From the Terry Fox Molecular Oncology Group, Lady
Davis Institute for Medical Research, and Departments of
§§ Microbiology & Immunology and
§ Medicine, McGill University, Montreal, H3T 1E2 Canada, the
** National Public Health Institute, Department of Virology,
Mannerheimintie 166, FIN-00300 Helsinki, Finland, and the
Istituto Superiore di Sanita, 00161 Rome, Italy
Received for publication, August 25, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Infection of host cells by viruses leads to the
activation of multiple signaling pathways, resulting in the expression
of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to
virus is interferon regulatory factor-3 (IRF-3) which is
post-translationally modified as a result of virus infection.
Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine
residues in the C-terminal region leads to dimerization, cytoplasmic to
nuclear translocation, association with the p300/CBP coactivator, and
stimulation of DNA binding and transcriptional activities. We now
demonstrate that IRF-3 is a phosphoprotein that is uniquely activated
via virus-dependent C-terminal phosphorylation.
Paramyxoviridae including measles virus and rhabdoviridae, vesicular
stomatitis virus, are potent inducers of a unique virus-activated
kinase activity. In contrast, stress inducers, growth factors,
DNA-damaging agents, and cytokines do not induce C-terminal IRF-3
phosphorylation, translocation or transactivation, but rather activate
a MAPKKK-related signaling pathway that results in N-terminal IRF-3
phosphorylation. The failure of numerous well characterized
pharmacological inhibitors to abrogate virus-induced IRF-3
phosphorylation suggests the involvement of a novel kinase activity in
IRF-3 regulation by viruses.
Virus infection of mammalian cells triggers multiple signal
transduction cascades involved in the activation of a diverse set of
immunoregulatory genes and proteins that together create the antiviral
state, an intracellular environment that antagonizes virus replication.
The type I interferon (IFN)1
family is essential to the development of the antiviral state and the
IFN gene family represents one of the best characterized models of
virus inducible gene activation (1). Once produced, these secreted
proteins induce gene expression in neighboring cells through cell
surface cytokine receptors and the JAK-STAT signaling pathways. STAT1/2
heterodimers, in conjunction with interferon-stimulated gene factor
3 The pathways involved in NF- The pathway(s) regulating IRF-3 phosphorylation and activation are also
the focus of considerable investigation. IRF-3 belongs to the family of
IRFs which include IRF-1 to IRF-7, interferon consensus
sequence-binding protein (IRF-8), and interferon-stimulated gene factor
3 Previous studies have demonstrated that treatment with dsRNA was
sufficient to trigger the nuclear accumulation of IRF-3 (17) and the
formation of an IRF-3 containing DNA binding complex (3, 16). Recent
studies also suggest that phosphorylation and activation of IRF-3 is
not restricted to viral infection, since LPS, DNA-damaging and
stress-inducing agents all stimulate nuclear accumulation of IRF-3, DNA
binding activity, and transactivation (23-25). Using a variety of
pharmacological and molecular approaches, we now demonstrate that IRF-3
is uniquely activated via C-terminal virus-dependent phosphorylation. In addition to Sendai virus and Newcastle disease virus (NDV), measles virus (MeV) and vesicular stomatitis virus (VSV)
are also identified as potent inducers of VAK activity. In contrast,
exposure of cells to stress inducers, growth factors, DNA-damaging
agents, and cytokines including doxorubicin and TNF- Reagents--
PDTC, sorbitol, LPS, and ribavirin were purchased
from Sigma and dissolved in distilled water or phosphate-buffered
saline. All other pharmacological inhibitors were from Calbiochem or
Biomol and resuspended in dimethyl sulfoxide or ethanol. Recombinant macrophage inflammatory protein 1 Plasmid Constructions and Mutagenesis--
CMVBL-IRF-3wt, -IRF-3
5A, -IRF-3 5D; pFlag-IRF-3 1-240, the reporter plasmids containing two
PRD II sites, pGL3-P2(2)tk-LUC and the IFN Cell Culture--
The rtTA-Jurkat, rtTA-Jurkat IRF-3wt, and
rtTA-Jurkat IRF-3-5D were described previously (27). Human embryonic
kidney (HEK) 293 cells and HeLa cells were grown in Transfections and Luciferase Assays--
All transfections were
carried out on subconfluent HEK 293 cells grown in 60-mm Petri dishes
or 24-well plates (luciferase assay). 5 µg of DNA constructs (per
60-mm dish) or 10 ng of pRLTK reporter (Renilla luciferase for internal
control), 100 ng of pGL3 reporter (firefly luciferase, experimental
reporter) and 250-500 ng of expression plasmids (24-well plate) were
introduced into target cells by calcium phosphate coprecipitation
method. At 24 h post-transfection, cells were infected with Sendai
virus for 12 h (80 hemagglutinating units (HAU)/ml) or treated
with the different inducers for the indicated times. At 36 h,
cells were collected, washed in ice-cold phosphate-buffered saline and assayed for reporter gene activities (Promega); whole cell extracts (WCE) were prepared in Nonidet P-40 lysis buffer (50 mM
Tris, pH 7.4, 150 mM NaCl, 30 mM NaF, 5 mM EDTA, 10% glycerol, 1.0 mM Na3VO4, 40 mM Immunoblot Analysis--
To verify the state of phosphorylation
of IRF-3 and to confirm the expression of the transgenes, WCE (30-60
µg) were subjected to electrophoresis on 7.5, 10, or 12% acrylamide
gels. Proteins were electrophoretically transferred to Hybond-C
nitrocellulose membranes (Amersham Pharmacia Biotech, Inc.) in
25 mM Tris, 192 mM glycine, and 20% methanol.
The membranes were blocked in TBS containing 5% nonfat dry milk and
0.1% Tween 20 for 1 h at 25 °C before incubation for 1.5 h at 25 °C with anti-IRF-3 (a kind gift from Dr. Paula Pitha),
anti-
For co-precipitation studies, WCE (200-1000 µg) were incubated with
1 µg of anti-CBP antibody A-22 (Santa Cruz) cross-linked to 30 µl
of protein A-Sepharose beads for 3 h at 4 °C (Amersham Pharmacia Biotech). The beads were washed five times with Nonidet P-40
lysis buffer, resuspended in denaturating sample buffer, and the eluted
IRF-3 proteins associated with CBP were analyzed by immunoblotting.
