From the Department of Pathology and Kaplan
Comprehensive Cancer Center, Molecular Oncology and Immunology Program,
New York University School of Medicine, New York, New York 10016 and
the ¶ Mount Sinai School of Medicine of New York University,
New York, New York 10029
Received for publication, September 22, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Induction of interferon- Type I interferon
(IFN),1 consisting of the
single IFN While several transcription factors required for IFN gene
induction have been identified, delineation of the signaling pathways stimulated by virus infection that lead to
phosphorylation-dependent activation remains incomplete.
Activation of NF Not only is the identity of the IRF3/IRF7 kinase unclear, the nature of
the activating component produced during virus infection that leads to
kinase activation remains unknown. One candidate has been dsRNA, a
common intermediate or by product of many viral infections that
directly activates PKR (23). Moreover, many viruses target inactivation
of PKR as a strategy to overcome host antiviral responses, by binding
dsRNA, interfering with the activation of PKR, blocking its recognition
of substrates, or inducing its degradation (24). However, recent
evidence suggests that dsRNA may not be the essential or at least not
the only viral component leading to IFN gene expression. For
instance, disruption of the gene for PKR abrogated the induction of IFN
in response to dsRNA, but did not prevent responsiveness to viral
infection (25-27). Moreover, cytomegalovirus, which is capable of
activating IRF-dependent gene expression (20, 28), can do
so from the cell surface without entering cells or replicating
(29).
We have investigated the mechanisms of phosphorylation of IRF7 and its
close relative, IRF3, during activation of IFN gene expression
following Newcastle disease virus (NDV) infection. By all parameters
tested, IRF3 and IRF7 appeared to be activated by the same mechanism.
Their phosphorylation occurred with similar kinetics which required
ongoing protein synthesis during viral infection, was blocked by the
broad-spectrum kinase inhibitor staurosporine but not by inhibitors
specific for individual kinases, and could be inhibited by the vaccinia
virus protein, E3L. However, IRF3 and -7 phosphorylation was unaffected
by the inactivation of the PKR gene, and IFN Cell Culture--
293T cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% bovine calf serum.
PKR(
For kinase inhibition studies, inhibitors were added to growth media
5 h post-infection, unless otherwise indicated, at the following
final concentrations: staurosporine, 500 nM; genistein, 300 µM; H7, 45 µM; H8, 45 µM;
acetylsalicylic acid, 5 mM; sodium salicylate, 5 mM. Dimethyl sulfoxide treatment was performed by adding
0.1% dimethyl sulfoxide by volume to growth media. Cycloheximide (75 µg/ml) was added to cells at the indicated time points
post-infection. Additional inhibitors were used at the concentrations
listed in Table I.
Plasmid Constructs--
Mouse IRF3 and IRF7 (3), and E3L and K3L
expression vectors (34), the kind gift of Robert Schneider (New York
University, NY), were driven by the cytomegalovirus
immediate-early promoter. The K167A point mutation in E3L was
created by PCR-mediated site-directed mutagenesis using the following
primers: sense,
5'-CGATAAGGCAGATGGAAAATCTGCTCGAGATGCTAAAAATAATGC-3'; antisense,
5'-GCATTATTTTTAGCATCTCGAGCAGATTTTCCATCTGCCTTATCG-3'. DRBP76 (35) and
Staufen expression vectors (36) were the kind gifts of Ganes Sen
(Cleveland Clinic, OH) and Juan Ortín (Centro Nacional de
Biotecnologia, Madrid, Spain), respectively. The adenovirus VA gene was
a kind gift of Robert Schneider (New York University, NY) and the
MKK7(D) construct was a kind gift of Jan Vilcek (New York University).
Transfections and Viral Infections--
293T cells were
transfected by the calcium phosphate method (37). 12 h after
transfection, plates of similarly transfected cells were pooled and
distributed onto 60-mm plates. Cells were infected 12 h later with
NDV, Manhattan strain (3), or influenza virus (38), as described.
Unless otherwise noted, cells were harvested 7 h post-infection,
and nuclear and cytoplasmic fractions were prepared, as described (39,
40).
Orthophosphate Labeling--
PKR( RNA Analysis--
Total RNA was prepared using 3 ml of Trizol
reagent (Life Technologies, MD) per 100-mm plate. Semi-quantitative
reverse transcriptase-PCR analysis was performed as previously
described (3).
