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INTRODUCTION |
IFNs1 are a family of
natural proteins serving as part of the defense systems against
infections. Cells can produce IFNs in response to virus infection, and
the newly synthesized IFNs are secreted extracelluarly, bind to IFN
receptors, and activate the Jak-Stat signaling pathway that leads to
the stimulation of expression of cellular genes generally called ISGs
(1-3). Some of these genes encode proteins that can inhibit viral
replication, thus conferring the antiviral state to the cells (3).
While the molecular mechanism involved in the induction of IFN and ISG
genes has been studied in great detail in vitro, it is
largely unknown how much this system is affected by other stress
factors. Of special concern is the potential impact of the bacterial
infection on this system because concomitant infection with both
bacteria and virus is a common clinical situation.
LPS is the major component of the outer membrane of Gram-negative
bacteria (4). Through activation of the target cells such as
macrophages and B cells, LPS induces innate immune response and
expression of cytokine genes which include IL-1, IFNB, and TNF
(5,
6). These cytokines are responsible for most of the biological effects
of LPS and deregulated production of these cytokines results in the
generalized inflammation or endotoxic shock. The signal transduction
pathway for LPS is initiated by its binding to LBP (LPS binding
protein) (7), an acute phase reactant produced by the liver, followed
by the binding to CD14, a glycosylphosphatidylinositol-anchored
membrane protein (8, 9) and Toll-like receptor (TLR2) (10). This
receptor is activated by LPS and the response depends on the binding of
LPS to LBP and is enhanced by CD14. After binding to the receptor, LPS
activates a number of tyrosine and serine kinases, including Raf-1, p42 and p44 isoforms of the MAP kinase, the p38 kinase, c-Jun kinase, ceramide activated kinase (CAK) and CD14 receptor-coupled kinase p56lyn (11-13), with consequent activation of
nuclear transcription factors such as NF-
B, Stat1 (signal transducer
and activator of transcription), Stat3, and NF-IL6 (C/EBP) (14-18).
LPS was also shown to activate nuclear factors binding to the
interferon stimulation responsible element although the identity of
these factors has not been elucidated (19, 20). Furthermore, functional
cooperation between LPS and IFNs, especially IFN
, was shown to
result in expression of a set of pro-inflammatory genes (21, 22).
Considering that IFNA and IFNB promoters contain cis-elements which are
highly homologous to the interferon stimulation responsible element, these findings indicate that LPS may potentially modulate the expression of IFN genes.
The signal transduction pathway leading to the induction of IFN genes
expression in virus-infected cells is largely unknown. The analysis of
the virus responsive element (VRE) of both IFNA and IFNB promoters has
identified highly conserved purine-rich sequence repeats that were
shown to bind the transcription factors of the IRF family (23-28). In
addition, IFNB gene promoter also contains a functional NF-
B site.
Three of the IRF factors, IRF-1, IRF-3, and IRF-7, were shown to
activate the promoters of IFNA or IFNB gene in transient transfection
assays (23, 29-35). However, the virus-mediated induction of IFNA and
IFNB genes was not impaired in mice or fibroblasts with homozygous
deletion of the IRF-1 gene (36, 37). The identification of IRF-3 and
characterization of its role as a signaling transducer in
virus-infected cells has provided a major step toward the understanding
of the virus-mediated signaling pathway leading to the expression of
type I IFN genes (38, 39). It was shown that IRF-3 is expressed
constitutively in a variety of tissues and cell lines and
synergistically cooperates with virus in the induction of both IFNA and
IFNB genes. Virus infection induces phosphorylation of IRF-3 at one
threonine and several serine residues at the carboxyl-terminal end. The
phosphorylated IRF-3 is then translocated from cytoplasm to nucleus
where it forms a complex with the transcription coactivator, p300/CBP
(29, 30). Recently, another IRF member, IRF-7, was identified that seems to play a critical role in the induction of IFNA genes (33-35). IRF-7 is also phosphorylated in infected cells and transported from
cytoplasm to nucleus. In contrast to IRF-3, IRF-7 is preferentially expressed in cells of lymphoid origin, and its transcription is stimulated by IFNA and virus infection (34). While the role of IRF-3
and IRF-7 in the induction of IFN genes has been gradually unveiled,
kinases that are responsible for the phosphorylation of IRF-3 and IRF-7
have not been identified yet.