Cytoplasmic and Nuclear Extracts Preparations--
To examine
subcellular localization of the IRF-3 protein, nuclear and cytoplasmic
extracts were prepared from HeLa cells after treatment with different
inducers for 8 h. The cells were washed in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM PMSF) and were resuspended in buffer A containing 0.1%
Nonidet P-40. The cells were then chilled on ice for 10 min before
centrifugation at 10,000 × g. This procedure was
performed twice to remove cytoplasmic contaminants in the nuclear
extracts. After centrifugation, supernatants were kept as cytoplasmic
extracts. The pellet were then resuspended in buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 5 µg/ml of
each leupeptin, pepstatin, aprotinin, spermine, and spermidine).
Samples were incubated on ice for 15 min before being centrifuged at
10,000 × g. Nuclear extract supernatants were diluted
with buffer C (20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 50 mM KCl, 0.5 mM dithiothreitol, 0.5 mM PMSF). Equivalent amounts of nuclear
and cytoplasmic extracts (20 µg) were subjected to SDS-PAGE in a 10% polyacrylamide gel. Proteins were electrophoretically transferred to
Hybond-C nitrocellulose membranes which were probed with IRF-3 antiboby
as described earlier.
Phosphatase Treatment--
HEK 293 cells were left untransfected
or transfected with expression plasmids encoding wild-type or mutated
forms of IRF-3. At 36 h post-transfection, cells were stimulated
and WCE were prepared. Endogenous IRF-3 (400 µg) or overexpressed
IRF-3 (150 µg) proteins were immunoprecipitated with anti-IRF-3
antibody (Santa Cruz) or anti-Flag antibody (Sigma) cross-linked to 30 µl of protein G-Sepharose beads for 4 h at 4 °C. Precipitates were washed two times in Nonidet P-40 lysis buffer followed by two
washes in phosphatase buffer (50 mM Tris, pH 9.0, 1 mM MgCl2, 0.1 mM ZnCl2,
1 mM spermidine, 0.5 mM PMSF, 5 µg/ml
aprotinin, and 5 µg/ml leupeptin). The phosphatase treatment was
started by resuspending the beads in a total volume of 40 µl of
phosphatase assay buffer containing 5 units of calf intestine alkaline
phosphatase (CIP; Amersham Pharmacia Biotech) in the absence or
presence of a phosphatase inhibitor mixture containing (final
concentration) 10 mM NaF, 1.5 mM
Na2MoO4, 1 mM Multiple Forms of IRF-3 Phosphoprotein--
C-terminal
phosphorylation of IRF-3 following paramyxovirus infection is a
prerequisite for its nuclear translocation, association with CBP/p300
co-activators, and transcriptional activation (13, 16, 17, 28). VAK
activity is relatively easy to detect in extracts from virus-infected
cells, since phosphorylated IRF-3 migrates slower in SDS-PAGE than
nonphosphorylated IRF-3 (13, 16, 17), a phenomenon observed with many
phosphoproteins. To characterize the different forms of phosphorylated
IRF-3 in virus-infected cells, IRF-3 specific immunoblotting was used
to reveal two forms of IRF-3 (designated forms I and II) in uninfected HEK 293, U937 and Jurkat cells (Fig.
1B, lanes 1, 3, and
7). These forms were also present in human epithelial HeLa
cells, human bronchial epithelial A549 cells, primary human monocytes
(see Figs. 3 and 6) and freshly isolated primary B cells (data not shown). Sendai virus infection resulted in the appearance of two slowly
migrating forms of IRF-3 (forms III and IV) in HEK 293, U937, and IRF-3
expressing Jurkat cells (Fig. 1B, lanes 2, 4, 5, 6, and 8). Forms III and IV represent IRF-3
phosphorylated at a cluster of serines near the C-terminal end of the
protein (Ref. 13 and see Fig. 4C). In addition, a net
decrease in the amount of IRF-3 was observed between 4 and 12 h
after virus infection of U937 cells, supporting the idea that
C-terminal phosphorylated IRF-3 is subject to proteasome-mediated
degradation (13). Overexpression of the constitutively active form of
IRF-3(5D) (13, 19, 26, 27) in Jurkat cells demonstrated that the
phosphomimetic form migrated slower in SDS-PAGE than endogenous IRF-3
protein, at a position similar to form IV observed in cells infected
with Sendai virus (Fig. 1B, lane 9). This initial
experiment, while largely confirming previous observations,
nevertheless clearly demonstrates that multiple forms of IRF-3
phosphoprotein exist in unstimulated and virus-infected cells.
Phosphatase treatment of immunoprecipitated IRF-3 isolated from cells
overexpressing IRF-3wt revealed that form II was also a phosphoprotein
(Fig. 1C, lanes 4-6). Phosphatase treatment resulted in the
disappearance of form II from the extract and an increase in IRF-3 form
I (Fig. 1C, compare lanes 4 and 5), an
effect that was blocked by addition of phosphatase inhibitors (Fig.
1C, lane 6). Interestingly, IRF-3(5A) and IRF-3(5D), in
which the five phosphoacceptor sites in the C terminus were mutated to
alanine (5A) and aspartic acid (5D) (see Fig. 1A), were
still expressed as two forms, form II (5A, lane 7) and form
V (Fig. 1C, 5D, lane 10) (see
also Fig. 1B, lane 9). These forms remained sensitive to
phosphatase treatment (Fig. 1C, lanes 8 and 11)
but were present when phosphatase inhibitors were used (lanes
9 and 12). Also, when endogenous IRF-3 was
immunoprecipitated from Sendai virus-infected HEK 293, forms III and IV
were readily detected (Fig. 1C, lane 13); CIP treatment
resulted in the conversion of forms III and IV to forms I and II (Fig.
1C, lane 14), an effect that was also blocked by phosphatase
inhibitors (Fig. 1C, lane 15). Based on these preliminary
observations, it appeared that multiple forms of IRF-3 phosphoprotein
could be detected and basal IRF-3 phosphorylation, represented by forms
II and V (for IRF-3(5D)), did not occur at the C-terminal
phosphoacceptor sites implicated in IRF-3 activation.
Pharmacological Inhibitors Fail to Block VAK Activity--
In the
effort to identify the pathway(s) activated by viral infection and
implicated in IRF-3 phosphorylation, the effect of well characterized
pharmacological inhibitors on IRF-3 phosphorylation following Sendai
virus infection was examined (Fig. 2 and
Table I). Use of specific pharmacological
inhibitors that targetted MEK1/2 (PD98059), p38 Activation of IRF-3 Is Restricted to Viral Infection--
The
antagonizing effect of ribavirin and UV treatment on
virus-dependent IRF-3 activation (Fig. 2, B and
C) indicated that C-terminal phosphorylation may be specific
to virus infection. Viruses from different families were tested for
their capacity to induce IRF-3 phosphorylation and activation. Two
paramyxoviridae family members, MeV and NDV, and one rhabdoviridae
family member, VSV, were also able to induce the generation of forms
III and IV in HEK 293 cells, primary monocytes and human bronchial
epithelial A549 cells, respectively (Fig.
