From the Departments of Neurology, § Medicine, Microbiology, and Immunology, University of Colorado Health Science Center, Denver, Colorado 80262 and Denver Veterans Affairs Medical Center, Denver, Colorado 80220
Received for publication, January 9, 2003, and in revised form, February 25, 2003
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ABSTRACT |
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Cellular transcription factors are often utilized
by infecting viruses to promote viral growth and influence cell fate.
We have previously shown that nuclear factor Experimental infection with mammalian reoviruses has provided a
classic model of viral pathogenesis (for review, see Ref. 1). Reovirus
induces apoptosis both in cultured cells and in target tissues (for
review, see Ref. 2). In the central nervous system and heart,
virus-induced apoptosis correlates with pathology and is a critical
mechanism by which disease is triggered in the host (3-5). Reovirus
induces apoptosis by a p53-independent mechanism that involves cellular
proteases, including calpains (3, 6) and caspases (5, 7, 8).
We have previously shown that in a variety of human epithelial cell
lines (7, 9) reovirus-induced apoptosis is mediated by tumor necrosis
factor (TNF)1-related
apoptosis-inducing ligand (TRAIL) (for review, see Ref. 10). However,
reovirus infection triggers apoptosis in both TRAIL-sensitive (7, 9)
and TRAIL-resistant cells (7). The question as to how reovirus induces
apoptosis in TRAIL-resistant lines has been answered in part by the
observation that reovirus can sensitize previously resistant cells to
killing by TRAIL (7, 9). Reovirus-induced sensitization of cells to
TRAIL requires caspase 8 activity and is associated with an increase in
the cleavage of pro-caspase 8 in cells treated with TRAIL and reovirus
compared with cells treated with TRAIL alone (9). The mechanism by
which reovirus induces increased caspase 8 activation in TRAIL-treated cells is, however, unknown.
The NF- The experiments described below investigate the role of NF- Cells, Viruses, and Reagents--
HEK293 (ATCC CRL1573) were
grown in Dulbecco's modified Eagle's medium supplemented with 100 units/ml each penicillin and streptomycin and containing 10% fetal
bovine serum. HeLa cells (ATCC CCL2) were grown in Eagle's minimal
essential medium supplemented with 2.4 mM
L-glutamine, nonessential amino acids, 60 units/ml each
penicillin and streptomycin and containing 10% fetal bovine serum
(Invitrogen). HEK293 cells expressing I Apoptosis Assays--
Cells were assayed for apoptosis by
staining with acridine orange for determination of nuclear morphology
and ethidium bromide to distinguish cell viability at a final
concentration of 1 µg/ml each (29). After staining, cells were
examined by epifluorescence microscopy (Nikon Labophot-2, B-2A filter;
excitation, 450-490 nm; barrier, 520 nm; dichroic mirror, 505 nm). The
percentage of cells containing condensed nuclei and/or marginated
chromatin in a population of 100 cells was recorded. The specificity of this assay has been previously established in reovirus-infected cells
using DNA laddering techniques and electron microscopy (9, 30).
Caspase 3 Activity Assays--
Caspase 3 activation assays were
performed using a kit obtained from Clontech. Cells
(1 × 106) were centrifuged at 200 × g for 10 min, supernatants were removed, and cell pellets
were frozen at Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from treated cells (5 × 106)
by washing cells in phosphate-buffered saline followed by incubation in
hypotonic lysis buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and a protease inhibitor mixture (Roche Applied Science)) at
4 °C for 15 min. One-twentieth volume 10% Nonidet P-40 was added to
the cell lysate, and the sample was vortexed for 10 s and
centrifuged at 10,000 × g for 5 min. The nuclear
pellet was washed once in hypotonic buffer, resuspended in high salt
buffer (25% glycerol, 20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitor
mixture), and incubated at 4 °C for 2-3 h. Samples were centrifuged
at 10,000 × g for 10 min, and the supernatant was used
as the nuclear extract.
Nuclear extracts were assayed for NF- Luciferase Gene Reporter Assays--
The
NF- Western Blot Analysis--
After infection with reovirus, cells
were pelleted by centrifugation, washed twice with ice-cold
phosphate-buffered saline, and lysed by sonication in 200 µl of a
buffer containing 15 mM Tris, pH 7.5, 2 mM
EDTA, 10 mM EGTA, 20% glycerol, 0.1% Nonidet P-40, 50 mM Reovirus Induces the Activation of NF-
We have previously shown that reovirus activates NF-
Luciferase reporter gene assays were also used to show that NF- NF- Reovirus Prevents the Activation of NF- Reovirus Blocks the Degradation of I Reovirus-induced Inhibition of Stimulus-induced I Reovirus-induced Apoptosis in HEK293 Cells Requires Viral RNA
Synthesis--
Having shown that reovirus-induced inhibition of I Viral RNA Synthesis Is Required for Reovirus-induced Sensitization
of Cells to TRAIL--
We have previously shown that reovirus
sensitizes cells to TRAIL-induced apoptosis (7, 9). The fact that
ribavirin blocks reovirus-induced apoptosis in TRAIL-resistant but not
TRAIL-sensitive cells suggests the mechanism by which reovirus
sensitizes cells to TRAIL requires viral RNA synthesis. HEK293 cells
were thus infected with reovirus with or without ribavirin. 24 h
post-infection cells were then treated with TRAIL, and apoptosis was
assayed after a further 24 h. Fig. 6C shows that
reovirus-induced sensitization of cells to TRAIL is inhibited in the
presence of ribavirin, indicating that reovirus-induced sensitization
of cells to TRAIL is dependent on viral RNA synthesis.