We have been interested in the influence of other pathogens such as
bacteria, fungus, or parasites on the virus-mediated induction of IFNs.
Mixed infection is a clinical condition that occurs with high frequency
in the immune-compromised people as a consequence of HIV-1 infection,
organ transplantation or cancer. As the initial step to address this
question, we used LPS as the model to mimic the bacterial infection and
examined whether it can influence the virus-mediated signaling pathway
and induction of IFN gene expression. We have found that LPS is a
potent suppressor of virus-mediated induction of IFN genes.
Characterization of the underlying molecular mechanism has correlated
the LPS-mediated suppression with the inhibition of the virus-mediated
phosphorylation and nuclear translocation of IRF-3 and IRF-7.
Furthermore, overexpression of IRF-3 partially reverted the LPS effect.
Our study illustrates the scenario in which bacterial infection
interferes with the virus-mediated induction of IFN genes expression.
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EXPERIMENTAL PROCEDURES |
Cells, Virus, and Reagents--
Raw cells were purchased from
ATCC (American Type Culture Collection) and grown in RPMI medium
supplemented with 10% fetal calf serum. NDV was propagated in the
allantoic cavity of 10-day-old eggs. Sendai virus was purchased from
Specific Pathogen-Free Avian Supply (Preston, CT). The antibody to
mouse IRF-3 was a gift from Dr. T. Fujita (The Tokyo Metropolitan
Institute of Medical Science). Antibodies to human IRF-3 were prepared
by immunization of rabbit with GST-IRF-3 fusion protein (32). CD14
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
LPS (Escherichia coli, serotype 055B5) was purchased from
Sigma (St. Louis, MO), and mouse IFN (mixture of IFNA and IFNB) was
purchased from Lee Biomolecular Research (San Diego, CA). The
Raw-CMV-IRF-3 cell line was established by transfecting the
pcDNA-IRF-3 into Raw cells and selecting for cells resistant to
G418. A pool of stably transfected colonies was used in the experiments.
Plasmids--
The huIRF-3 expression plasmid, pcDNA-IRF-3,
was constructed by cloning the full-length huIRF-3 cDNA into the
pcDNA3 (Invitrogen, San Diego, CA) at the
HindIII/NotI sites immediately 3' to the CMV
promoter. The plasmids used for synthesis of IFNA, IFNB, and IL-6
riboprobes have been described previously (40, 41). The plasmid
containing the NDV-encoded nucleocapsid (NP) gene of NDV was obtained
from Dr. T. Morrison (University of Massachusetts, Worchester, MA). The
plasmids containing the murine IFNA4 promoter and its deletion mutants
(IFNA4-(-464), IFNA4-(-118)) inserted in front
of the CAT gene have been described before (42). The GFP vector was
obtained from CLONTECH (Palo Alto, CA), and the GFP-IRF-3 and GFP-IRF-7 expression plasmids were described previously (29, 34).
Northern Blot Hybridization--
Total RNA was isolated with
TRIzol reagent (Life Technologies, Gaithersburg, MD) and purified
according to the protocol of the manufacturer. Ten micrograms of
purified RNA was analyzed on 0.8% formaldehyde-agarose gel and
followed by transfer to nitrocellulose paper. The filters were
prehybridized in the hybridization buffer (50% formamide, 5× SSC, 150 µg/ml herring sperm DNA, 5× phosphatidylethanolamine) for 1 h
and then hybridized with the 32P-labeled riboprobes in the
same buffer in 65 °C (for cDNA probe, 50 °C) overnight. Blots
were washed sequentially with the following buffers: 2× SSC,0.1% SDS;
0.5× SSC, 0.1%SDS; 0.1× SSC, 0.1%SDS until clear background was
achieved. The ethidium bromide-stained gel was used as the loading control.