3A, lanes 2, 4, and
7). These viruses resulted in a
phosphorylation-dependent degradation of IRF-3 which was no
longer detected in primary monocytes after infection with NDV for
18 h (Fig. 3A, lane 5, and data not shown). Induction of IRF-3 forms III and IV by MeV infection also resulted in
transactivation of IFN- Stress Inducers, DNA Damaging Agents, Growth Factors, and NF-
DNA damaging agents activate the classical stress pathway MKK4/SEK1 and
JNK (37-39); furthermore, the catalytic activity of MKK4/SEK1 is
regulated by MAPKKK family members of which MEKK1 is the best described
member (40). Anisomycin, epidermal growth factor, and hyperosmolarity
are also good inducers of MEKK1 activity (data not shown and Refs.
41-43). Therefore, the effect of overexpressing MEKK1 on IRF-3
phosphorylation was examined. Fig. 4B demonstrates that
Flag-tagged IRF-3 was expressed as nonphosphorylated form I and
phosphorylated form II (Fig. 4B, lane 1). Importantly,
coexpression of MAPKKKs MEKK1 and Cot, a member of the MAPKKK family
recently implicated in NF-
Partial mapping of the region of IRF-3 phosphorylated by these agents
or by MAPKKKs overexpression revealed that phosphorylation did not
occur in the C-terminal region (Fig. 4C). Overexpression of
IRF3wt showed the accumulation of form II following sorbitol treatment
or when MEKK1 and Cot were co-transfected (Fig. 4C, lanes 8, 12, and 16). However, when the Ser-Thr cluster at aa 396-405 was mutated to Ala (5A), the shift from form I to form II
still occurred under the same conditions (Fig. 4C, lanes 9, 14, and 17). Overexpression of IRF-3wt and 5A showed
that the cluster of serine residues in the C-terminal region was
essential for Sendai virus-induced generation of forms III and IV (Fig. 4C, compare lanes 4 and 6). In
addition, no accumulation of form II was observed in Sendai
virus-infected cells overexpressing IRF-3 (5A) (Fig. 4C, lane
6). Therefore an independent pathway leading to IRF-3
phosphorylation, distinct from the virus inducible C-terminal specific
pathway, appears to be stimulated by stress inducers, DNA-damaging
agents, and growth factors.
To further pinpoint the region of IRF-3 targeted for phosphorylation by
stress inducers and DNA damaging agents, a series of IRF-3 deletion
mutants were evaluated. As illustrated in Fig. 5A, truncation of full-length
IRF-3 to a protein of 240 or 198 aa did not alter the generation of
forms I and II (Fig. 5A, lanes 4-6); however, truncation to
a protein of 186 aa resulted in a single form of IRF-3 (Fig. 5A,
lane 3), indicating that the modification occurred between aa
186-198. With IRF-3-(1-198), anisomycin resulted in the conversion of
form I to form II (Fig. 5B, compare lanes 1 and
13); CIP treatment reverted form II to form I in a manner that was sensitive to phosphatase inhibitors (Fig. 5B, lanes
14 and 15). The 150, 174, and 186 aa IRF-3 truncations
were expressed as a single form in both control and anisomycin-treated
cells and were insensitive to phosphatase (Fig. 5B, lanes
4-12 and 16-24). As shown above for full-length
IRF-3, stress inducers, DNA damaging agents, and growth factors such as
doxorubicin and PMA as well as MEKK1 and Cot1 overexpression resulted
in the complete or partial conversion of Flag-tagged IRF-3-(1-198)
from form I to form II (Fig. 5C), thus indicating that the
phosphorylation site was located between aa 186 and 198. Analysis of
this region of IRF-3 revealed a single potential site of Ser
phosphorylation located within the sequence
186GPSENPLKRLLVP198. In addition, Sendai virus
infection did not induce accumulation of form II (Fig. 5C, lane
2, and Fig. 4C, lanes 5 and 6), suggesting that the modification of IRF-3 by stress inducers and DNA-damaging agent was not used by virus to induce the activated forms of IRF-3 (form III and IV).
N-terminal Phosphorylation Does Not Alter IRF-3 Function--
To
examine the functional consequences of N-terminal phosphorylation on
IRF-3 activity, cells were stimulated with stress inducers and
evaluated for IRF-3 functions such as CBP association, nuclear
accumulation of IRF-3, DNA binding, and transactivation activity. Fig.
6A shows that PMA,
doxorubicin, stress inducers, such as anisomycin, sorbitol, and NaCl,
and TNF-
Since association of IRF-3 with CBP coactivator is a critical step in
IRF-3 activation (13, 16, 17, 28), the relationship between the
conversion of form I to form II and association with CBP coactivator
was evaluated. Co-immunoprecipitation analysis demonstrated that in
HeLa, HEK 293, and U937 cells, the association between IRF-3 and CBP
was only detected in Sendai-infected cells when forms III and IV are
present (Fig. 6B, lane 2); similarly when cytoplasmic and
nuclear partitioning was evaluated, only virus-induced IRF-3
translocated from the cytoplasm to the nucleus of HeLa cells (Fig.
6C, lanes 2 and 7).