Inhibition of NF-
Fig. 4 shows that TNF Virus Infection Sensitizes HEK293 Cells to Apoptosis Induced by
TNF Reovirus-induced apoptosis in human epithelial HEK293 cells and in
several human cancer cell lines is mediated by TRAIL and is blocked by
the presence of soluble TRAIL receptors and by anti-TRAIL antibodies
(7, 9). However, reovirus can induce apoptosis in both TRAIL-sensitive
and TRAIL-resistant cells. Reovirus, therefore, has the ability to
sensitize TRAIL-resistant cells to TRAIL-induced apoptosis (7, 9). We
have previously shown that in TRAIL-sensitive HeLa cells reovirus
infection results in the activation of NF- Although required for virus-induced apoptosis, NF- Although the inhibition of NF- Reovirus-induced activation of NF- NF- In neurons TNF Reovirus-induced apoptosis is mediated by TRAIL and involves the
release of TRAIL from infected cells (7). Thus, the supernatant from
reovirus-infected cells contains TRAIL and can induce apoptosis in
TRAIL-sensitive cells (7). This apoptosis is blocked in the presence of
soluble TRAIL receptors, indicating that it is specific to TRAIL and is
not blocked in the presence of a neutralizing reovirus antibody,
indicating that it is not due to residual virus in the supernatant.
TRAIL released from reovirus-infected cells, thus, induces apoptosis by
inducing receptor-mediated activation of caspase 8 (10). These results
show that reovirus regulation of NF- The ability of TRAIL to induce apoptosis in a variety of human cancer
cells but not in normal cells has triggered the investigation of this
reagent as a potential therapeutic agent for human cancers. However,
many cancer cells are resistant to TRAIL-induced apoptosis. We have
previously shown that reovirus can sensitize TRAIL-resistant human
cancer cell lines to TRAIL-induced apoptosis. The results presented
here suggest that the mechanism for this sensitization results from the
ability of reovirus to block NF- The NF- The results demonstrated here indicate that reovirus both activates and
then inhibits NF-B (NF-
B) is
activated after reovirus infection and that this activation is required for virus-induced apoptosis. In this report we identify a second phase
of reovirus-induced NF-
B regulation. We show that at later times
post-infection NF-
B activation is blocked in reovirus-infected cells. This results in the termination of virus-induced NF-
B activity and the inhibition of tumor necrosis factor
and
etoposide-induced NF-
B activation in infected cells.
Reovirus-induced inhibition of NF-
B activation occurs by a mechanism
that prevents I
B
degradation and that is blocked in the presence
of the viral RNA synthesis inhibitor, ribavirin. Reovirus-induced
apoptosis is mediated by tumor necrosis factor-related apoptosis
inducing ligand (TRAIL) in a variety of epithelial cell lines. Herein
we show that ribavirin inhibits reovirus-induced apoptosis in
TRAIL-resistant HEK293 cells and prevents the ability of reovirus
infection to sensitize TRAIL-resistant cells to TRAIL-induced
apoptosis. Furthermore, TRAIL-induced apoptosis is enhanced in HEK293
cells expressing I
B
N2, which blocks NF-
B activation. These
results indicate that the ability of reovirus to inhibit NF-
B
activation sensitizes HEK293 cells to TRAIL and facilitates
virus-induced apoptosis in TRAIL-resistant cells. Our
findings demonstrate that two distinct phases of virus-induced NF-
B
regulation are required to efficiently activate host cell apoptotic
responses to reovirus infection.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B family of cellular transcription factors promotes the
expression of a variety of cellular genes, including genes that have
either pro- or anti-apoptotic effects, and it is thought that the
balance of expression of NF-
B-regulated genes may determine cell
fate (12). The prototypical form of NF-
B exists as a heterodimer of
proteins p50 and p65 (RelA) (13, 14). NF-
B is normally sequestered
in the cytoplasm by its binding to a family of inhibitor proteins,
collectively known as I
B (15, 16). In response to a variety of
stimuli, I
B is phosphorylated, resulting in its ubiquitination and
subsequent degradation (17-20). This allows the release of NF-
B,
which translocates to the nucleus (21), where it stimulates cellular
gene transcription (for review, see Refs. 22 and 23). In a variety of
cell types, the binding of reovirus to the cell surface receptors
junctional adhesion molecule and sialic acid induces the activation of
NF-
B (24, 25). In TRAIL-sensitive HeLa cells this activation is
detected 2-12 h post-infection (pi), involves both the p65 and p50
subunits of NF-
B, and is required for reovirus-induced apoptosis
(26). In HeLa cells reovirus-induced NF-
B activation and apoptosis require viral disassembly but not subsequent events of reovirus replication and are not inhibited by the viral RNA synthesis inhibitor ribavirin or by replication incompetent viruses (27).
B in
reovirus-induced apoptosis in TRAIL-resistant HEK293 cells. These
studies show that reovirus infection of HEK293 cells results in an
initial, transient phase of NF-
B activation that is required for
reovirus-induced apoptosis in these cells. This is followed by a later
phase of virus-induced NF-
B inhibition. Reovirus-induced inhibition
of NF-
B activation is associated with impaired degradation of I
B
and is inhibited by the viral RNA synthesis inhibitor, ribavirin. In
contrast to findings in TRAIL-sensitive HeLa cells, in
which ribavirin blocks reovirus replication but not apoptosis, both
these events are blocked by ribavirin in TRAIL-resistant HEK293 cells.