Protein Analysis--
Electromobility shift assays (EMSA) were
performed using nuclear extracts, as described (6, 40). Western blots
were performed by standard techniques (41), except that E3L proteins
were transferred to polyvinylidene difluoride with a 0.2-µm pore
size. Rabbit antisera to IRF3 and IRF7 (Zymed Laboratories
Inc.) were used at 0.5 µg/ml. Chicken anti-NDV (SPAFAS,
North Franklin, CT) was used at a dilution of 1:1000. Monoclonal
anti-E3L antibody Tw2.3 (42) was an ammonium sulfate fraction from
supernatants of hybridoma cultures, the kind gift of Jonathan Yewdell
(National Institutes of Health, Bethesda, MD). DRBP76 and Staufen
proteins were detected using antibodies to epitope tags. In
vitro kinase assays for IKK were performed by immunoprecipitating
IKK from mouse fibroblasts, and incubating the recovered protein with 1 µg of GST fusion protein substrates in 15 µl of kinase buffer (20 mM Hepes, pH 7.6, 20 mM magnesium chloride, 20 mM IRF3 and IRF7 Can Be Phosphorylated with Equivalent Kinetics during
NDV Infection--
IRF3 is constitutively expressed in cells while
IRF7 is induced in response to an early wave of IFN production
following viral infection (44). This differential expression
complicates the question of whether these two proteins can be activated
by the same kinase. To alleviate this technical shortcoming, we
ectopically expressed IRF3 or IRF7 by transient transfection so that
expression would be normalized under the control of an artificial
promoter and phosphorylation could be measured independent from protein induction. Infection of transfected cells with NDV led to
phosphorylation, as judged by a mobility shift following SDS-PAGE that
is dependent on phosphorylation (3, 6, 14, 16), of both IRF7 (Fig. 1A) and IRF3 (Fig.
1B). Moreover, both proteins were activated with equivalent
kinetics, becoming phosphorylated at 5 h post-infection and
showing maximal phosphorylation 7-8 h post-infection that remained
high for several hours (data not shown). Acquisition of DNA binding
activity, as judged by EMSA, correlated precisely with phosphorylation
for both proteins (data not shown). We validated this transfection
system by examining the kinetics of phosphorylation of endogenous IRF3
(Fig. 1D) which was phosphorylated with the same kinetics as
the ectopically expressed protein. Therefore, the sequential activation
of IRF3- and IRF7-target genes normally observed in untransfected cells
following NDV infection can be accounted for by the requirement for
induction of the IRF7 protein rather than by utilization of distinct
kinases.
Considerable viral protein synthesis occurred within the first 7 h
of infection, as judged by immunoblotting for viral proteins using
antisera directed against NDV virions (Fig. 1C). This
observation prompted us to consider whether protein synthesis,
including viral protein synthesis, might be involved in IRF activation.
To test this notion, protein synthesis was blocked by addition of
cycloheximide at various times after infection. Inhibition of protein
synthesis at any point up until ~3 h post-infection prevented
phosphorylation of IRF7 (Fig. 2,
lanes 3-5). However, addition of cycloheximide after that
point was without effect (Fig. 2, lanes 6-9). Consistent with the requirement of phosphorylation of IRF3 (16) and IRF7 for
acquisition of DNA binding (6), cycloheximide added early in infection
also blocked IRF7 activity as measured by EMSA (data not shown). These
data suggest that proteins accumulating during the first 3 h of
NDV infection are required for activation of the IRF7 kinase. Since no
serine kinases are encoded by NDV (45), it is likely that viral
proteins are involved either directly or indirectly in activation of a
cellular kinase that targets IRF7. Alternatively, cycloheximide might
block synthesis of a short-lived cellular protein.
No Specific Kinase Inhibitor Blocks IRF7
Phosphorylation--
Cellular kinases potentially involved in IRF
phosphorylation were investigated initially by use of pharmacological
inhibitors. To distinguish a direct effect of inhibition on an IRF
kinase from a possible indirect effect caused by, for instance,
inhibition of necessary viral protein synthesis, pharmacological
inhibitors were added late in infection (5 h post-infection), just
prior to the observed appearance of phosphorylated protein and at a time point at which ongoing protein synthesis was no longer required. IRF7 phosphorylation was blocked by staurosporine (Fig.
3A, lane 4), a broad-spectrum
kinase inhibitor previously shown to block phosphorylation of IRF1 in
virus-infected cells (46). IRF7 phosphorylation was also reduced by the
related inhibitor, K252a (Table I).