Next, the effect of DNA-damaging and stress-inducing agents on the
transactivating potential of IRF-3 was measured using a reporter gene
assay with the IRF-3 responsive In the present study, we describe a series of pharmacological and
molecular experiments designed to further characterize the signaling
pathway(s) leading to IRF-3 phosphorylation and activation following
virus infection or treatment with a variety of activating agents. IRF-3
phosphoprotein exists as two forms in uninfected cells: form I
represents non-phosphorylated IRF-3, while form II represents a basally
phosphorylated form of IRF-3 that is sensitive to phosphatase
treatment. Mapping studies using IRF-3 deletions and point mutations
demonstrate that phosphorylation of form II does not occur within the
previously characterized cluster of serine residues at the C terminus
of IRF-3 (13, 17). Rather, the phosphoacceptor site involved in the
generation of form II appears to map to the N-terminal domain of IRF-3
between aa 186 and 198. Treatment with stress inducers, DNA-damaging
agents, cytokines, and growth factors, does not induce C-terminal IRF-3 phosphorylation, translocation, or transactivation but rather activates
a MAPKKK-related signaling pathway that increases the proportion of
N-terminal phosphorylated IRF-3 resulting in the accumulation of form
II. Following viral infection, two additional slowly migrating forms of
IRF-3 are detected, designated form III and IV, that are sensitive to
phosphatase treatment and represent C-terminal phosphorylation of
IRF-3. Only forms III and IV translocate to the nucleus of
virus-infected cells, and only C-terminal phosphorylated IRF-3
possesses DNA binding potential, CBP coactivator association, and
transcriptional activity. Several well characterized, specific pharmacological inhibitors failed to block virus-induced C-terminal phosphorylation, thus apparently ruling out many known signaling pathways in the virus activation cascade. Furthermore, in
vitro kinase assays demonstrated that extracellular-activated
kinases (ERK 1/2), JNK, p38 The results of this study contradict a number of recent investigations
demonstrating that stress inducers and DNA damaging agents functionally
activate IRF-3. Navarro and David (24) reported that LPS treatment of
human U373 astrocytoma cells resulted in IRF-3 activation, via a
Toll-receptor and p38 dependent pathway. The authors demonstrated
nuclear translocation and DNA binding of IRF-3 but did not examine the
phosphorylation state of IRF-3 or the functional activity of the
LPS-induced complex. Also the IRF-3·DNA complex that was identified
migrated rapidly in EMSA at a position consistent with a complex that
did not include CBP/p300 coactivator. The functionally active complex
contains minimally IRF-3, CBP/p300, and DNA, resulting in a high
molecular weight virus-induced complex (45) or virus-activated factor
(3). In light of the present findings, an interpretation consistent with these observations is that LPS-induced IRF-3 phosphorylation occurs in the N-terminal domain. In U373 cells, LPS appears to be
sufficient to induce translocation of IRF-3 into the nucleus followed
by enhanced IRF-3 DNA binding (24). However, because of the absence of
C-terminal phosphorylation, IRF-3 was unable to engage CBP/p300
coactivator. Furthermore, in our hands with several cell types, LPS was
unable to induce functional IRF-3 activation (data not shown).
Kim et al. (23, 46) in a series of recent papers,
demonstrated that stress inducers and genotoxic agents such as DNA
damaging agents doxorubicin and UV radiation stimulated IRF-3 (and
IRF-7) phosphorylation, nuclear translocation, CBP association, and
transcriptional activation of an IRF-3 responsive promoter. These
experiments raise the exciting possibility that IRF-3 activation may be
central to the innate host response to environmental stress. However, the analysis of IRF-3 phosphorylation by Kim et al. (23, 46) was not resolved sufficiently to delineate the different
IRF-3-phosphorylated forms. Furthermore, the construct used to measure
IRF-3 functional activity consisted of an artificial construct
containing five Gal4-binding sites to measure the activity of a
Gal4-IRF-3 fusion construct. As detailed in the present manuscript, DNA
damaging agents did stimulate IRF-3 phosphorylation at the N-terminal
site but failed to induce nuclear accumulation through CBP association or transcriptional activation of a natural IRF-3 responsive
promoter-RANTES, even using the identical HeLa cell model. At this
stage, we believe that overexpression of IRF-3 coupled with a sensitive
but artificial transcriptional readout may lead to IRF-3 activation in
response to genotoxic stress.
Interestingly, a small molecule CG18 that stimulates MEKK1 activity was
used to activate the stress-mediated signaling pathway and was shown to
stimulate the formation of the IFN- The IRF-3 function regulated by N-terminal phosphorylation remains to
be elucidated. However, based on the present study, several scenarios
are possible. N-terminal phosphorylation by the stress-induced pathway
may alter IRF-3 conformation, thus making the C-terminal Ser-Thr
cluster more accessible to VAK (Fig. 7,
pathway 1). This possibility was also proposed by Kim
et al. (25). The two-step mechanism is, however,
questionable since viral infection did not induce N-terminal
phosphorylation of IRF-3 (Figs. 4C and 5C),
indicating that VAK activity does not require this modification to
activate IRF-3. Another possibility is that N-terminal phosphorylation
may control IRF-3 activity at a step preceding nuclear translocation,
such as relief of autoinhibition or dimerization (Fig. 7, pathway
2). Finally, the possibility exists also that N-terminal
phosphorylation has no major effect on IRF-3 activity as a
transcription factor, but may rather be involved in a distinct function
of IRF-3 based on the observation that IRF-3 interacts with regulatory
proteins that are not involved in transcription control (data not
shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
bind to interferon-stimulated response elements found in
numerous IFN-induced genes such as 2'-5' oligoadenylate synthase and
the double stranded RNA (dsRNA) activated kinase (PKR), resulting in
the induction of proteins which impair viral gene expression and
replication (1). Molecular regulation of IFN gene transcription is
tightly regulated by extra- and intracellular signals induced at the
site of infection. One of the best characterized models of such
regulation is the virus-inducible promoter/enhancer of the
IFN-
gene (2-4). This promoter includes an overlapping
set of regulatory elements designated positive regulatory domains
(PRDs) I to IV, which interact with several signal-responsive
transcription factors including NF-
B (p50-p65), ATF-2/c-Jun
heterodimers, and interferon regulatory factors (IRF) that bind to PRD
II, PRD IV, and PRD I-III, respectively. Together with the
chromatin-associated HMG I(Y) proteins, these transcription factors
form a stereospecific transcriptional enhancer complex, termed the
enhanceosome (2-4) that stimulates the high level, transient
activation of IFN-
transcription.
B and ATF-2/c-Jun activation have been
well characterized. Following viral infection, treatment with
proinflamatory stimuli like tumor necrosis factor (TNF)-
, interleukin-1, (IL-1), or exposure to dsRNA, these transcription factors are activated through stimulation of distinct kinase cascades. In unstimulated cells, the NF-
B factors are retained in the
cytoplasm in association with inhibitory subunits, I
Bs;
virus-induced phosphorylation at conserved N-terminal residues is
accomplished by the I
B kinase (IKK) complex. Phosphorylation
triggers a signal that induces ubiquitin-dependent
degradation of I
B, and subsequent nuclear translocation of the
NF-
B dimers (reviewed in Ref. 5). The rate-limiting step in this
process is the activation of IKK which is composed of two catalytic
subunits IKK
and
and one regulatory subunit IKK
/NEMO.