Ribavirin also inhibits reovirus-induced sensitization of HEK293 cells
to TRAIL-induced apoptosis. We further show that HEK293 cells are
sensitized to TRAIL-induced apoptosis by the expression of I
B
N2,
which blocks the activation of NF-
B. This suggests that the ability
of reovirus to block NF-
B activation at later times post-infection
sensitizes HEK293 cells to TRAIL-induced apoptosis and is critical
for apoptosis in TRAIL-resistant cells. The demonstration that multiple
levels of virus-induced NF-
B regulation are required to efficiently
activate host cell apoptotic responses to reovirus infection represents
a novel mechanism of viral-induced apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
N2, a strong dominant negative I
B mutant lacking the NH2-terminal
phosphorylation sites that regulate I
B degradation and the
consequent activation of NF-
B, were a kind gift from Dr. G. Johnson). Reovirus strain Type 3 Abney (T3A) was used for all
experiments. T3A is a laboratory stock that has been plaque-purified
and passaged (twice) in L929 (ATCC CCL1) cells to generate working
stocks (28). TRAIL was obtained from Upstate Biotechnology and Sigma,
TNF
was obtained from Invitrogen, and etoposide and ribavirin were
obtained from Sigma. Ribavirin was used at a concentration of 200 µM.
70 °C until all time points were collected. Assays
were performed in 96-well plates and analyzed using a fluorescent plate
reader (CytoFluor 4000, PerSeptive Biosystems). Cleavage of
DEVD-aminofluoromethylcoumarin, a synthetic caspase-3 substrate, was
used to measure caspase 3 activation in reovirus-infected cells.
Cleavage after the second Asp residue produces free
aminofluoromethylcoumarin that can be detected using a fluorescent
plate reader. The amount of fluorescence detected is directly
proportional to the amount of caspase 3 activity.
B activation by EMSA using a
32P-labeled oligonucleotide consisting of the NF-
B
consensus binding sequence (Santa Cruz Biotechnology). Nuclear extracts
(5-10 µg of total protein) were incubated with a binding reaction
buffer containing 2 µg of poly(dI·dC) (Sigma) in the presence of 20 mM HEPES (pH 7.9), 60 mM KCl, 1 mM
EDTA, 1 mM dithiothreitol, and 5% glycerol at 4 °C for
20 min. Radiolabeled NF-
B consensus oligonucleotide (0.1-1.0 ng)
was added, and the mixture was incubated at room temperature for 20 min. For competition experiments, a 10-fold excess of unlabeled
consensus oligonucleotide or an oligonucleotide containing the SP-1
consensus site (Santa Cruz Biotechnology) were added to reaction
mixtures. Nucleoprotein complexes were subjected to electrophoresis on
native 5% polyacrylamide gels at 180 V, dried under vacuum, and
exposed to Biomax MR film (Eastman Kodak Co.).
B-dependent luciferase reporter construct was a gift
from Dr. B. Sugden. The construct contains four NF-
B binding sites
upstream of the luciferase gene. HEK293 cells (1.5 × 105) in 6-well tissue culture plates (Costar) were
incubated for 24 h before being transfected with 1 µg of the
luciferase reporter construct and 1 µg of a
cytomegalovirus-
-galactosidase reporter construct
(Clontech) using LipofectAMINE (Invitrogen). After
an additional 24-h incubation, cells were either mock-infected or infected with T3A at an m.o.i. of 100 plaque-forming units per cell and
incubated at 37 °C for various intervals. Cells were then harvested
and resuspended in 1 ml of sonication buffer (91 mM
dithiothreitol, 0.91 M
K2HPO4 (pH 7.8), centrifuged at
2000 × g for 10 min, and resuspended in 100 µl of
sonication buffer. Cells were vortexed, frozen (
20 °C) and thawed
three times, and centrifuged at 14,000 × g for 10 min.
Samples (10 µl) were assessed for luciferase activity after the
addition of 350 µl of luciferase assay buffer (85 mM
dithiothreitol, 0.85 M
K2HPO4 (pH 7.8), 50 mM ATP,
15 mM MgSO4) by determining optical density in
a luminometer (Monolight 2010, Analytical Luminescence Laboratory).
Samples were assayed for
-galactosidase activity using standard
procedures (32) to normalize for transfection efficiency.
-mercaptoethanol, 100 µg/ml
leupeptin, 2 µg/ml aprotinin, 40 µM
Z-Asp-2,6-dichlorobenzoyloxime, and 1 mM
phenylmethylsulfonyl fluoride. The lysates were then cleared by
centrifugation at 16,000 × g for 5 min, normalized for
the protein amount, mixed 1:1 with SDS sample buffer (100 mM Tris, pH 6.8, 2% SDS, 300 mM
-mercaptoethanol, 30% glycerol, and 5% pyronine Y), boiled for 5 min, and stored at
70 °C. Proteins were electrophoresed by
SDS-PAGE (10% gels) and probed with antibodies directed against
I
B
(Santa Cruz No. 203). All lysates were standardized for
protein concentration with antibodies directed against actin (Oncogene
No. CP01). Autoradiographs were quantitated by densitometric analysis
using a Fluor-S MultiImager (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in TRAIL-resistant
HEK293 Cells--
Reovirus-induced apoptosis in a variety of
epithelial cell lines, including HEK293 cells and HeLa cells, is
mediated by TRAIL (7, 9). However, whereas reovirus induces similar
levels of apoptosis 48 h pi in HeLa and HEK293 cells (Fig.