However, none of the more specific kinase inhibitors tested were
capable of preventing IRF phosphorylation (Fig. 3A and Table
I). These included inhibitors of PKR (2-amino purine (47)) and of IKK (acetylsalacylic acid and salacylic acid (48)), kinases known to be
activated in response to virus infection and involved in activation of
NF
Another technique used to probe the nature of the IRF kinase involved a
search for agents that might block a virally activated cellular
signaling step, or, alternatively, that might activate IRF7
phosphorylation in the absence of viral infection. We treated cells
with DNA damaging agents, calcium chelators, proteosome inhibitors,
phosphatase inhibitors, and activators of the unfolded protein
response. We also overexpressed a constitutively active form of MKK7,
an activator of the JNK pathway (49, 50). None of these treatments
caused IRF7 phosphorylation in the absence of viral infection nor
prevented IRF7 activation when added to virus-infected cells, allowing
us to rule out involvement of the known stress- and virus-activated
signaling pathways in stimulating IRF7 phosphorylation (Table
I).
Genetic Evidence for Lack of Involvement of PKR and
IKK--
Because both PKR and IKK are known to be activated in
response to viral infection, we sought additional evidence for their apparent lack of involvement in IRF phosphorylation by examining mutant
cell lines lacking these enzyme activities. Embryo fibroblasts were
prepared from mice devoid of the PKR gene due to gene
targeting (26). Induction of type I IFN genes
following NDV infection was assessed as a measure of endogenous IRF
phosphorylation. IFN
Phosphorylation of IRF3 and IRF7 was directly tested in virus-infected,
PKR-null cells. Flag epitope-tagged IRF3 or IRF7 were transfected into PKR-null cells which were subsequently
infected with NDV and labeled with [32P]inorganic
phosphate. Protein extracts were immunoprecipitated and analyzed by
SDS-PAGE and autoradiography. Phosphorylated IRF3 or IRF7 was recovered
from PKR-mutant cells (Fig. 4B), demonstrating the PKR independence of this process. It should be noted that both IRF3
and IRF7 are basally phosphorylated in the absence of viral infection
and become additionally phosphorylated in response to infection, as
indicated by their change in electrophoretic mobility. Neither basal
nor induced phosphorylation was inhibited by the absence of the
PKR gene.
Another enzyme known to be activated in response to virus infection and
required for activation of NF
Several genes have been isolated recently that bear homology to
PKR. One possibility raised by the ability of
PKR-null cells to phosphorylate IRF proteins in response to
virus infection while being deficient in response to dsRNA might be the
existence of PKR-related enzymes that are activated by a
viral component other than dsRNA. To test this notion, we examined
IFN gene expression in response to virus infection of cells
deficient for individual PKR-related genes due to gene
targeting. Cells deficient for PERK, an eIF2 IRF7 Phosphorylation Is Blocked by Vaccinia Virus E3L
Protein--
Viruses maintain numerous strategies to evade host innate
immune defenses, including inhibition of PKR (55). Three such viral
inhibitors that target inhibition of PKR are vaccinia virus E3L and K3L
proteins and adenovirus VA RNA (56-62). To test the potential of these
genes as inhibitors of IRF phosphorylation, cells were co-transfected
with E3L, K3L, or VA expression constructs along with IRF3 or IRF7,
infected with NDV, and assayed for IRF phosphorylation by SDS-PAGE.
Expression of K3L or VA were without effect (Fig.
6A, and data not shown); in
contrast, expression of E3L blocked IRF7 phosphorylation in a
dose-dependent manner (Fig. 6A, top panel).
Coexpression of E3L with IRF7 also blocked its ability to bind DNA
following isolation from virus-infected cells (Fig. 6A, lower
panel), likely a reflection of the lack of phosphorylation.
Coexpression of K3L was without effect in either assay (Fig.
6A).
E3L binds dsRNA and has been thought of as a competitive inhibitor of
PKR that sequesters dsRNA (56, 60) and directly inhibits PKR through
protein-protein interaction following binding to dsRNA (63, 64). We
tested the requirement of dsRNA binding for the inhibition of IRF7
phosphorylation by E3L using a point mutant version of E3L, in which
lysine 167 was converted to alanine (K167A), and is incapable of
binding dsRNA (65). Loss of RNA binding by E3L disrupted its ability to
inhibit IRF7 phosphorylation (Fig. 6B, lane 6). To evaluate
whether direct sequestering of dsRNA might be the sole mechanism of the
inhibitory function of E3L, we tested two additional dsRNA-binding
proteins. Expression of neither DRBP76 (35) nor mammalian Staufen (66)
prevented IRF7 phosphorylation, nor did they block the inhibitory
action of E3L (Fig. 6C and data not shown), despite these
proteins being implicated in regulation of PKR and viral infection (35,
36, 66). Additionally, E3L but not K3L effectively inhibited IRF3 phosphorylation following NDV infection (Fig. 6D).
Therefore, it is likely that E3L directly inhibits a cellular kinase
that targets both IRF3 and IRF7 rather than functioning merely to
sequester dsRNA, although its ability to bind dsRNA is necessary for
this inhibitory function, as it is for inhibition of PKR.