Numerous studies now suggest that the IKK
catalytic subunit is
required for IKK and NF-
B activation by TNF-
, interleukin-1,
lipopolysaccharide (LPS), dsRNA, and viral infection (6-10). Unlike
NF-
B, the heterodimers ATF-2/c-Jun are expressed as nuclear proteins
that are activated by phosphorylation of their activation domains by
c-Jun amino-terminal kinases (JNKs) which are downstream of a well
defined stress-activated kinase cascade (11)
(IRF-9) (12). IRF-3 is expressed constitutively in a variety of
tissues, and the relative levels of IRF-3 mRNA do not change in
virus-infected or IFN-treated cells. IRF-3 demonstrates a unique
response to viral infection. Phosphorylation of latent cytoplasmic
IRF-3 on serine and threonine residues in the C-terminal region leads
to a conformational change, dimerization, cytoplasmic to nuclear
translocation, association with the p300/CBP coactivator, stimulation
of DNA binding and transcriptional activities (3, 13-17). Activated
IRF-3 can in turn induce a specific subset of type 1 IFN genes in
response to viral infection including IFN-
and human
IFN-
1 (murine
4), as well as the CC-chemokine RANTES and the interleukin-15 (15, 18-22). As with NF-
B activation, the
rate-limiting step in this process is C-terminal phosphorylation of
IRF-3 by an uncharacterized virus activated kinase (VAK) activity.
, resulted in
N-terminal phosphorylation but not C-terminal IRF-3 phosphorylation by
a mitogen-activated protein kinase kinase kinase (MAPKKK)-related
signaling pathway. N-terminal phosphorylation was not sufficient to
promote nuclear translocation, transactivation, or degradation of
IRF-3. The fact that numerous well characterized pharmacological
inhibitors failed to block VAK activity suggests the involvement of a
novel kinase in IRF-3 regulation by viruses.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, macrophage inflammatory protein 1
, and RANTES were from R&D Systems. Pertussis Toxin, epidermal growth factor, platelet-derived growth factor-BB, insulin, and thrombin
were kind gifts from Dr. Sylvain Meloche.
promoter,
pGL3-IFN-
-LUC were described previously (13, 19, 26). The
B-mutated RANTES promoter, pGL3-
Bm-RANTES-LUC, was prepared by
cloning the BglII-SalI fragment (
397 to +5,
filled in with the Klenow enzyme) from the
Bm-RANTES-CAT reporter
plasmid (19) into the NheI site (filled in with the Klenow
enzyme) of the pGL3-basic vector. The expression constructs encoding
different C-terminal IRF-3 truncations, pFlag-IRF-3-(1-198),
-(1-186), -(1-174), and -(1-150) were generated by overlap
polymerase chain reaction mutagenesis using Vent DNA polymerase.
Constructs encoding for MAPKKKs, PCDNA3-MEKK1-HA, and pRK5-MYC-Cot
were kind gifts from Drs. Richard Gaynor and Warner Greene, respectively.
-minimal
essential medium and Dulbecco's modified Eagle's medium,
respectively, supplemented with 10% fetal bovine serum, glutamine, and
antibiotics. The monocytic cell line U937 was cultured in RPMI
supplemented with 5% fetal bovine serum. The human bronchial lung
carcinoma cell line A549 was purchased from ATCC (CCL-185) and cultured
in F12K supplemented with 10% fetal bovine serum. Extracts of primary
monocytes uninfected or infected with NDV were a kind gift of Dr.
Sandra Gessani, ISS, Rome.
-glycerophosphate,
10
4 M phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml of each leupeptin, pepstatin, and aprotinin, and 1% Nonidet
P-40) and stored at
80 °C.
-actin (Sigma), or anti-Flag M2 (Sigma) (1:500 to 1:1000) in
blocking solution. After washing four times in TBS, 0.1% Tween 20, the
membranes were incubated for 1 h with horseradish
peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:10000) in
blocking solution. Immunoreactive bands were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Inc).
-glycerophosphate,
0.4 mM Na3VO4, and 0.1 µg of
okadaic acid per ml. The reactions were incubated at 37 °C for
2 h and stopped by washing the beads once with Nonidet P-40 lysis
buffer and addition of 50 µl of 2 × denaturating sample buffer.
The samples were resolved by SDS-gel electrophoresis and analyzed by
immunoblotting using anti-IRF-3 and anti-Flag antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Multiple forms of IRF-3 phosphoprotein.
A, schematic representation of IRF-3. The DNA-binding
domain, the NES element, the proline-rich region, and the C-terminal
IRF association domain are indicated. The region between aa 382 and 414 are expanded below the schematic. The amino acids targeted for alanine
or aspartic acid substitutions are shown in large letters.
The point mutations are indicated below the sequence: 5A, S396A, S398A,
S402A, T404A, S405A; 5D, S396D, S398D, S402D, T404D, S405D. cDNAs
encoding for IRF-3 lacking the C-terminal region (IRF-3-(1-240),
-(1-198), -(1-186), -(1-174), and -(1-150)) and the DNA-binding
domain ( NIRF-3-(133-427)) are also shown. B,
phosphorylation of IRF-3 in HEK 293, U937, and rtTA-Jurkat, rtTA-Jurkat
IRF-3wt, and rtTA-Jurkat IRF-3(5D) cells. Jurkat cells were induced
with Dox (1 µg/ml) for 16 h. Then HEK 293, U937, and rtTA-Jurkat
IRF3wt were infected with Sendai virus (80 HAU/ml) for 4, 8, or 12 h or left uninfected (
). Endogenous IRF-3 proteins were detected in
whole cell extracts (55 µg) by immunoblotting using anti-IRF-3
antibody (from Dr. Paula Pitha). C, forms II, III, and IV
are sensitive to phosphatase treatment. HEK 293 cells were transfected
with vector alone pBSCMV (pBS) or constructs encoding for IRF-3(wt),
IRF-3(5A), and IRF-3(5D) or left untransfected (Sendai virus). At
36 h post-transfection or 8 h after infection with Sendai
virus (80 HAU/ml), whole cell extracts were prepared and subjected to
immunoprecipitation using IRF-3 antibody covalently linked to protein
A-Sepharose beads. Immunoprecipitated IRF-3 was then used in a
phosphatase assay as described under "Materials and Methods." The
resulting immunoprecipitated proteins were resolved by 7.5% SDS-PAGE.
IRF-3 phosphorylated forms were analyzed by immunoblotting using
anti-IRF-3 antibody. PI, phosphatase inhibitors. Lanes
13-15 are derived from the experiment shown in Fig.