1A), these cell types differ
substantially in their sensitivity to TRAIL-induced apoptosis (Fig.
1B).
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Fig. 1.
Reovirus and TRAIL-induced apoptosis in HeLa
and HEK293 cells. HEK293 and HeLa cells were treated with reovirus
(m.o.i. 0, 10, 100) for 48 h (A) or TRAIL (0-200
ng/ml) for 24 h (B). The graph shows the
mean percentage apoptosis obtained from three independent experiments.
Error bars represent S.E.
B in
TRAIL-sensitive (HeLa) cells and that this activation is required for
reovirus-induced apoptosis in these cells (15). To determine the role
of NF-
B in reovirus-induced apoptosis in TRAIL-resistant cells we
first investigated whether NF-
B is activated after reovirus infection of these cells. HEK293 cells were infected with reovirus (m.o.i. 100), and at various times pi nuclear extracts were prepared and incubated with a 32P-labeled oligonucleotide probe
comprising NF-
B binding sequences. After incubation with nuclear
extracts from reovirus-infected cells the mobility of the
oligonucleotide probe during electrophoresis was retarded, indicating
the binding of activated NF-
B to the probe sequences (Fig.
2A). Activated NF-
B-probe
complexes in reovirus-infected cells were present 2-4 h pi and were
undetectable at later times pi. Binding specificity was demonstrated by
the fact that an excess of cold NF-
B, but not SP-1, sequences
prevented the appearance of both the reovirus-induced (Fig. 2,
upper band) and non-stimulus-induced (Fig. 2, lower
band) NF-
B-probe complexes (Fig. 2B).
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Fig. 2.
Activation of NF- B
after reovirus infection of HEK293 cells. A, EMSA of
reovirus-infected HEK293 cells. Nuclear extracts were prepared at
various times after infection with reovirus (m.o.i. 100) and were
incubated with a 32P-labeled oligonucleotide consisting of
the NF-
B consensus binding sequence. Incubation mixtures were
resolved by acrylamide gel electrophoresis, dried, and exposed to film.
Reovirus-induced NF-
B·DNA complexes are indicated.
B, specificity of reovirus-induced NF-
B·DNA complexes.
HEK293 cells were infected with reovirus or were mock-infected. Nuclear
extracts were prepared 4 h after infection, and EMSA analysis was
performed using a 32P-labeled oligonucleotide consisting of
the NF-
B consensus binding sequence. The gel shows NF-
B·DNA
complexes present in nuclear extracts prepared from mock (lane
1) and reovirus (lane 2)-infected cells. Also shown are
reactions from reovirus-infected cells incubated with an excess of cold
oligonucleotide sequences comprising SP1 (lane 3) and
NF-
B (lane 4) consensus binding sequences. C,
NF-
B-dependent luciferase expression in
reovirus-infected HEK293 cells. HEK293 cells (1.5 × 105) expressing wild type (WT) I
B or
I
B
N2 were transfected with 1 µg of a luciferase reporter
construct containing NF-
B binding sites. After 24 h, cells were
infected with T3A (m.o.i. 100) and incubated at 37 °C for the times
shown. Cell extracts were then prepared, and luciferase activity was
determined. The results are expressed as the mean luciferase units for
three independent experiments. Error bars indicate
S.E.
B is
activated after infection of HEK293 cells with reovirus infection.
Cells were transfected with a construct containing the luciferase gene
under the control of NF-
B binding sequences. After transfection,
cells were infected with reovirus (m.o.i. 100), and at various times pi
cells were harvested and assayed for luciferase activity. Fig.
2C shows that luciferase gene expression is increased after
infection with reovirus. A 3-fold increase in reporter gene activity
was detected as early as 6 h pi, peaked at 12 h pi (5-fold
increase), and then declined (Fig. 2C). Luciferase reporter
gene activity was not detected 12 h after reovirus infection of
cells expressing a dominant negative form of I
B
N2, which lacks
the sites necessary for I
B phosphorylation. The subsequent ubiquitination and degradation of I
B, which is necessary for NF-
B
activation, is thus blocked in these cells. These results indicate that
NF-
B is activated in a transient manner after reovirus-infection of
HEK293 cells.
B Activation Is Required for Reovirus-induced
Apoptosis--
After having shown that NF-
B is activated after
reovirus infection of TRAIL-resistant cells we next wished to determine
whether NF-
B is required for reovirus-induced apoptosis in
these cells. HEK293 were infected with reovirus, and at various times
post-infection were harvested and assayed for apoptosis. Compared with
mock-infected cells, reovirus infection resulted in a significant
increase in the number of apoptotic cells at both 24 and 48 h
pi. However, reovirus-induced apoptosis was blocked in cells expressing
I
B
N2 (Fig. 3A). Reovirus-induced
apoptosis was also assayed by measuring caspase 3-activity using a
fluorogenic substrate assay. Increased caspase 3 activity, compared
with mock-infected cells, was detected at 18 h (3-fold) and
24 h (7.5-fold) pi. Again, reovirus-induced caspase 3-activity was
blocked in cells expressing I
B
N2 (Fig. 3B). These
results indicate that NF-
B activation is required for
reovirus-induced activation of caspase 3 and apoptosis.
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Fig. 3.
NF- B activation is required for
reovirus-induced apoptosis and caspase 3 activity.