Activation of IFN gene expression is a cellular
response resulting from innate immune recognition of virus infection,
but how cells sense viral replication is only poorly characterized. While production of viral products such as dsRNA play a role in activation of IKK and possibly of JNK (12), the data reported here
suggest that additional signals stimulate phosphorylation of IRF3 and
IRF7, essential events in induction of the protective IFN response.
This evidence is severalfold. First, the known target of dsRNA in
mammalian cells, PKR, was not required for stimulation of IRF3 or -7 phosphorylation. Neither pharmacological inhibitors of PKR nor ablation
of the PKR gene resulted in impaired IRF phosphorylation. Second, while PKR-mutant cells were defective in responses
to dsRNA, they retained responsiveness to viral infection,
demonstrating that infection provided additional signals beyond simply
dsRNA. Finally, protein synthesis during viral infection was required for IRF phosphorylation, and the kinetics of phosphorylation and of the
required protein synthesis correlated well with the major synthesis of
viral proteins. Therefore, it is likely that newly synthesized viral
proteins play a necessary role in activation of the cellular IRF
kinase. A model for regulation of IRF phosphorylation is shown in Fig.
7. Viral infection leads to production of
dsRNA and activation of PKR. Infection also leads to an independent activation event in a protein synthesis-dependent manner,
targeting a novel cellular kinase (X) and leading to IRF
phosphorylation. dsRNA-activated PKR might directly phosphorylate IRF
or might activate the same kinase X, leading to IRF phosphorylation. We cannot rule out the possibility that the required viral protein synthesis induces a secondary product (possibly an RNA) that is the
ultimate activator of the IRF kinase. It is also possible that
synthesis of a cellular protein is necessary during viral infection. In
either case, these events are independent of PKR and its close
homologues PERK, IRE1 (IFN
) gene
expression in virus-infected cells requires phosphorylation-induced
activation of the transcription factors IRF3 and IRF7. However, the
kinase(s) that targets these proteins has not been identified. Using a
combined pharmacological and genetic approach, we found that none of
the kinases tested was responsible for IRF phosphorylation in cells infected with Newcastle disease virus (NDV). Although the
broad-spectrum kinase inhibitor staurosporine potently blocked IRF3 and
-7 phosphorylation, inhibitors for protein kinase C, protein kinase A,
MEK, SAPK, IKK, and protein kinase R (PKR) were without effect. Both
I
B kinase and PKR have been implicated in IFN induction, but cells genetically deficient in I
B kinase, PKR, or the
PKR-related genes PERK, IRE1, or
GCN2 retained the ability to phosphorylate IRF7 and induce
IFN
. Interestingly, PKR mutant cells were defective for
response to double-stranded (ds) RNA but not to virus infection, suggesting that dsRNA is not the only activating viral component. Consistent with this notion, protein synthesis was required for IRF7
phosphorylation in virus-infected cells, and the kinetics of
phosphorylation and viral protein production were similar. Despite
evidence for a lack of involvement of dsRNA and PKR, vaccinia virus E3L
protein, a dsRNA-binding protein capable of inhibiting PKR, was an
effective IRF3 and -7 phosphorylation inhibitor. These results suggest
that a novel cellular protein that is activated by viral products in
addition to dsRNA and is sensitive to E3L inhibition is responsible for
IRF activation and reveal a novel mechanism for the anti-IFN effect of
E3L distinct from its inhibition of PKR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gene and a family of IFN
genes, is
rapidly induced by infection of mammalian cells by a broad spectrum of
viruses (1, 2). Gene induction is rapid, and can be divided into two
distinct phases initiated by induction of IFN
and
IFN
4 expression followed by induction of other members of
the IFN
gene family (3, 4). Differences in the gene
expression profiles of the distinct type I IFN genes can be accounted
for by unique properties of their promoter/enhancer structures.
IFN
expression is controlled by an enhanceosome that
binds three distinct transcription factor complexes in the context of
chromatin-organizing proteins (5). Each of these complexes,
c-jun/ATF2 (AP1), IRF, and NF
B, become active following
protein phosphorylation events induced in response to virus infection.
IFN
promoters are also activated by IRF proteins, and the
distinct DNA binding characteristics and patterns of induction and
activation of IRF3 and IRF7 confer the differential expression of the
IFN
gene family (3, 6-9).