Transfection and CAT Assay--
Raw cells (3×10 6)
were seeded on 60-mm dishes 1 day before transfection. Five micrograms
of reporter IFNA4/CAT plasmids and 100 ng of
-galactosidase were transfected into Raw cells with Superfect
reagent (Qiagen, Chatsworth, CA). When indicated, cells were infected
with NDV or treated with LPS for the indicated time periods as
described in the respective figure legend. The results from the CAT
assay were normalized to an equal amount of
-galactosidase.
Western Blot Analysis--
Raw cells were treated with LPS or
infected with Sendai virus as described in the figure legends. Whole
cellular extracts were prepared by incubation of cell pellets in cell
lysis buffer (20 mM HEPES, pH 7.9, 50 mM NaCl,
10 mM EDTA, 2 mM EGTA, 0.1% Nonidet P-40, 10%
glycerol, 1 mM dithiothreitol, 50 mM sodium fluoride, 5 mM sodium orthovanadate) on ice for 30 min. The
extracts were cleared by ultracentrifugation at 15,000 rpm for 30 min, and proteins (30 µg) were separated by electrophoresis in 7.5% acrylamide, SDS gel and transferred to nitrocellulose paper. The Western blotting was performed by following the protocols of the manufacturer (ECL method, Amersham Pharmacia Biotech)
Fluorescence Microscopic Study--
Raw cells
(5×105 cells) were seeded in the chambered cover glass
(Nunc, Naperville, IL) 24 h before transfection with 0.5 µg of
GFP/IRF-3 (29) or GFP/IRF-7 (34) expression plasmids. Transfection was
done with Superfect reagent (Qiagen), and 16 h after transfection, cells were treated with LPS at the time points described in the figure
legends and infected with Sendai virus for 6 h. Cells were examined under fluorescence microscope at the wavelength of 507 nM. The pictures presented were recorded under the same
magnification power and the same exposure time.
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RESULTS |
Pretreatment of Raw Cells with LPS Differentially Modulates the
NDV-mediated Induction of Cytokines--
To examine the effect of LPS
treatment on the virus-mediated induction of IFNA and IFNB genes, the
mouse macrophage cell line Raw 264.7 was treated with LPS (1 µg/ml)
for 1 h and then infected with NDV (m.o.i. 5) for 6 h.
Analysis of the relative levels of IFNA and IFNB mRNA has shown
that IFNA mRNA could only be detected in NDV-infected cells but not
in cells that were pretreated with LPS (Fig.
1A). The relative levels of
IFNB mRNA were also suppressed in LPS-treated cells, but the level
of suppression was lower than that of IFNA. In contrast, induction of
IL-6 gene expression by NDV was enhanced by LPS pretreatment. The dose
response experiments (Fig. 1B) demonstrated that as low as
10 ng/ml of LPS was sufficient to suppress the levels of IFNA mRNA
by about 80%, whereas the same amount of LPS suppressed the levels of
IFNB mRNA only by about 30%. The kinetics study shown in Fig.
1B demonstrated that the suppression of IFN genes expression
by LPS was very fast; treatment with LPS for 10 min was already able to
suppress virus-mediated induction of IFNA and IFNB genes. In an
independent experiment, the effective dose of LPS to mediate the
suppression of IFNA gene induction has been determined to be as low as
3 ng/ml (data not shown). It was shown that the effect of LPS can be
divided into low (~ 1 ng/ml) and high dose effects and that the low
dose effect was CD14-dependent (7). Because the inhibition
of IFNA and IFNB genes expression could be observed with low levels of
LPS, it seemed to indicate that LPS may employ a
CD14-dependent pathway to suppress the virus-mediated
induction of IFN. However, co-incubation of anti-CD14 antibody (Santa
Cruz Biotechnology) with LPS neither blocked the LPS inhibition nor
modulated the virus-mediated induction of IFNA and IFNB genes (data not
shown).

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Fig. 1.
Pretreatment of Raw cells with LPS
differentially modulates NDV-mediated induction of cytokines.