HEK293 cells expressing wild type (WT) I
B or I
B
N2
were infected with reovirus (m.o.i. 100) and assayed for apoptosis
(A) and caspase 3 activity (B) at various times
pi. The results are expressed as the mean percentage apoptosis or
fold-activation obtained from three independent experiments.
Error bars indicate S.E.
B by TNF
and
Etoposide--
Although reovirus induces NF-
B after infection of
HEK293 cells, this activation is transient in nature. We next
investigated whether the transient nature of reovirus-induced NF-
B
activation resulted from a block in NF-
B activation at later times
pi. Both TNF
(100 ng/ml) and etoposide (100 µM) are
classic inducers of NF-
B and cause a rapid and robust activation of
NF-
B in HEK293 cells as determined by the appearance of a shifted
probe band after EMSA in treated, but not untreated cells (Fig.
4A). NF-
B binding was seen
as early as 1 h after treatment with TNF
and etoposide and was
persistent. Prior infection of cells with reovirus blocked the
appearance of the TNF
and etoposide-induced-shifted probe band
compared with that seen in mock-infected cells (Fig. 4B). In
contrast, there was no difference in the intensity of the lower
NF-
B-probe complex after etoposide or TNF
treatment in either
mock or reovirus-infected cells. These results indicate that
reovirus infection blocks the activation of NF-
B after treatment of
HEK293 cells with TNF
or etoposide, indicating that reovirus infection both induces and then inhibits the activation of NF-
B.
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Fig. 4.
Reovirus prevents TNF
and etoposide-induced activation of
NF-
B. A, time course of
NF-
B activation after treatment with TNF
and etoposide. HEK293
cells were treated with TNF
(100 ng/ml) or etoposide (100 µM) for the indicated times. Nuclear extracts were then
prepared, and EMSA analysis was performed using an oligonucleotide
probe comprising NF-
B binding sequences. Shifted bands,
corresponding to activated NF-
B·DNA complexes, are indicated.
B, prior infection with reovirus prevents TNF
- and
etoposide-induced NF-
B activation. HEK293 cells were infected with
reovirus or were mock-infected. 12 h pi cells were treated with
TNF
(100 ng/ml) or etoposide (100 µM)
(Etop.). Nuclear extracts were prepared after treatment at
the times indicated, and EMSA was performed using an oligonucleotide
probe comprising NF-
B binding sequences. Stimulus-induced
NF-
B·DNA complexes are indicated.
B after Treatment of Cells
with Etoposide and TNF--
Activation of NF-
B results from the
stimulus-induced degradation of the inhibitor family of proteins,
collectively known as I
B. Treatment of HEK293 cells with the
NF-
B-inducing stimuli etoposide (100 µM) and TNF (100 ng/ml) thus causes the degradation of I
B as detected by Western blot
analysis using an antibody directed against I
B
(Fig.
5A). Degradation of I
B
is detectable around 1 h after treatment with both TNF and
etoposide, and levels gradually decline over a 24-h period. In
contrast, no changes in levels of I
B
were detected after reovirus
infection (Fig. 5B). We next determined whether
reovirus blocked etoposide- and TNF-induced activation of NF-
B by
inhibiting I
B
degradation. Cells were infected with reovirus
(m.o.i. 100). Then, at various times pi cells were treated with
etoposide (100 µM) or TNF (100 ng/ml). After a further
3 h, to allow etoposide and TNF-induced I
B
degradation,
cells were harvested and assayed for the presence of I
B
by
Western blot analysis. When etoposide was added 2 h after reovirus
infection etoposide induced the degradation of I
B
as expected.
However, by 4 h pi the ability of etoposide to induce the
degradation of I
B
was inhibited, and at 12 h pi there was no
degradation of I
B
after etoposide treatment (Fig. 5B).
Similar results were obtained after TNF treatment of reovirus-infected cells (Fig. 5B). These results indicate that the mechanism
by which reovirus inhibits NF-
B activation at later times pi
involves inhibition of I
B degradation in reovirus-infected
cells.
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Fig. 5.
Reovirus prevents etoposide-induced
degradation of
I B
.
A, time course of etoposide- and TNF-induced I
B
degradation. HEK293 cells were treated with etoposide
(Etop., 100 µM) or TNF (100 ng/ml). After the
indicated times cells were harvested for Western blot analysis. After
SDS-PAGE, blots were probed with an anti-I
B
antibody.
B, time course of levels of I
B
in reovirus-infected
cells. HEK293 cells were infected with reovirus (m.o.i. 100). After the
indicated times cells were harvested for Western blot analysis. After
SDS-PAGE, blots were probed with an anti-I
B
antibody.
C, reovirus prevents etoposide- and TNF-induced degradation
of I
B
. HEK293 cells were infected with reovirus (m.o.i. 100). At
the times indicated pi cells were treated with etoposide (100 µM) or TNF (100 ng/ml). After a further 3 h, to
allow these reagents to induce I
B
degradation, cells were
harvested for Western blot analysis. After SDS-PAGE, blots were probed
with an anti-I
B
antibody. Also shown are the results from
densitometric analysis.
B
Degradation Requires Viral RNA Synthesis--
The ability of reovirus
to inhibit stimulus-induced I
B
degradation and subsequent NF-
B
activation occurs somewhat later than would be expected for the initial
events of viral infection, including receptor binding, viral entry, and
disassembly, and is more concurrent with the time at which viral
proteins are produced in reovirus-infected HEK293 cells (not shown).
Therefore we next investigated whether viral replication was required
for reovirus-induced inhibition of I
B degradation. Ribavirin is a
viral RNA synthesis inhibitor that inhibits reovirus replication (27).