B requires phosphorylation-induced degradation of
its inhibitor, I
B, through the action of the I
B kinase (IKK)
complex composed of IKK
, IKK
, and IKK
/NEMO (10). The catalytic
activity of IKK is activated by a wide variety of inducers, including
viral infection and double-stranded RNA (dsRNA), a common by product of
viral infection (11). Activation of IKK by dsRNA and viral infection
may depend on a second enzyme, protein kinase R (PKR); however, in this
role PKR may function noncatalytically and serve as an adaptor protein
(12), although even this requirement has been recently called into
question (13). The AP1 transcription factor, also required for
IFN
gene induction, is likewise activated by
phosphorylation through the action of c-Jun kinase (JNK), which is also activated in virus-infected cells (12). The third transcription factor complex required for IFN gene induction, composed of IRF3 and/or
IRF7, is also activated by phosphorylation specifically in
virus-infected cells (3, 6, 14-22). However, the kinase responsible
for its activation remains to be identified.
gene expression was still induced in virus-infected
PKR(
/
) and IKK
/
) cells, as well as in
cells deficient for several PKR-related genes. These data
suggest that IRF7 activation can be stimulated by a viral component
other than or in addition to dsRNA, that a novel cellular kinase
distinct from PKR is responsible for its phosphorylation, and that the E3L protein exerts its anti-IFN effect by inhibiting not only PKR but
also the distinct IRF3/7 kinase(s).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) and wild type control fibroblasts were
immortalized by the 3T3 process (30) from mouse embryo fibroblasts
obtained from PKR gene-targeted animals (26), the kind gift
of Joan Durbin (Ohio State University) and John Bell (University of
Ottawa, Canada), and were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum.
Perk(
/
) (31), IRE1
/
(
/
) (32),
GCN2(
/
) embryonic stem cells (33) and wild type control
cells were the kind gift of David Ron (New York University, NY).
GCN2(
/
) teratoma cells were derived from
GCN2(
/
) embryonic stem cells by passage in nude mice and
selection in culture in the presence of G418 (400 µg/ml). NEMO
(IKK
)-deficient 1.3E2 and control 70Z/3 cells (11) were the kind
gift of Gilles Courtois (Pasteur Institute, Paris, France) and were
maintained in RPMI medium supplemented with 10% fetal bovine serum and
50 µM
-mercaptoethanol.
/
) fibroblasts
were transfected with epitope-tagged IRF7 or IRF3 using LipofectAMINE
2000 (Life Technologies, MD). After 24 h, cells were infected with
NDV, and 2 h post-infection were washed twice with
phosphate-buffered saline, incubated in phosphate-free medium for
3 h, and then incubated for 2 h with 2.5 mCi/ml
[32P]orthophosphate. Cells were washed twice and lysed in
whole cell lysis buffer (300 mM NaCl, 50 mM
HEPES, pH 7.6, 1.5 mM MgCl2, 10% glycerol, 1%
Triton X-100, 10 mM
Na4P2O7, 20 mM NaF, 1 mM EGTA, 0.1 mM EDTA, 1 mM
dithiothreitol, 1 mM Na4VO3, and
protease inhibitors). Following cell lysis, the transfected protein was isolated by immunoprecipitation overnight at 4 °C using 1 µg of M2
anti-Flag antibody (Sigma), and the immunoprecipitated protein was
analyzed by 8% SDS-PAGE.
glycerophosphate, 20 mM
p-nitrophenyl phosphate, 1 mM
Na4VO3, 1 mM EDTA, plus protease
inhibitors) in the presence of 10 µCi of [
-32P]ATP
at 30 °C for 30 min, followed by analysis by 10% SDS-PAGE and
autoradiography. Antibody against IKK was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). GST-IRF7 was expressed in bacteria from
the pGEX-2T vector (43). GST-I
B and mutant I
B were kind gifts of
Jan Vilcek (New York University).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Kinetics of IRF phosphorylation.
A, kinetics of IRF7 phosphorylation. 293T cells transfected
with 10 µg of an IRF7 expression construct were infected with NDV
12 h later, and nuclear extracts harvested at the indicated times
post-infection were analyzed for IRF7 phosphorylation by SDS-PAGE and
immunoblotting. B, kinetics of IRF3 phosphorylation. Cells
were transfected with 10 µg of IRF3 and were analyzed as described in
A. C, kinetics of NDV protein accumulation.
Nuclear extracts from NDV-infected 293T cells were analyzed by
immunoblotting for virion proteins. The 0-h time point represents
uninfected cell extract. D, kinetics of phosphorylation of
endogenous IRF3 parallel those for ectopically expressed protein. Whole
cell extracts from mouse 3T3 cells infected with NDV for various times,
as indicated, were analyzed by SDS-PAGE and immunoblotting.
h.p.i., hours post-infection.
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Fig. 2.