A, raw cells were either untreated or treated with LPS (1 µg/ml) for 1 h, washed, and infected with NDV (m.o.i. 5) for
6 h when indicated. B, the dose-dependent
LPS-mediated suppression of NDV induction of IFNA and IFNB genes. Raw
cells were either untreated or treated with different concentrations of
LPS as indicated for 1 h, cells were then washed and infected with
NDV for 6 h. Total RNA was isolated as described under
"Experimental Procedures," was size-separated on 0.8%
formaldehyde-agarose gel, and was analyzed by Northern hybridization
with IFNA, IFNB, and IL-6 specific probes. The ethidium bromide-stained
RNA on agarose gel before transfer to the nitrocellulose filter is
shown.
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The LPS-mediated Suppression of IFN Induction by Virus Is Not a
Consequence of Suppression of Viral Replication--
To determine
whether the observed inhibition of IFN genes expression by LPS is a
result of the inhibition of viral replication, we examined NDV
replication in LPS-treated cells and untreated controls by analyzing
the NP gene transcripts. An earlier study has observed that the levels
of NDV NP transcripts could be correlated with the induction level of
IFN mRNA (43). As shown in Fig. 2, at
6 h post-NDV infection, the high levels of NP transcripts could be
detected (Fig. 2A, lane 2), which were not
modulated by LPS treatment applied at 1, 2.5, or 3.5 h after NDV
infection (Fig. 2A, lane 3-5). The relative
levels of NP transcripts were, however, lower in cells infected with
NDV in the presence of CHX (5 µg/ml) (Fig. 2A, lane
7). Only when cells were treated with high levels of LPS (1 µg/ml) 1 h before virus infection (Fig. 2A,
lane 6) was viral replication inhibited, and the relative levels of NP transcripts were lower than in the infected, untreated controls. Pretreatment with LPS at 10 ng/ml and lower has not affected
NP synthesis. Therefore, in the following experiments, we have used LPS
at the concentration of 10 ng/ml for pretreatment and 1 µg/ml for LPS
treatments initiated after virus infection.

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Fig. 2.
Comparison of the relative levels of the NDV
nucleocapsid (NP) mRNA and the respective cytokine
mRNA in LPS-treated cells and NDV-infected Raw cells.
A, Raw cells were infected with NDV for 6 h, and LPS
was added either 1 h before (designated as 1) or at
1, 2.5, or 3.5 h post-infection (designated as +1, + 2.5, and +3.5, respectively). When indicated, CHX
(5 µg/ml) was added to the medium 80 min before NDV infection. Total
RNA (10 µg) was analyzed by Northern hybridization with the NP probe.
B, LPS can suppress virus-mediated induction of the IFN
genes when LPS was added shortly after virus infection. Raw cells were
infected with NDV for 6 h, and LPS was added at the indicated time
points post-infection. Ten micrograms of total RNA was then analyzed by
Northern hybridization with the cytokine probes as indicated.
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LPS was shown to induce a low level of IFNB in the monocytes and
macrophages (44, 45). A previous study has disclosed that some of the
effects of LPS on the macrophages, such as induction of nitric oxide,
is due to the production of IFNB (46). Although LPS treatment (1 µg/ml, 4 h) was unable to induce IFNA gene expression in Raw
cells, IFNB mRNA could be detected by reverse
transcriptase-polymerase chain reaction under the same treatment (data
not shown). While LPS pretreatment (1 h) totally suppressed the
virus-mediated induction of the IFNA gene, the exogenously added IFN
(250 units/ml, 1 h) slightly enhanced the virus-mediated induction
of the IFNA gene (data not shown). Furthermore, LPS-mediated
suppression of IFNA genes expression was not affected by the presence
of CHX (5 µg/ml) (data not shown). This concentration of CHX blocked
the transcription of viral nucleocapsid gene by 80% (Fig.
2A, lane 7). These data demonstrated that the
effect of LPS pretreatment was not caused by IFN or any other
LPS-induced protein.
The study of the interaction between LPS and virus in the context of
IFN induction was further extended by treating Raw cells with LPS at
different time points after the virus infection to determine at which
stages of viral infection the LPS-mediated inhibition is still
effective. As shown in Fig. 2B, the inhibition of IFNA and
IFNB genes expression was less effective if LPS treatment was started
later than 3 h after NDV infection. In contrast, the induction of
IL-6 was enhanced by LPS treatment even when started as late as
3.5 h post-infection. The LPS-mediated inhibition was also not
specific for NDV infection and also could be demonstrated in Raw cells
infected with Sendai virus (data not shown).