4A.
and
2 (SB203580),
phosphatidylinositol 3-kinase (wortmannin and LY294002), and mTOR/FRAP
(rapamycin) (29-35) did not affect the generation of the two
hyperphosphorylated forms of IRF-3 (III and IV) by Sendai virus (Fig.
2A, lanes 3-7). Pretreatment of cells with the
intracellular calcium chelating agent BAPTA-AM (Fig. 2A, lane
10) did, however, induce a shift from forms I to II. It also
completely blocked virus-induced IRF-3 phosphorylation (Fig. 2A,
lane 11, and Table I), suggesting that a
calcium-dependent phosphatase might be involved in the
generation of form I and more importantly, that a
calcium-dependent pathway may be upstream of IRF-3
activation. Many other pharmacological inhibitors also failed to block
IRF-3 phosphorylation (Table I). Interestingly, ribavirin, a selective
inhibitor of the RNA polymerase of paramyxoviruses (36) had a
dose-dependent inhibitory effect on IRF-3 phosphorylation
(Fig. 2B, lanes 3-8, and Table I), possibly due to its
ability to inhibit the replication of Sendai virus (data not shown).
Furthermore, UV-treated Sendai virus was unable to induce C-terminal
IRF-3 phosphorylation (Fig. 2C), suggesting that complete
IRF-3 activation through C-terminal phosphorylation requires
replication competent virus.
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Fig. 2.
Pharmacological inhibitors fail to block VAK
activity. A and B, HEK 293 cells were
pretreated with different pharmacological inhibitors for 30 min and
then were left untreated ( ) or infected with Sendai virus (80 HAU/ml)
for 8 h (+) in the continuous presence of inhibitors. Whole cell
extracts (75 µg) were prepared from infected and control cells and
were analyzed for the presence of phosphorylated forms of IRF-3 by
immunoblotting with anti-IRF-3 antibody. The concentration of
inhibitors used were: PD98059 (PD), 50 µM;
SB203580 (SB), 30 µM; wortmannin
(Wt), 100 nM; LY294002 (LY), 50 µM; rapamycin (Rp), 15 ng/ml; BAPTA-AM
(BA), 15 µM; dimethyl sulfoxide
(D), 0.1%; ribavirin (Riba), 250-1000 µg/ml.
C, HEK 293 cells were left untreated (
) or infected with
UV-treated virus (80 HAU/ml) or untreated virus (40 and 80 HAU/ml) for
8 h. Whole cell extracts and immunoblotting were preformed as
described above.
List of pharmacological inhibitors, their cellular targets, and effects
on Sendai virus-induced IRF-3 phosphorylation in HEK 293 cells
and NF-
B mutated RANTES promoters
(
Bm-RANTES) (Fig. 3B).
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Fig. 3.
Activation of IRF-3 is restricted to virus
infections. A, phosphorylation of IRF-3. Whole cell
extracts (75 µg), prepared from HEK 293 cells, freshly isolated
primary monocytes, and A549 cells uninfected ( ) or infected with MeV
(multiplicity of infection of 1.0), NDV (100 HAU/ml), and VSV
(multiplicity of infection of 10) for different time points, were
resolved by 7.5% SDS-PAGE and transferred to nitrocellulose. IRF-3 was
analyzed by immunoblotting for the presence of phosphorylated IRF-3
forms (II to IV) with anti-IRF-3 antibody. B,
transactivation of PRD I-III- and interferon-stimulated response
elements containing promoters. HEK 293 cells were transiently
transfected with reporter constructs containing IFN-
enhancer
(IFN-
-LUC) and the
B-mutated RANTES promoter (
Bm-RANTES-LUC).
At 24 h post-transfection, cells were treated as indicated in the
figure and LUC activity was analyzed 12 h later. Relative LUC
activity was measured as fold activation as described under
"Materials and Methods." Each value represents the mean ± S.E. of triplicate determinations. The data are representative of at
least two different experiments with similar results. The concentration
of viruses used was: Sendai virus (Sv), 80 HAU/ml; measles
virus (MeV), multiplicity of infection of 1.0.
B
Inducers Stimulate N-terminal IRF-3 Phosphorylation--
Recent
studies showed that DNA-damaging agents and stress inducers activated
IRF-3 in HeLa cells (23, 25). To determine which forms of IRF-3 were
activated by this diverse array of agents, HEK 293 cells were induced
with Sendai virus, stress inducing stimuli sorbitol and anisomycin,
DNA-damaging agent doxorubicin, and the growth factor/NF-
B inducer
phorbol 12-myristate 13-acetate (PMA) (Fig.
4A). Treatment with
anisomycin, sorbitol, doxorubicin, PMA, and also epidermal growth
factor (data not shown) resulted in the accumulation of form II without
the generation of forms III and IV (Fig. 4A, lanes 7-18),
as observed with Sendai virus (Fig. 4A, lanes 4-6). The
conversion of forms I to II using growth factor, stress, and
DNA damaging agents was sensitive to CIP treatment and, as shown above,
CIP was sensitive to phosphatase inhibitors (Fig. 4A, lanes 2, 5, 8, 11, 14, and 17), indicating that the phosphorylation
elicited by these agents was distinct from the virus-induced
phosphorylation. Previous studies demonstrated also that dsRNA
treatment was sufficient to trigger the nuclear accumulation of IRF-3
and the formation of a functionally active IRF-3 containing DRAF
complex (3, 16, 17). LPS treatment of U373 astrocytoma cells was also
shown to induce IRF-3 nuclear translocation and DNA binding activity
(24). Surprisingly dsRNA treatment of HEK 293 cells and LPS treatment
of U937 and HeLa cells did not induce any phosphorylation of IRF-3, as
detected by immunoblot analysis (data not shown). Other cytokines and
growth factors such as CC-chemokines (macrophage inflammatory protein
1-
, macrophage inflammatory protein 1-
, and RANTES) thrombin,
insulin, platelet-derived growth factor-BB also had also no effect on
IRF-3 phosphorylation (data not shown).
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Fig. 4.