Cycloheximide (CHX)
inhibition of IRF7 phosphorylation. 293T cells were transfected
with 10 µg of an IRF7 expression construct, infected with NDV as
indicated, and cycloheximide (75 µg/ml) was added to growth media at
the indicated times (h) post-infection. Lanes 1, 2, and
9 did not receive cycloheximide treatment. Cells were
harvested at 7 h post-infection and nuclear extracts were analyzed
for IRF7 phosphorylation (upper panel) and viral protein
accumulation (lower panel). Lane 1 (lower
panel) represents mock-transfected cells infected with NDV for
7 h.
B. Other agents ineffective in blocking IRF phosphorylation included inhibitors of PKC, PKA, p38, extracellular kinase,
phosphatidylinositol 3-kinase, and tyrosine kinases. The
phosphorylation status of IRF7 correlated with its ability to bind DNA,
as detected by EMSA (Fig. 3A, lower panel). Specificity of
DNA binding was confirmed by antibody and DNA competition reactions
(Fig. 3B) using anti-Flag (lane 3), anti-IRF7
(lane 4), or homologous DNA competitor (lane 5).
IRF3 phosphorylation was also blocked by staurosporine treatment (Fig.
3C, lane 3) but not by any other inhibitors tested, and none
of the pharmacologic agents tested led to IRF phosphorylation in the
absence of infection (data not shown). Phosphorylation of ectopically
expressed IRF3 paralleled that of the endogenous protein whose
phosphorylation was blocked by staurosporine and cycloheximide (Fig.
3C, lanes 6 and 7) but not by genistein, H7, H8,
or wortmanin (lanes 8-11).
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Fig. 3.
IRF phosphorylation is inhibited by
staurosporine. A, 293T cells were transfected with 10 µg of an IRF7 expression construct, infected with NDV, and the
indicated treatment was added to growth media at 5 h
post-infection. Cells were harvested at 7 h post-infection and
nuclear extracts were analyzed for IRF7 by SDS-PAGE (upper
panel) and EMSA (lower panel). B, IRF7-DNA
complex formed in response to NDV infection of transfected 293T cells
(lane 2) was specifically competed by antibody against an
epitope tag (anti-Flag, lane 3) or against native IRF7
(lane 4) and by excess binding site competitor (lane
5). C, 293T cells (lanes 1-4) were
transfected with 10 µg of an IRF3 expression construct and treated as
described in A. Inhibition by staurosporine (S)
and not by genistein (G) is shown. 3T3 cells (lanes
5-11) were infected with NDV without (lane 5) or in
the presence of staurosporine (lane 6), cycloheximide
(lane 7), genistein (lane 8), H7 (lane
9), H8 (lane 10), or wortmanin (lane 11),
and then analyzed for endogenous IRF3 phosphorylation. All inhibitors
were added 5 h post-infection, with the exception of cycloheximide
which was added simultaneously with virus.
Inhibitors tested
4 and IFN
expression,
dependent on IRF3 phosphorylation, and expression of additional members
of the IFN
gene family (non-IFN
4 genes), dependent on IRF7 phosphorylation, were detected in approximately equal
abundance in both wild type and PKR-null cells (Fig.
4A). Similar results were
obtained using PKR-null cells derived from a different mouse
strain that sustained a distinct mutation (27), corroborating the
results obtained with inhibitors (data not shown). However,
IFN
induction in response to dsRNA as opposed to virus infection was impaired in PKR-null cells (data not shown),
as previously reported (25).
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Fig. 4.
PKR and IKK are not responsible for
IFN gene induction or IRF
phosphorylation. A, PKR(
/
) and wild type
cells were infected with NDV for 9 h, as indicated, and expression
of IFN genes was measured by RT-PCR, as indicated. B,
PKR(
/
) mouse fibroblasts were transfected with 20 µg
of IRF3 or IRF7 expression constructs, as indicated, labeled with
[32P]orthophosphate, and IRF proteins were recovered by
immunoprecipitation with anti-Flag antibodies and analyzed by SDS-PAGE
and autoradiography. Positions of basal and activated IRF isoforms are
indicated. C, wild type and NEMO
(IKK
)-deficient cells were infected with NDV as indicated and
assayed for IFN
and IFN
gene expression by
RT-PCR, as indicated. D, activated IKK was recovered from
interleukin 1 (IL1)-treated (lane 1) or
NDV-infected mouse fibroblasts (lanes 2-4) by
immunoprecipitation and incubated with GST-I
B (lanes 1 and 2), mutant I
B (lane 3), or GST-IRF7
(lane 4) in the presence of [32P]ATP, followed
by analysis by SDS-PAGE and autoradiography. Gpdh,
glyceraldehyde-3-phosphate dehydrogenase.