LPS Suppresses the Virus-mediated Activation of IFNA
Promoter--
To determine whether the LPS-mediated inhibition of the
IFN genes induction is regulated at the transcriptional level, we analyzed the effect of LPS on the inducible expression of a reporter gene in transiently transfected Raw cells. The cells were transfected with a plasmid containing the CAT gene under the control of the IFNA4
promoter, infected with NDV, and treated with LPS as indicated in Fig.
3. It can be seen that LPS treatment,
either before or after virus infection, suppressed virus-mediated
induction of IFNA/CAT plasmid by 2-5-fold, indicating that LPS
suppression occurs at the transcriptional level. The suppression was
more effective in the cells treated with LPS before or 1 h after
the infection than at a later time post-infection. This is consistent with the results obtained when the expression of the endogenous IFNA
gene was analyzed. To determine whether the LPS inhibits the
virus-mediated transcriptional activation of IFNA promoter by induction
of phosphatases, we treated cells with okadaic acid, a known inhibitor
of phosphatases (PP1 and PP2A) before and simultaneously with LPS
treatment. However, the inhibition of virus-mediated induction of
IFN/CAT plasmid was not affected by okadaic acid.

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Fig. 3.
LPS suppresses the NDV-mediated induction of
IFNA4 CAT plasmids. Raw cells were cotransfected with 5 µg of
plasmids containing the IFNA4-(-464) promoter in front of the CAT gene
and 100 ng of pCMV- -galactosidase. Sixteen h later, cells were
infected with NDV for 8 h. Treatment with LPS was either at 1 h before or at 1, 2, and 4 h after virus infection. Total cells
extracts were harvested at the end of infection, and the extracts were
assayed for the CAT activity as described under "Experimental
Procedures." The CAT activity was further normalized to equal levels
of -galactosidase. Average value of three independent experiments is
shown.
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To further define the cis-elements of the IFNA4 promoter
that are critical for LPS-mediated suppression, plasmids containing deletion mutants of IFNA4 promoters linked to the CAT
reporter gene were used for transfection. We have found that as short
as
118 bp of the IFNA promoter, which contains a 35-bp long
virus-inducible element (IE), is able to confer the LPS-mediated
inhibition (data not shown).
LPS Suppresses the Virus-mediated Phosphorylation of IRF-3 and
Impedes Its Translocation from Cytoplasm to Nucleus--
We and others
have recently described that IRF-3 serves as a transducer of
virus-mediated signaling from cytoplasm to nucleus and plays a critical
role in the induction of IFNA and IFNB genes (29, 30, 34). It was shown
that IRF-3 is phosphorylated in infected cells and consequently
translocated to the nucleus, where it interacts with the transcription
coactivator CBP/p300. To determine whether the LPS-mediated inhibition
of IFNA and IFNB genes expression is the result of interference with
IRF-3 function, we analyzed the effect of LPS on the virus-mediated
phosphorylation of IRF-3. IRF-3 is constitutively present in uninfected
Raw cells and infection of Raw cells with Sendai virus for 6 h
resulted in a decrease of relative levels of IRF-3 and the
phosphorylation of IRF-3, which was reflected by the appearance of a
slow migrating band (Fig. 4) on Western
blot analysis. These results are in agreement with our previous
findings in which we demonstrated that phosphorylated IRF-3 is targeted
for degradation presumably by the ubiquitin pathway (29). In infected,
LPS-treated cells, phosphorylation of IRF-3 was prevented as indicated
by the absence of the slow migrating IRF-3 band. The absence of this
slow migrating band was most clear in cells treated with LPS 1 h
before or 1 h after the infection (Fig. 4). Thus, for both the
suppression of IRF-3 phosphorylation as well as for the inhibition of
expression of the IFN genes, LPS treatment has to be initiated before
or soon after virus infection. LPS treatment alone has not induced
phosphorylation of IRF-3 protein (data not shown).

