loading of kinases was monitored by Western blot with the respective
antibody. To quantify kinase activities in E, band
intensities of phosphorylated c-Jun-(1-135) were normalized to the
corresponding amount of immunoprecipitated JNK/SAPK
of each
sample.
, which is the rat isoform of JNK2, as a
substrate. Both wild-type forms of SEK/MKK4 (Fig. 5B,
lane 4) and MKK7 (Fig. 5C,
lane 4) were activated upon infection, which was
not the case in the control samples with the dominant negative kinases
(Fig. 5, B and C, lane 2).
To analyze whether the observed phosphorylation of GST-SAPK
was on
the functional relevant sites, we performed a coupled assay by adding
in recombinant GST-c-Jun-(1-135) as a substrate for GST-SAPK
to the
sample. Indeed, GST-c-Jun-(1-135) was phosphorylated selectively in
the samples corresponding to the infected cells (Fig. 5D,
lanes 2 and 4), indicating that
virus-induced activation of both MKK4 and -7 resulted in a functional
phosphorylation of GST-SAPK
. For a further proof that SEK/MKK4 and
MKK7 are upstream of JNK upon virus infection, the activity of the
kinase was assessed in the presence of dominant negative forms of both
MKKs. Fig. 5E shows that expression of both dominant
negative MKK4/SEK (lane 3) and dominant negative
MKK7 (lane 4) results in an inhibition of
virus-induced activation of coexpressed HA-tagged JNK/SAPK. A
concentration-dependent inhibition was not only observed by expression of SEK(K>R) but also of the JNK-binding domain of JIP-1 (Fig. 5F). Overexpressed JIP-1 inhibits JNK by competitive
binding and retention in the cytoplasm (22). On the other hand,
transient expression of wild-type MKK7 acts synergistically with virus
infection to induce JNK/SAPK activity (Fig. 5G,
lane 4). This indicates that the amounts of
cellular MKK7 are limited and that the signal transmission is greatly
enhanced if a surplus of the kinase is provided. Expression of an
active mutant of MKK7 (MKK7(S3E)) resulted in a strong JNK activation
already in the absence of virus, which is again significantly enhanced
after infection (Fig. 5G, lanes 5 and
6), presumably due to the additional action of
virus-activated MKK4/SEK. Taken together, virus infection induces the
activity of both MKK4/SEK and MKK7, which act synergistically to
activate JNK/SAPK.
View larger version (28K):
[in a new window]
Fig. 6.
Influenza A virus-induced
AP-1-dependent gene expression is regulated by the JNK
signaling pathway. U937 (A-C) and MDCK cells
(C) were transfected with a 5× AP-1 luciferase construct
and either cotransfected with empty expression vector (KRSPA or pCS3)
or plasmids expressing dominant negative JNK/SAPK
(SAPK (KK>RR)) (A),
dominant negative c-Jun (Tam67) (A), wild-type
MKK7 (MKK7 wt) (B), constitutively active MKK7
(MKK7(S3E)) (B), dominant negative MKK4/SEK
(SEK(K>R)) (C), or the JNK-binding
domain of JIP-1 (C). Cells were infected 24 h later
with the influenza strain FPV at a MOI of 5 and harvested 4 h
postinfection (p.i.). Luciferase activities in the lysates
were subsequently determined as described and are expressed as -fold
activation compared with the uninfected control (A and
B). In C, virus-induced AP-1 promoter activity in
cells transfected with empty vectors is arbitrarily set as 100%, and
relative luciferase activities are given in percentage of the
control.
B (36). However, expression of these proteins resulted in
neither the activation of JNK (data not shown) nor the activation of
AP-1-dependent promoters (36). These studies were extended
to the viral NS1 and NEP/NS2 proteins, which also did not induce JNK
activity when overexpressed (data not shown). Another component of the
virus is the single-stranded vRNA, which is transcribed and replicated by the viral RDRP complex consisting of the PB2, PB1, and PA proteins (1). The vRNA is transcribed into mRNA and amplified via a complementary RNA replication intermediate. To assess whether dsRNA has
the capacity to induce JNK signaling, we treated MDCK cells with the
dsRNA analog, poly(IC). Indeed, poly(IC) stimulation induced a time-
and concentration-dependent activation of JNK (Fig.