Cells were infected with reovirus (m.o.i. 100) in the presence or
absence of ribavirin. 12 h after infection cells were treated with
etoposide (100 µM) for 3 h. They were then harvested
and analyzed by Western blot analysis using an I
B antibody. Fig.
6A shows that etoposide treatment of mock-infected cells in the presence or absence of ribavirin, results in the degradation of I
B. As expected, in reovirus-infected cells the ability of etoposide to induce the degradation of I
B is blocked. However, etoposide does induce I
B
degradation in cells treated with both reovirus and ribavirin, indicating that viral RNA synthesis is required for reovirus-induced inhibition of stimulus-induced I
B degradation.
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Fig. 6.
In HEK293 cells reovirus replication is
required for reovirus-induced inhibition of
NF- B activation by etoposide, reovirus-induced
apoptosis, and reovirus-induced sensitization of cells to TRAIL.
A, ribavirin blocks the ability of reovirus to inhibit
etoposide-induced I
B
degradation. Cells were infected with
reovirus or were mock-infected in the presence or absence of ribavirin.
12 h after infection, cells were treated with etoposide or were
left untreated for 3 h before cells were harvested for Western
blot analysis using an anti-I
B
antibody. B,
ribavirin blocks reovirus-induced apoptosis in HEK293 cells. HEK293
cells and HeLa cells were infected with reovirus (m.o.i. 100) or were
mock-infected (m.o.i. 0) in the absence (black bars) or
presence (shaded bars) of ribavirin. After 48 h cells
were harvested and assayed for apoptosis. The graph shows
the mean percentage apoptosis obtained from three independent
experiments. Error bars represent S.E. C,
ribavirin blocks reovirus-induced sensitization of cells to TRAIL.
HEK293 cells were infected with reovirus (m.o.i. 10) or were
mock-infected in the absence (black bars) or presence
(shaded bars) of ribavirin. 24 h after infection, cells
were treated with TRAIL (20 ng/ml). After a further 24 h cells
were harvested and assayed for apoptosis. The graph shows
the mean percentage apoptosis obtained from three independent
experiments. REO, reovirus. Error bars represent
S.E.
B
degradation requires viral replication and is blocked in the presence of ribavirin, we investigated the effect of ribavirin on
reovirus-induced apoptosis. HEK293 cells were infected with reovirus in
the presence or absence of ribavirin. After 48 h cells were
harvested and assayed for apoptosis. Ribavirin significantly inhibited
reovirus (m.o.i. 100)-induced apoptosis in HEK293 cells (Fig.
6B), indicating that reovirus-induced inhibition of
apoptosis of HEK293 cells requires viral RNA replication. In contrast,
ribavirin did not inhibit reovirus-induced apoptosis in TRAIL-sensitive
HeLa cells, as has previously been shown (27).
B Activation Sensitizes Cells to Apoptosis
Induced by TRAIL and TNF
--
Our results indicate that ribavirin
blocks both reovirus-induced apoptosis in TRAIL-resistant cells and
reovirus-induced sensitization of TRAIL-resistant cells to
TRAIL-induced apoptosis. Because ribavirin also blocks the ability of
reovirus to inhibit NF-
B activation in infected cells at later times
pi we wished to determine whether inhibition of NF-
B activation was
the mechanism by which reovirus sensitizes TRAIL-resistant cells to
TRAIL-induced apoptosis. HEK293 cells expressing I
B
N2
were treated with various concentrations of TRAIL. At 24 h after
treatment cells were harvested and assayed for apoptosis. High
concentrations of TRAIL (200 ng/ml) did not induce significant levels
of apoptosis in HEK293 cells expressing vector alone
compared with untreated cells. In contrast, both 20 and 200 ng/ml TRAIL
induced significant apoptosis in HEK293 cells expressing I
B
N2
(Fig. 7A).
Apoptosis was also determined using caspase 3 activity assays. Cells
were treated with similar concentrations of TRAIL and were harvested
4 h after treatment for caspase 3 activity assays. At 4 h pi
TRAIL (20 and 200 ng/ml) induced caspase 3 activity in HEK293 cells
expressing I
B
N2 but not in cells expressing vector alone (Fig.
7A). An 8-fold increase in caspase 3 activity was seen in
TRAIL (20 ng/ml)-treated cells expressing I
B
N2 compared with
cells expressing vector alone, and at 200 ng/ml TRAIL induced a 20-fold
increase. The expression of I
B
N2, thus, sensitizes HEK293 cells
to TRAIL-induced apoptosis, suggesting that reovirus-induced inhibition
of NF-
B activation is the mechanism by which reovirus sensitizes of
cells to TRAIL.
View larger version (16K):
[in a new window]
Fig. 7.
Expression of
I B
N2 sensitizes cells
to TRAIL and TNF
-induced apoptosis.