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Fig. 4.
LPS suppresses the virus-mediated
phosphorylation of IRF-3. Raw cells were infected with Sendai
virus, and LPS treatment was started at the times indicated. Cell
extracts were prepared at 6 h post-infection as described under
"Experimental Procedures." Thirty micrograms of whole cell extract
was resolved in 7.5% SDS-acrylamide gel, and proteins were transferred
onto the nitrocellulose paper and probed with mouse IRF-3 antibody.
Western blotting was performed as described under "Experimental
Procedures."
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To determine whether the suppression of phosphorylation of IRF-3 by LPS
results in the inhibition of translocation of cytoplasmic IRF-3 into
nucleus, we analyzed the effect of LPS treatment on nuclear
translocation of the GFP-tagged IRF-3. In a transfection experiment
with IFN/CAT plasmid, the GFP-tagged IRF-3 was able to stimulate
expression of this plasmid in infected cells with the efficiency of
about 45% of the wild type IRF-3 (data not shown). Raw cells were
transfected with the expression plasmid encoding the GFP/IRF-3 fusion
protein (29), and 24 h later, transfected cells were infected with
Sendai virus for 6 h. It can be seen that, in uninfected cells,
GFP/IRF-3 is located only in cytoplasm (Fig.
5a), whereas it is efficiently
translocated into the nucleus at 6 h post-infection (Fig.
5b). Treatment with LPS 1 h before or 1 h after
Sendai virus infection efficiently suppressed the nuclear translocation
of GFP/IRF-3 (Fig. 5, c and d). When treatment with LPS was initiated 4 h after virus infection (5e),
some cells showed the presence of GFP/IRF-3 only in cytoplasm, whereas
others had the GFP/IRF-3 dispersed both in the cytoplasm and nucleus. These data clearly show that LPS treatment of the cells initiated either before or within the first hour after viral infection interferes with the phosphorylation of IRF-3 and its transportation from the
cytoplasm to the nucleus.

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Fig. 5.
LPS suppresses virus-mediated nuclear
translocation of IRF-3. Raw cells were transfected with 0.5 µg
of GFP/IRF-3, and 16 h later, cells were infected with Sendai
virus (b) or left uninfected (a). Some of the
infected cells were treated with LPS either 1 h before
(c) or 1 (d) or 4 (d) h after the
infection. The migration of GFP/IRF-3 was examined 6 h after the
infection by fluorescence microscopy as described under "Experimental
Procedures."
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Overexpression of IRF-3 Reverts the LPS-mediated Suppression of IFN
Induction--
To further determine whether IRF-3 is the target for
LPS suppression of the virus-mediated induction of IFNA and IFNB genes expression, we generated a Raw cell line, Raw-CMV-IRF3, which constitutively overexpressed human IRF-3. Using Western blot analysis with antibodies to human IRF-3, we could detect expression of human
IRF-3 in Raw-CMV-IRF3 cells but not in the parental Raw cells (Fig.
6A). Infection of the
Raw-CMV-IRF3 cells with Sendai virus resulted in the expression of IFNA
genes, as determined by the presence of IFNA mRNA. As shown in Fig.
6B, treatment of Raw cells with LPS initiated 2 or 4 h
after virus infection resulted in a decrease in the relative levels of
IFNA mRNA (70 and 50% suppression, respectively), while no
LPS-mediated suppression could be seen in the infected CMV-IRF-3 cells.
These results indicate that overexpression of IRF-3 can overcome the
LPS-mediated inhibition of IFNA genes expression in infected cells.

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Fig. 6.
Overexpression of IRF-3 reverts the
LPS-mediated suppression of IFNA induction by virus. Raw-CMV-IRF-3
cell line was established as described under "Experimental
Procedures." A, the expression of IRF-3 in these cells was
demonstrated by Western blot analysis. B, the relative
levels of IFNA mRNA detected in Sendai virus-infected Raw cells (6 h) and Raw-CMV-IRF-3 cells treated with LPS either at 2 or 4 h
after infection were determined by Northern blot hybridization as
described under "Experimental Procedures." The levels of IFNA and
glyceraldehyde-3-phosphate dehydrogenase mRNAs were quantitated by
using PhosphorImager (Molecular Dynamics), and the levels of IFNA
mRNA were normalized to the level of glyceraldehyde-3-phosphate
dehydrogenase mRNA. The results are expressed as the ratio of
relative levels of IFNA mRNA induced in the presence of LPS over
that in the absence of LPS.