Stress inducers, DNA-damaging agent, and
NF- B inducers stimulate N-terminal IRF-3
phosphorylation. A and B, phosphatase
treatment. HEK 293 cells were left untreated (
) or treated for 8 h with the indicated agents (A) or co-transfected with
Flag-IRF3wt and two MAPKKKs, MEKK1, and Cot (B). At 36 h post-transfection or following different treatments, whole cell
extracts were prepared and subjected to immunoprecipitation using IRF-3
antibody covalently linked to protein A-Sepharose beads or Flag
antibody immobilized onto protein-G Sepharose beads. The
immunoprecipitated IRF-3 proteins were then used in a phosphatase
assay, as described under "Materials and Methods." The resulting
immunoprecipitated proteins were resolved by 7.5% SDS-PAGE and
transferred to nitrocellulose. Phosphorylation of IRF-3 was analyzed by
immunoblotting with anti-IRF-3 antibody. PI, phosphatase
inhibitors. C, phosphorylation or IRF-3 by stress inducers
does not occur in the C-terminal end of the protein. HEK 293 cells were
transfected with vector alone pBSCMV (pBS) or constructs
encoding for IRF-3wt (WT) and IRF-3 5A (5A) or
co-transfected with filling vector (
) or MEKK1 and Cot expression
plasmids (+). At 30 h post-transfection, where indicated, cells
were left untreated (
) or treated (+) for 8 h with Sendai virus
(80 HAU/ml) or sorbitol (0.3 M). Whole cell extracts (30 µg) were analyzed for IRF-3 phosphorylation by immunoblotting with
anti-IRF-3 antibody.
B activation following T cell
receptor engagement (44), induced the accumulation of form II in
transfected cells (Fig. 4B, lanes 4 and 7). This
form represented the phosphatase-sensitive form of IRF-3 (Fig.
4B, lanes 5, 6, 8, and 9) as observed above with
stress inducers and DNA-damaging agents (Fig. 4A). In
contrast to Sendai virus infection (see Fig. 1B, lanes 5 and
6), no degradation of IRF-3 was observed in cells
overexpressing MEKK1/Cot or treated with stress inducers and
DNA-damaging agents after 16 to 24 h of treatment (data not shown).
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Fig. 5.
Mapping the site of N-terminal
phosphorylation of IRF-3. A, C-terminal IRF-3
truncation results in the expression of one form of IRF-3. HEK 293 cells were transfected with various IRF-3 expression plasmids as
indicated above the lanes. At 30 h post-transfection,
whole cell extracts were prepared (20 µg) and analyzed for IRF-3
expression using anti-Flag antibody. Brackets show the two
forms of IRF-3, asterisks show the expression of only one
form of IRF-3 following truncation (B). HEK 293 cells were
transfected with constructs as indicated in A. At 36 h
post-transfection or 3 h after treatment with 1 µM
anisomycin, whole cell extracts were prepared and subjected to
immunoprecipitation using Flag antibody linked to protein G-Sepharose
beads. Immunoprecipitated IRF-3 was then used in a phosphatase assay as
described under "Materials and Methods." The resulting
immunoprecipitated proteins were resolved by 12% SDS-PAGE.
IRF-3-phosphorylated forms were analyzed by immunoblotting using
anti-Flag antibody. PI, phosphatase inhibitors.
C, HEK 293 cells were transfected with Flag-tagged truncated
version of IRF-3 (Flag-IRF-3-(1-198)) or co-transfected with filling
vectors pCDNA3 (pC) and pRK5 (pR) or Cot and
MEKK1 (M) expression plasmids where indicated. At 30 h
post-transfection, Flag-IRF-3-(1-198)-transfected cells were
stimulated for 8 h with Sendai virus (Sv, 80 HAU/ml),
PMA (P, 100 ng/ml), sorbitol (S, 0.3 M); doxorubicin (D, 1 µg/ml), and anisomycin
(A, 1 µM). 30 µg of whole cell extracts were
then resolved by SDS-gel electrophoresis on 12% acrylamide gel and
transferred to nitrocellulose membrane. Phosphorylation of Flag-IRF-3
was analyzed by immunoblotting with anti-Flag antibody.
induced a shift from form I to form II (Fig. 6A,
lanes 3-8) without inducing the slowly migrating forms of IRF-3
observed when cells were infected by Sendai virus (Fig. 6A, lane
2). The effect of TNF-
on the conversion of form I to form II
was transient, with maximal conversion to form II occurring after 30 min and returning to equal proportions of form I and II after 2 h
(data not shown).2
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Fig. 6.
N-terminal phosphorylation does not alter
IRF-3 subcellular localization or function. A, IRF-3
phosphorylation. Whole cell extracts prepared from HEK 293 cells
untreated ( ) or treated with different agents or infected with Sendai
virus (Sv; 80 HAU/ml) for 8 h (except for
TNF-
-treated cells where a 30-min stimulation is shown) were
prepared. Protein extracts (75 µg) were analyzed by immunoblotting
for the presence of phosphorylated IRF-3 (II to IV) with anti-IRF-3
antibody. The concentration of agents used were: sorbitol
(S), 0.3 M; NaCl (N), 0.25 M; anisomycin (A), 1 µM;
doxorubicin (D), 1 µg/ml; PMA (P), 100 ng/ml;
TNF-
(T), 25 ng/ml. B, interaction between
IRF-3 and CBP coactivator. HeLa, HEK 293, and U937 cells were treated
as described in A. Whole cell extracts (500 µg) were
immunoprecipitated with anti-CBP antibody A22, covalently linked to
protein A-Sepharose beads. The immunoprecipitated proteins were
resolved by SDS-gel electrophoresis on 7.5% acrylamide gel and
transferred to nitrocellulose membrane. The membrane was probed with
anti-IRF-3 antibody. As indicated, only forms III and IV were found to
bind CBP. Lane 10, WCE (30 µg) prepared from uninfected
HEK 293 cells were used to show the position of forms I and II. The
concentration of agents used are described in A. LPS
(L), 10 µg/ml. C, cytoplasmic to nuclear
translocation of IRF-3. Hela cells were treated as indicated in
A and B. Cytoplasmic and nuclear fractions were
prepared as described under "Materials and Methods." Each isolated
fraction was subjected to 10% SDS-PAGE, transferred to nitrocellulose
membrane, and probed with anti-IRF-3 antibody. Lower panels,
membranes were stripped and reblotted with an anti-
-actin antibody.
D, transactivation of interferon-stimulated response
elements and PRD II containing promoters. HEK 293 cells were
transfected with the
B-mutated RANTES promoter (
Bm-RANTES-LUC) or
P2(2)tk-LUC reporter plasmids and the MEKK1 (M, 250 ng) or
NIRF-3 (
N, 500 ng) expression plasmids when indicated.
At 24 h post-transfection, cells were treated as indicated
below the bar graph and LUC activity was analyzed
12 h later. Relative LUC activity was measured as fold activation.