HEK293 cells expressing WT I
B or I
B
N2 were treated with the
indicated concentrations of TRAIL (A), TNF
(B), and etoposide (Etop.,
C). After treatment cells were harvested and assayed for
apoptosis or caspase 3 activity. The graph shows the mean
percentage apoptosis and fold-increase in caspase 3 activity obtained
from three independent experiments. Error bars represent
S.E.
and etoposide induce the activation of NF-
B
in HEK293 cells. TRAIL also induces NF-
B activation in these cells
(31). Having shown that the expression of I
B
N2 sensitizes cells
to TRAIL-induced apoptosis, we next determined whether the expression
of I
B
N2 would also sensitize HEK293 cells to TNF and
etoposide-induced apoptosis. Neither TNF
(200 ng/ml) nor etoposide
(100 µM) induced apoptosis in HEK293 cells. However, whereas levels of TNF
as low as 2 ng/ml induced significant
apoptosis in cells expressing I
B
N2 (Fig. 7B), the
expression of I
B
N2 did not sensitize cells to etoposide (100 µM)-induced apoptosis (Fig. 7C). Similarly a
12-fold increase in caspase 3 activation was seen 24 h after
TNF
(20 and 200 ng/ml) treatment of cells expressing I
B
N2 but
not after TNF
treatment of cells expressing vector alone (Fig.
7B). Again, caspase-3 activation was not enhanced in
etoposide-treated cells expressing I
B
N2 compared with cells expressing vector alone (Fig. 7C). These results indicate
that the expression of I
B
N2 sensitizes HEK293 cells to TRAIL and TNF
but not etoposide-induced apoptosis.
and TRAIL--
We have previously shown that reovirus infection
sensitizes HEK293 cells to TRAIL-induced apoptosis. Results described
above show that blocking NF-
B activation sensitizes cells to both
TRAIL- and TNF
-induced apoptosis. Because reovirus-induced
inhibition of NF-
B activation is the mechanism by which reovirus
sensitizes cells to TRAIL-induced apoptosis, we wanted to determine
whether the inhibition of NF-
B activation after reovirus infection
would also sensitize these cells to TNF
-induced apoptosis. Cells
were incubated with reovirus (m.o.i. 10). 24 h post-infection
cells were then treated with TRAIL (20 ng/ml) or TNF
(20 ng/ml).
Apoptosis was then determined after a further 24 h. Fig.
8 shows that TRAIL and TNF
alone do
not induce apoptosis in HEK293 cells, as previously shown (Fig. 7).
Infection of HEK293 cells with reovirus (m.o.i. 10) also induces only
low levels (22%) of apoptosis in HEK293 cells (see Fig. 1). However,
treatment of cells with TRAIL or TNF
in the presence of reovirus
produces high levels (70 and 63%) of apoptosis in HEK293 cells. These
values are significantly greater than the sum of apoptosis induced by
TRAIL and reovirus or TNF
and reovirus when these agents are used
alone, indicating that reovirus infection acts synergistically with
TRAIL and TNF
to induce apoptosis. In contrast, reovirus did not
sensitize cells to etoposide-induced apoptosis, in agreement with the
observation that the expression of I
B
N2 also does not sensitize
these cells to etoposide.
View larger version (11K):
[in a new window]
Fig. 8.
Reovirus sensitizes HEK293 cells to TRAIL and
TNF -induced apoptosis. HEK293 cells were
infected with reovirus (m.o.i. 10) or were mock-infected. 24 h
after infection, cells were treated with TRAIL, TNF
, or etoposide
(etop) or were left untreated. After a further 24 h,
cells were harvested and assayed for apoptosis. The graph
shows the mean percentage apoptosis obtained from three independent
experiments. Error bars represent S.E. Reo,
reovirus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and that this activation
is required for reovirus-induced apoptosis (26). The results presented
in this report describe the role of NF-
B in reovirus-induced
apoptosis in TRAIL-resistant (HEK293) cells. We show that
reovirus-induced NF-
B activation is highly regulated in these cells.
At early times pi (2-4 h) reovirus activates NF-
B, as demonstrated
by the presence of NF-
B in the nucleus of reovirus-infected cells
and by the ability of this NF-
B to bind to radiolabeled
oligonucleotide probe sequences comprising NF-
B binding sites.
Activation of NF-
B-responsive genes after reovirus infection of
HEK293 cells is also demonstrated by luciferase reporter gene assays.
NF-
B activation is required for reovirus-induced apoptosis since
reovirus-infection does not result in caspase 3 activity or
apoptosis-associated changes in nuclear morphology in HEK293 cells
expressing I
B
N2. These results are similar to those observed for
TRAIL-sensitive HeLa cells and suggest that reovirus-induced apoptosis
in TRAIL resistant cells also requires the expression of pro-apoptotic
NF-
B-regulated genes.
B activation is
transient in both reovirus-infected TRAIL-sensitive and TRAIL-resistant
cells. The transient nature of NF-
B activation in HEK293 cells
results from the inhibition of NF-
B activation at later times pi
since reovirus can block the ability of both etoposide and TNF
to
induce NF-
B activation. This inhibition of NF-
B activation
results from the inhibition of stimulus-induced I
B degradation and
is time-dependent. Thus, at early times post-infection (2, 4 h) etoposide or TNF are still able to induce the degradation of
I
B
. However, at later times post-infection (8-12 h) neither reagent induces I
B
degradation. These results are consistent with
the fact that reovirus only activates NF-
B at early times pi.
B activation in reovirus-infected
cells might be expected to induce a concordant increase in levels of
I
B
at later times pi, we were unable to detect such a change. We
predict that this is because of the low levels of NF-
B that are
activated in reovirus-infected cells and the relative insensitivity of
Western blotting compared with EMSA.
B is not dependent on viral
replication and occurs in the presence of ribavirin in both HeLa (27)
and HEK293 cells.2 Because
the ability of reovirus to inhibit stimulus-induced NF-
B activation
occurs somewhat later than the initial infection events (receptor
binding, viral entry, and disassembly) and occurs around the time that
viral proteins are produced in reovirus-infected HEK293 cells (not
shown), we next investigated whether viral RNA synthesis was required.