B is IKK (12). To confirm the inhibitor
studies that suggested the lack of its involvement in IRF
phosphorylation, we took advantage of a mutant cell line lacking the
IKK
/NEMO subunit that is therefore completely defective for IKK
activity (11). Consistent with a lack of requirement for IKK activity
for IRF phosphorylation and no requirement of NF
B function for
IFN
gene transcription, IFN
gene expression was induced normally in NDV-infected mutant cells (Fig. 4C).
As expected, IFN
induction was impaired (Fig. 4C,
lane 4), due to its demonstrated requirement for NF
B (51). The
modest reduction in IFN
gene induction is likely due to
the absence of IFN
-dependent positive feedback (3, 52).
Finally, we asked whether IKK was capable of phosphorylating IRF7, even
though it was not required. Activated IKK was immunoprecipitated from
extracts of virus-infected cells and tested for its ability to
phosphorylate IRF7 in vitro. Although phosphorylation of
I
B was readily detected with virus-activated IKK (Fig. 4D,
lane 2) to levels similar to that achieved with IKK isolated from
interleukin 1
-treated cells (Fig. 4D, lane 1), no
phosphorylation of IRF7 was observed using the virus-activated kinase
(Fig. 4D, lane 4). A mutant form of I
B in which serine residues 23 and 25 were changed to alanine (I
Bm) served as
specificity control for activation of IKK (Fig. 4D, lane
3).
kinase (31),
for IRE1
and -
, enzymes involved in activation of JNK
(32), or for mouse GCN2, another eIF2
kinase (33, 53,
54), were all capable of producing IFN
,
IFN
, and the non-IFN
4 subset of
IFN
genes in response to viral infection (Fig.
5). Therefore, none of these PKR-related
kinases is individually required for IFN gene induction and
is unlikely to be a required IRF kinase.
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Fig. 5.
PKR related kinases are not required for
IFN gene induction by NDV infection. Cells from
the indicated mouse cell lines were infected with NDV, and total
cellular RNA prepared at the indicated hours post-infection
(h.p.i.) was analyzed for IFN gene induction by
RT-PCR. 0 h represents uninfected cells. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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Fig. 6.
IRF7 phosphorylation is inhibited by
E3L. A, dose-dependent inhibition of IRF7
phosphorylation by E3L. 293T cells were transfected with an IRF7
expression vector and the indicated amount (µg) of E3L or K3L
expression constructs and infected with NDV, as indicated. Nuclear
extracts were analyzed for activation of IRF7 by SDS-PAGE (upper
panel). Nuclear extracts from uninfected (lane 1) or
NDV-infected cells (lanes 2-8) that had been co-transfected
with IRF7 and the indicated amounts of E3L or K3L were analyzed by EMSA
(lower panel). B, dsRNA binding is necessary but
not sufficient to inhibit IRF7 phosphorylation. 293T cells were
transfected with 10 µg of the following expression constructs: IRF7
(all lanes), E3L (lanes 3 and 4 and 11 and 12) and E3L-K167A (lanes 5 and 6),
infected with NDV, as indicated, and nuclear extracts harvested 7 h post-infection were analyzed for IRF7 (upper panel), E3L
(lower panel). Lanes 1-3 of the lower left
panel correspond to the NDV infected lanes in the upper
panel (lanes, 2, 4, and 6). C,
DRBP76 does not inhibit IRF7 phosphorylation. Cells were analyzed as
described for panel B following transfection with
Flag-tagged DRBP76, as indicated. D, IRF3 phosphorylation is
inhibited by E3L. 293T cells were transfected with 10 µg of an IRF3
expression vector and 10 µg of E3L (lanes 3 and
4) or K3L (lanes 5 and 6), infected
with NDV, as indicated, and nuclear extracts were analyzed for IRF3 by
immunoblotting.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
, and GCN2.
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[in a new window]
Fig. 7.
Proposed model of IRF3 and -7 phosphorylation. Virus-induced dsRNA activates PKR while a second,
protein synthesis-dependent step activates an unknown
kinase X which phosphorylates IRF3 and -7. Activated PKR either
directly or indirectly through kinase X also leads to IRF3 and IRF7
phosphorylation. E3L protein blocks both PKR and kinase X in a
dsRNA-dependent fashion.
Our data are consistent with a single kinase targeting both IRF3 and IRF7. By studying transfected cells, we avoided the requirement for IRF7 induction that normally limits its action to a secondary wave of IFN production (3). Under these experimental conditions, both IRF3 and IRF7 were phosphorylated with the same kinetics, and endogenous IRF3 was phosphorylated with similar kinetics, validating the transfection system. Moreover, IRF3 and IRF7 phosphorylation showed the same sensitivity and resistance to a panel of pharmacological agents. Indeed, only the broad-spectrum kinase inhibitors staurosporine and to a lesser extent K252a were effective IRF kinase inhibitors. Although staurosporine was first characterized as an inhibitor of PKC, other inhibitors of PKC or down-regulation of PKC protein by prolonged exposure to phorbol ester were ineffective in preventing IRF phosphorylation. Other cellular stress responses, such as induction of the unfolded protein response or stimulation of MAP kinase cascades, did not appear to play a role in the IRF response to virus, suggesting that a novel cellular signaling pathway allows the innate immune system to recognize viral infection. Hiscott and colleagues have recently reached a similar conclusion (67).