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LPS Interferes with the Nuclear Transport of IRF-7 in Infected
Cells--
We and others have recently shown that IRF-7 plays an
important role in the expression of IFNA genes (33-35). Similarly as IRF-3, IRF-7 is transported from cytoplasm to nucleus in infected cells, and the phosphorylation of serine residues in the carboxyl terminus of this protein is important for its transactivating activity.
We have, therefore, examined whether LPS treatment also affects the
nuclear transport of GFP/IRF-7 fusion protein in infected cells. The
results in Fig. 7a show that,
in transfected Raw cells, GFP/IRF-7 can be detected predominantly in
the cytoplasm. In contrast, at 7-h post-infection, GFP/IRF-7 starts to
accumulate in the nucleus although the transport is not completed yet
(Fig. 7b). In cells which were either pretreated with LPS or
treated with LPS 1 h after the infection, GFP/IRF-7 is localized
only in cytoplasm (Fig. 7, c and d). When LPS
treatment was started at 4-h post-infection, some cells showed the
presence of GFP/IRF-7 in both nucleus and cytoplasm, while others
showed only the cytoplasmic localization (Fig. 7e). These
data indicate that LPS treatment, when started before or early after
infection, prevents transport of IRF-7 from cytoplasm to nucleus.
However, these data also show that when overexpressed, low levels of
GFP/IRF-7 can be detected in the nucleus even in the absence of viral
infection.

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Fig. 7.
LPS suppresses virus-mediated nuclear
translocation of IRF-7. Transfection with GFP/IRF-7 into Raw
cells, infection, and treatment with LPS were done as described in Fig.
5. a, uninfected transfected cells; b, cells
infected with Sendai virus; c, cells treated with LPS 1 h before infection or 1 (d) or 4 h (e) after
the infection. The migration of GFP/IRF-7 was examined under a
fluorescence microscope as described in Fig. 5.
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DISCUSSION |
Two families of transcription factors play a critical role in the
virus-mediated induction of IFNA and IFNB gene expression. The IRFs
factors, especially IRF-3 and IRF-7, can serve as direct transducers of
virus-mediated signaling from cytoplasm into the nucleus and activate
transcription of IFNA and IFNB genes. In addition, viral infection also
leads to the phosphorylation and degradation of I
B and subsequent
transport of NF-
B (p50/p65 heterodimer) into the nucleus where it
participates in the activation of transcription of the IFNB gene. In
the present study, we have demonstrated that LPS is a potent suppressor
of virus-mediated stimulation of IFN genes expression. The suppression
of IFN genes expression in Raw cells could be demonstrated when cells
were treated with LPS before or soon after virus infection at
concentrations as low as 3 ng/ml. Although LPS suppressed both the
induction of IFNA and IFNB genes expression in infected cells, the
suppression of IFNA gene expression was stronger than that of IFNB
gene. One reason for the lower sensitivity of IFNB to the LPS-mediated
inhibition could be the presence of functional NF-
B site in the
promoter of the IFNB gene. In macrophages and monocytes, LPS treatment was shown to activate IKK kinases (47), resulting in the
phosphorylation of I
B and release of NF-
B factor (p50/p65
heterodimer) into nucleus. Indeed, we have found that treatment of Raw
cells with LPS for 15 min led to the accumulation of p65/RelA in the
nucleus and stimulated the binding of NF-
B to the positive
regulatory domain II (PRDII) of the IFNB promoter (data not shown). The
role of NF-
B in virus-mediated induction of the IFNB gene has been well demonstrated (48-50). Thus, these data indicate that the
LPS-mediated modulation of the transcriptional activity of IFNB gene
involves both positive and negative regulations.