7A), which was not inhibited
by actinomycin D or cycloheximide (data not shown). This indicates that
activation occurs directly and does not involve new RNA or protein
synthesis. To analyze whether accumulation of influenza-like RNAs gives
rise to JNK activation, we used a plasmid-based replication system for
vRNA-like RNA polymerase I (Pol I) transcripts. Briefly, cells were
transfected with expression plasmids for the RDRP constituents PB2,
PB1, PA, and NP and for a vRNA-like Pol I transcript carrying the
conserved 5' and 3' promoter structures of influenza vRNA segments
flanking an antisense reporter gene. Such constructs have been
successfully used to provide vRNA-like templates for the RDRP in
plasmid-derived reverse genetics approaches (37). In these studies, it
was demonstrated that all three polymerase genes as well as the NP gene
have to be cotransfected to allow transcription and amplification of
the reporter gene template (37). Using GFP as a reporter gene (pPol I
GFP) ~5-10% of transfected cells showed green fluorescence after cotransfection of plasmids expressing PB2, PB1, PA, and NP, while no
green cells were detectable if either the plasmids for NP or the
template vRNA were replaced by empty vectors (data not shown). This
indicates that only 5-10% of transfected cells carry all of the
plasmids required for transcription and amplification of the template
vRNA. Nevertheless, we observed a significant activation of GST-SAPK
in the samples cotransfected with all essential replicase genes and
either pPol I GFP or pPol I Luc plasmids expressing template vRNAs
(Fig. 7B, lanes 3 and 6).
No SAPK
activation was detectable if either NP (Fig. 7B,
lane 2) or the template RNAs were omitted (Fig.
7B, lane 4) or replaced by empty pPol
I vectors (Fig. 7B, lanes 5 and
7). These findings demonstrate that JNK/SAPK is activated
during influenza virus-specific transcription and replication of an
influenza-like RNA template by the viral RDRP complex, suggesting that
vRNA accumulation is the trigger for activation of the JNK signaling
pathway.
View larger version (39K):
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Fig. 7.
Poly(IC) treatment of cells or amplification
of an influenza vRNA-like segment by a reconstituted RDRP complex
results in JNK activation. A, MDCK cells were treated
with the dsRNA analog poly(IC) at the concentrations and for the times
indicated. JNK activity was assessed in the cell lysates as previously
described. B, MDCK cells were transfected with expression
plasmids for GST-SAPK , PB2, PB1, PA, and NP. Equal DNA load was
adjusted with empty expression vector. To provide template RNAs for the
RDRP plasmids expressing Flu-like RNA containing a GFP or luciferase
antisense reporter gene (pPol I GFP and pPol I Luc) were cotransfected.
In the samples indicated, the RNA template constructs were replaced by
the corresponding empty vector (pPol I vector). Proper function of the
influenza replicase complex was monitored by the count of GFP-positive
cells. Note that only 5-10% of transfected cells contained all
plasmids for transcription and expression of GFP. 24 h
posttransfection, cells were harvested, and lysates were assayed for
GST-SAPK
activity as described.
Promoter--
IFN-
is one of the most potent
antiviral cytokines. The IFN-
promoter/enhanceosome was shown to
bind c-Jun/ATF-2 heterodimers, which are essential for the
virus-induced transcription of the cytokine gene (31). To finally
analyze whether virus-induced IFN-
expression is dependent on
the JNK signaling pathway, we cotransfected dominant negative
mutants of JNK/SAPK (SAPK
(KK>RR)) or c-Jun (TAM67) with an IFN-
promoter luciferase construct. Both, treatment with poly(IC) and
infection with influenza A virus, resulted in a strong transactivation
of the IFN-
promotor, which was efficiently blocked in the presence
of dominant negative JNK/SAPK or c-Jun (Fig.
8, A and B). These
data demonstrate that JNK/SAPK is essential for RNA- or influenza
virus-induced transcription of the cytokine gene. Since the JNK pathway
appears to regulate the expression of an efficient antiviral cytokine,
inhibition of the pathway should increase the ability of the virus to
replicate in cells. Indeed, higher virus yields were obtained in
infected MDCK expressing dominant negative mutants of either MKK7,
JNK/SAPK, or c-Jun (Fig. 8C). Taken together, these data
suggest that activation of the JNK signaling pathway and
AP-1-dependent gene expression upon influenza virus
infection are part of the innate immune response of the cell,
e.g. by triggering expression of antiviral cytokines such as
IFN-
.