Despite the lack of evidence for dsRNA and PKR involvement in IRF
phosphorylation by virus, we found that the vaccinia virus PKR
inhibitor E3L, but not K3L, was an effective IRF phosphorylation inhibitor (Fig. 6). Moreover, the ability of E3L to bind RNA was necessary for its anti-IRF action. These data suggest that dsRNA is one
and possibly an essential aspect of the recognition of viral infection.
However, while the action of dsRNA alone is dependent on the activity
of PKR, the action of dsRNA in the context of other components of viral
replication acted in a PKR-independent manner, although it remained
sensitive to E3L inhibition. These findings suggest that an enzyme
related to PKR could be a likely candidate for the virus-activated IRF
kinase; however, none of the known PKR-related genes tested
was individually required, including PERK, IRE1,
IRE1
, and GCN2. It remains a possibility that
another PKR-related enzyme, for instance, HRI (68) or an as yet to be
identified related kinase, is the IRF kinase. Alternatively, a
combination of these genes operating in a redundant fashion could have
obscured the requirement for any one individual enzyme.
Whether IRF phosphorylation depends on a novel gene or a combination of
PKR-related enzymes, it is likely that E3L protein directly inhibits
the catalytic function of such an enzyme, as it does PKR. Inhibition by
E3L likely required the presence of dsRNA, possibly serving to activate
the inhibitory function of E3L. However, sequestering of dsRNA alone is
probably insufficient to inhibit IRF phosphorylation since other
RNA-binding proteins were unable to mimic this action of E3L.
Interestingly, the non-RNA binding amino terminus of E3L is required
for full inhibition of PKR (63, 64) and must interact with PKR, not
simply sequester its activator dsRNA to block catalytic activity. The
amino terminus of E3L also induces multimerization (69) that may be
necessary for its function. In contrast, the K3L protein inhibits PKR
catalysis by acting as a psuedo-substrate, mimicking the PKR substrate
eIF2 and blocking the catalytic site of the enzyme. The finding that K3L was not an effective inhibitor of the IRF3/7 kinase suggests a
substrate specificity distinct from PKR. The ability of these two viral
proteins to target independent aspects of the IFN pathway may explain
why both genes have been maintained in the vaccinia virus genome.
The action of E3L to inhibit IRF3 and IRF7 phosphorylation in response
to NDV infection is reminiscent of the recently reported action of the
influenza viral protein NS1 to inhibit IRF3 nuclear translocation (70).
Indeed, we also observed inhibition of IRF7 phosphorylation in the
presence of NS1 protein (Table I). We would speculate that both E3L and
NS1, and possibly additional, virally-encoded inhibitors of IFN
induction, function by binding and inhibiting a cellular IRF protein
kinase which is activated in response to dsRNA plus additional viral components.
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ACKNOWLEDGEMENTS |
---|
We thank David Ron and Heather Harding (New
York University School of Medicine) for gifts of cell lines and for
many helpful discussions, Joan Durbin (Ohio State University) and John
Bell (University of Ottawa) for PKR(/
) mouse embryo
fibroblasts, Gilles Courtois (Institut Pasteur, Paris, France) for
NEMO-deficient cells, Jonathan Yewdell (National Institutes of Health,
Bethesda, MD) for anti-E3L hybridoma cells, Robert Schneider, Jan
Vilcek, Madrid, Spain), Ganes Sen (Cleveland Clinic, OH), Bernard
Jacobs (University of Arizona), and Stuart Shuman (Memorial
Sloan-Kettering, New York) for gifts of plasmids and reagents and for
helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants AI28900, AI46503, and AI46954 and by a fellowship from the Arthritis Foundation.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.
§ Present address: Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, Cedex 15, France.
To whom correspondence should be addressed: Dept. of
Pathology, MSB 556, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-8192; Fax: 212-263-8211; E-mail: levyd01@med.nyu.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M008717200
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ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
IKK, IB kinase;
dsRNA, double-stranded RNA, PKR, protein
kinase R;
NDV, Newcastle disease virus;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
EMSA, electrophoretic mobility
shift assay;
PAGE, polyacrylamide gel electrophoresis;
JNK, c-Jun
NH2 kinase;
GST, glutathione S-transferase.
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