The observation that the LPS-mediated suppression of IFN genes
expression is most effective at the early stages of viral infection indicates that LPS interferes with the initial stages of virus-induced signaling pathway. The suppression of IFN induction by LPS is not a
result of an inhibition of virus replication because LPS can suppress
IFN induction without affecting the transcription of the nucleocapsid
genes. While LPS efficiently suppressed virus-mediated induction of
IFNA and IFNB genes, induction of the IL-6 gene in infected cells was
stimulated by LPS. LPS alone was found to induce expression of several
early inflammatory genes in macrophages, including TNF
, TNFR-2,
IP-10, IL-1, IFNB, and IRF-1. This activation is dependent on the
LPS-induced tyrosine phosphorylation of the MAP kinases (51, 52). Thus,
the selective susceptibility of IFN induction to LPS may indicate that
LPS suppresses activation of factors crucial for the induction of IFNs,
but not for IL-6 and other cytokines.
As discussed above, IRF-3 and IRF-7 were recently identified as
transducers of virus-mediated signaling pathway (29, 30, 34, 53). IRF-3
was shown to be post-translationally phosphorylated at Ser-385 and
Ser-386 (30) and at Ser-396 and Ser-398 (29). Phosphorylation was shown
to facilitate translocation of IRF-3 into the nucleus and stimulate
transcription of both IFNA and IFNB genes in infected cells. Our data
clearly show that LPS suppresses virus-mediated phosphorylation of
IRF-3 and its consequent translocation to the nucleus. Complete
inhibition of nuclear transport of IRF-3 in infected cells was observed
in cells that were pretreated or treated with LPS within 1 h after
the infection. When the LPS treatment was initiated 4 h after
virus infection, IRF-3 could be detected both in cytoplasm and in the
nucleus. These data correlate the inhibition of virus-mediated IRF-3
phosphorylation and nuclear translocation with the inhibition of IFNA
and IFNB gene expression in LPS-treated cells. Further evidence of the
involvement of IRF-3 in the LPS-mediated inhibition came from
experiments using a cell line constitutively overexpressing IRF-3. In
these cells, suppression of virus-mediated induction of the IFNA gene
by LPS was partially reverted when cells were pretreated with LPS (data
not shown) and no suppression was observed when the cells were treated
with LPS at 2 or 4 h after virus infection. These data indicate
that LPS interferes with the function of IRF-3 which results in the impairment of IFNA gene induction by virus. In addition to IRF-3, IRF-7
was recently demonstrated to stimulate expression of IFNA genes in
infected cells (33-35) and together with IRF-3 to be a part of the
transcriptional enhansosome binding to the promoter region of IFNB gene
in infected cells (31). Similarly as IRF-3, IRF-7 is transported from
cytoplasm to nucleus (34, 35) in infected cells. Phosphorylation of
IRF-7 on two carboxyl-terminal serines was shown to be required for
induction of the IFNA genes (33). Our data indicate that LPS treatment
also interferes with the nuclear transport of GFP/IRF-7 in infected
cells. Additional experiments have to determine whether LPS also
inhibits virus-mediated phosphorylation of IRF-7.
While the signaling pathway(s) activated in NDV or Sendai
virus-infected cells is being unfolded, it is still unclear which serine/threonine kinase(s) phosphorylates IRF-3 or IRF-7. The similarity in the phosphorylation sites present on IRF-3 and IRF-7 as
well as similar kinetics of the transport of these two factors to the
nucleus in infected cells suggests that a similar kinase is
phosphorylating IRF-3 and IRF-7. It was shown previously that double-stranded RNA-dependent protein kinase (PKR)
can be activated in infected cells; however, it is unlikely that this
kinase phosphorylates IRF-3 or IRF-7 because homozygous deletion of the
PKR gene does not abolish virus-mediated induction of the IFN genes
(54). LPS induces a complex signaling pathway which includes tyrosine phosphorylation of several targets, activation of G protein (55), PKC
(56), as well as activation of Stat3 (17). LPS was also found to
post-transcriptionally regulate the transcriptional transactivator C/EBP (15).Which one of these pleiotropic responses to endotoxin abrogates the virus-induced signaling and consequent phosphorylation of
IRF-3 and IRF-7 remains to be established.