View larger version (16K):
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Fig. 8.
Influenza A virus-induced expression of
IFN- is regulated by the JNK signaling
pathway. A and B, MDCK cells were
transfected with an IFN-
promoter/enhanceosome luciferase construct
and cotransfected with either empty expression vector or plasmids
expressing dominant negative JNK/SAPK
(SAPK
(KK>RR)) or dominant negative
c-Jun (TAM67). 24 h posttransfection, cells were either
treated with poly(IC) (50 µg/ml) (A) for 6 h or
infected with FPV for 4 h (B). Luciferase activity in
the cell lysates was determined as described under "Experimental
Procedures." C, MDCK cells were transfected with empty
expression vector or plasmids expressing dominant negative JNK/SAPK
(SAPK
(KK>RR)), dominant negative
c-Jun (TAM67), or dominant negative MKK7
(MKK7(K>M)). Transfection efficiency was
monitored by GFP expression to be 50-60%. 9 h postinfection,
supernatants were analyzed for the amount of infectious virus particles
released in conventional plaque assays. Virus yield in vector
transfected cells was arbitrarily set as 100%. Experiments were
performed twice in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Thus, virus-induced activation of JNK and AP-1 appears to
be part of the innate antiviral response of the host cell.
-subunit of the eukaryotic initiation factor 2 (44). It was
recently shown that virus-induced activation of I
B kinase and
NF-
B is dependent on RNA-dependent kinase activity;
however, JNK was activated independently of RNA-dependent
kinase in these studies (41). Our experiments indicate that influenza A
virus-induced AP-1 activation is mediated by JNK and its upstream
activators MKK4/SEK and MKK7. A variety of kinases that are direct
upstream activators of MKK7 and MKK4/SEK are known (45) and may be
involved in virus-induced JNK activation, but none of these kinases had
so far been shown to be activated by dsRNA. Thus, the search is open
for a dsRNA-regulated enzyme upstream of MKK4/SEK and MKK7.
promoter occur before the
protein-synthesis block. The late virus-induced activity of JNK at
8-10 h postinfection may only poorly contribute to gene expression
events. Although this late activation phase is not counteracted by the
virus, it may not positively interfere with virus replication, since
cells in which JNK signaling is impaired allowed a more efficient virus propagation (Fig. 8C).
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ACKNOWLEDGEMENTS |
---|
We thank Heide Häfner and Irina Böhle for excellent technical assistance and Alex McLellan, Gaby Neumann, Oliver Planz, Christoph Scholtissek, and Thorsten Wolff for critical reading of the manuscript. We are very grateful to P. Holland, J. Kyriakis, P. Palese, J. Pavlovic, and L. Zon for providing plasmids.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant Lu 477/4-3 and a grant from the Fonds der Chemischen Industrie (to S. L.).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.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Institut für Medizinische Strahlenkunde und Zellforschung, Julius-Maximilians Universität Würzburg, Versbacher Strasse 5, D-97078 Würzburg, Germany. Tel.: 49 931 201 3851; Fax: 49 931 201 3835; E-mail: s.ludwig@mail.uni-wuerzburg.de.
** Present address: SmithKline Beecham Pharma, Sächsische Serumwerke, D-01069 Dresden, Germany.
§§ Present address: Institut für Virologie, Fachbereich Veterinärmedizin, Justus-Liebig Universität, D-35392 Giessen, Germany.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M009902200
2 C. Ehrhardt and S. Ludwig, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: RDRP, RNA-dependent RNA polymerase; AP-1, activating protein-1; ATF, activating transcription factor; JNK, Jun-N-terminal kinase; MDCK, Madin-Darby canine kidney; MKK, MAPK kinase; vRNA, viral RNA; dsRNA, double-stranded RNA; IL, interleukin; IFN, interferon; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase, SEK, SAPK/ERK kinase; JIP-1, JNK-interacting protein-1, GFP, green fluorescent protein; MOI, multiplicity of infection; GST, glutathione S-transferase; HA, hemagglutinin; MES, 4-morpholineethanesulfonic acid; EMSA, electrophoretic mobility shift assay; Pol I, polymerase I.
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