Each value represents the mean ± S.E. of triplicate
determinations. The data are representative of at least three different
experiments with similar results. The concentration of agents used
were: Sendai virus (Sv), 80 HAU/ml; PMA (P), 100 ng/ml; LPS (L), 10 or 100 µg/ml; TNF-
(T),
10 or 100 ng/ml; doxorubicin (D), 1 µg/ml; anisomycin
(A), 1 µM; and sorbitol (S), 0.20 M.
Bm-RANTES-LUC (19). Sendai virus
infection resulted in a 25-fold induction of RANTES activity in HEK 293 cells (Fig. 6D). Virus-inducible expression of the RANTES
promoter was inhibited by co-transfection with a dominant-negative
mutant of IRF-3 (
NIRF-3) (19, 27), demonstrating that the
inducibility of the RANTES promoter was essentially dependent on IRF-3
activation (Fig. 6D) (19). Under the same conditions, stimulation for up to 15 h with NF-
B inducers (PMA and TNF-
at 100 ng/ml), DNA-damaging agent (doxorubicin), and stress inducers (LPS at 100 µg/ml, anisomycin, and sorbitol) failed to stimulate RANTES activity. Moreover, a pretreatment of cells for 1 h with 10 µg/ml LPS did not affect Sendai virus-induced RANTES activity (Fig.
6D). Co-transfection with a MEKK1 expression construct also had no effect on RANTES activity, whereas both TNF-
treatment and
MEKK1 stimulated NF-
B dependent LUC activity 7- and 12-fold, respectively. These experiments demonstrate that stress inducing agents, DNA-damaging agents, and cytokines such as doxorubicin and
TNF-
and growth factors stimulate a MAPKKK-related pathway that
phosphorylates IRF-3 in the N-terminal part of the protein. However,
this phosphorylation event appears to have no readily discernible
consequence on IRF-3 translocation, association with CBP coactivator,
DNA binding activity, or transactivation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, IKK
/
, and PKR were unable to
phosphorylate the C-terminal end of IRF-3 (data not shown). Full
activation of IRF-3 appears to be restricted to viral infection
including paramyxoviruses (MeV, Sendai, and NDV) and rhabdoviruses
(VSV) which are potent inducers of VAK activity. Our data thus provide evidence of an uncharacterized virus-regulated kinase pathway involved
in C-terminal IRF-3 phosphorylation and activation.
enhanceosome (25). All the
enhancer binding activities, ATF, c-Jun, IRF-3, and NF-
B were
activated. MEKK1 activated IRF-3 through the JNK pathway but not
through p38 or IKK pathways. These experiments imply that MEKK1 can
induce IRF-3 and ATF2/c-Jun through the JNK pathway and NF-
B through
the IKK pathway, resulting in the integration of multiple signal
transduction pathways leading to the proper assembly of the IFN-
enhanceosome. The phosphorylation sites targeted by the MEKK1-related
pathway are distinct from the C-terminal sites, since the IRF-3(5A)
protein was still phosphorylated in response to CG18 and MEKK1.
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Fig. 7.
Schematic representation of IRF-3 activation
following N- and C-terminal phosphorylation. In uninfected cells,
intramolecular association between the C terminus and the DBD maintains
IRF-3 in a latent state in the cytoplasm by masking both DBD and IAD
regions of the protein (form I). Basal activities of both N-terminal
kinase and phosphatase may affect the overall ratio between IRF-3 forms
I and II. Treatment of cells with stress inducers, DNA-damaging agents,
and growth factors activates a MAPKKK-related pathway involved in the
positive regulation of the N-terminal kinase, resulting in an increase
in form II. N-terminal phosphorylation may induce a conformational
change that reveals phosphoacceptor sites for VAK in the C-terminal end
of IRF-3 (pathway 1). C-terminal phosphorylation by VAK then
relieves the intramolecular association between DBD and IAD leading to
homodimerization of IRF-3. C-terminal autoinhibition could also be
relieved through N-terminal phosphorylation (pathway 2)
resulting in homodimerization of IRF-3 before C-terminal
phosphorylation by VAK. IRF-3 can then accumulate in the nucleus and
activate genes through DNA binding and CBP association. Ultimately,
IRF-3 is degraded by the proteasome pathway. DBD,
DNA-binding domain; IAD, IRF association domain.
Finally, these experiments demonstrate for the first time that
replication competent virus is required for full activation of IRF-3
since UV inactivation or ribavirin inhibition of virus replication
blocked IRF-3 activity. Furthermore, the paramyxovirus MeV and the
rhabdovirus VSV may be added to the growing list of viruses capable of
activating IRF-3 function. Interestingly, influenza virus (as well as
other viruses of different classes) was unable to activate
IRF-3.3 Consistent with this
observation, a recent study has demonstrated that the influenza virus
NS1 protein, a dsRNA-binding protein, specifically inhibited IRF-3
(47), although the mechanism of inhibition remains to be elucidated.
These studies demonstrate that, as with many other viruses, the ability
to interfere with the IFN antiviral cascade may contribute
significantly to the virulence and pathogenicity of viral infection.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Paula Pitha, Brian Ward, Warner Greene, Richard Gaynor, and Sylvain Meloche for reagents used in this study and members of the Molecular Oncology Group, Lady Davis Institute for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council of Canada and the Cancer Research Society Inc.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 a Medical Research Council Fellowship.
Supported by a Fraser Monat McPherson Fellowship from McGill University.
¶¶ Supported by a Medical Research Council Senior Scientist award. To whom reprint requests should be addressed: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, H3T 1E2 Canada. Tel.: 514-340-8222 (ext. 5265); Fax: 514-340-7576; E-mail: mijh@musica.mcgill.ca.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007790200
2 R. Lin, unpublished data.
3 B. ten Oever, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IFN, interferon;
IRF, interferon regulatory factor;
PRD, positive regulatory domain;
IKK, IB kinase;
dsRNA, double stranded RNA;
LPS, lipopolysaccharide;
MAPKKK: mitogen-activated protein kinase kinase kinase, PMA, phorbol
12-myristate 13-acetate;
VAK, virus-activated kinase;
HEK, human
embryonic kidney;
CIP, calf intestine alkaline phosphatase;
TNF, tumor
necrosis factor;
MeV, measle virus;
NDV, Newcastle disease virus;
VSV, vesicular stomatitis virus;
JNK, c-Jun N-terminal kinase;
PAGE, polyacrylamide gel electrophoresis;
aa, amino acid(s);
PMSF, phenylmethylsulfonyl fluoride;
HAU, hemagglutinating units;
WCE, whole
cell extracts;
RANTES, regulated on activation normal T cell expressed;
BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid.
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