In the presence of the viral RNA synthesis inhibitor ribavirin the
ability of etoposide to induce the degradation of I
B was completely
blocked, indicating that viral RNA synthesis is required for this process.
B regulates genes with both pro- and anti-apoptotic effects. The
ability of reovirus to block NF-
B activation at later times
post-infection enhances virus-induced apoptosis in TRAIL-resistant cells, as demonstrated by three lines of investigation. First, TRAIL-induced apoptosis is enhanced in HEK293 cells expressing I
B
N2. This indicates that blocking NF-
B activation sensitizes cells to TRAIL-induced apoptosis. Second, ribavirin, which blocks the
ability of reovirus to inhibit stimulus-induced I
B degradation, blocks reovirus-induced apoptosis in TRAIL-resistant, but not TRAIL-sensitive cells. Finally, ribavirin also blocks the ability of
reovirus to sensitize cells to TRAIL-induced apoptosis.
may be more important in mediating
reovirus-induced apoptosis than TRAIL (5). The results presented here indicate that reovirus can also sensitize cells to TNF
-induced apoptosis by inhibiting NF-
B activation at later times pi, which may
have important consequences for the ability of reovirus to induce
apoptosis in these cells and to cause disease of the central nervous
system in infected animals. The expression of I
B
N2 was not found
to sensitize cells to etoposide-induced apoptosis. This suggests that
etoposide induces apoptosis by a mechanism that is different from that
induced by TRAIL and TNF
. Previous studies show that NF-
B
activation is required for etoposide-induced apoptosis (32), supporting
our observation that the expression of I
B
N2 does not sensitize
HEK293 cells to etoposide-induced apoptosis.
B is also critical for
virus-induced apoptosis. NF-
B is first activated at early times
after reovirus infection, an event that is required for apoptosis in
both TRAIL-sensitive and TRAIL-resistant cells and which presumably
acts to up-regulate the expression of pro-apoptotic NF-
B-regulated
genes. Both TRAIL and its receptors are regulated by NF-
B (32-34).
It is, thus, likely that the pro-apoptotic effects of NF-
B
activation that are required for reovirus-induced apoptosis include the
up-regulation of these genes. TRAIL, DR4, and DR5 are up-regulated
after reovirus-infection (7), although the involvement of
NF-
B in this process has yet to be established. At later times pi,
reovirus inhibits the activation of NF-
B in infected cells. This has
the effect of blocking stimulus-induced NF-
B activation. In
uninfected HEK293 cells TRAIL induces the activation of NF-
B (31).
Our results suggest that TRAIL-induced NF-
B activation has an
inhibitory effect on TRAIL-induced apoptosis in these cells. Thus, the
ability of reovirus to block TRAIL-induced NF-
B activation will
sensitize cells to TRAIL-induced apoptosis, therefore allowing both
TRAIL and reovirus-induced apoptosis in TRAIL-resistant cells. The
timing of reovirus-induced inhibition of stimulus-induced NF-
B
activation is in accordance with TRAIL release from reovirus-infected
cells, which occurs at later times pi (7). Thus, it appears that
NF-
B activation is turned off in reovirus-infected cells before
TRAIL is released to facilitate reovirus-induced apoptosis in
TRAIL-resistant cells.
B activation. Other studies also
indicate that blocking NF-
B activation can sensitize human cancer
cells to TRAIL-induced apoptosis (35-37). Together these findings
could have an important impact on the use of TRAIL as a potential
cancer therapeutic in combination with other agents that inhibit
NF-
B.
B pathway provides an attractive target to viral pathogens
for modulating host cell events. NF-
B promotes the expression of
more than 100 genes that participate in the host immune response, oncogenesis, and regulation of apoptosis. In addition, activation of
NF-
B is a rapid immediate early response that occurs within minutes
after exposure to a relevant inducer, does not require de
novo protein synthesis, and results in a strong transcriptional stimulation of several early viral as well as cellular
genes. NF-
B is, thus, activated by multiple families of viruses,
including human immunodeficiency virus type 1 (HIV-1) (38), human
T-cell lymphotrophic virus- (39), hepatitis B virus (40), hepatitis C
virus (41, 42), Epstein-Barr virus (43), rotavirus (44), and influenza
virus (45) to promote viral replication, prevent virus-induced
apoptosis, and mediate the immune response to the invading pathogen
(for review, see Ref. 46). In contrast, activation of NF-
B by
Sindbis (47, 48) and Dengue virus (49) is associated with the induction
of apoptosis, which may increase viral spread. In still other cases,
proteins encoded by adenovirus (50), hepatitis C virus (51), and
African swine fever virus (52) inhibit NF-
B activity to enhance
replication or contribute to viral pathogenicity.
B activity to efficiently induce apoptosis in
infected cells. This is the first time that two phases of NF-
B
regulation have been shown to be required to modulate viral-host
interactions within a specific cell type. We propose that the complex
regulation of NF-
B by reovirus is critical for TRAIL- and
TNF
-induced apoptosis in reovirus-infected cells. Death receptor
ligands are commonly used by viruses to induce apoptosis. For example,
HIV infection increases the expression of TRAIL and sensitizes T-cells
to TRAIL-mediated apoptosis (53). In addition, alteration of the cell
surface expression of Fas may be involved in virus-induced or viral
regulation of apoptosis in cells infected with influenza virus (54,
55), herpes simplex virus type 2 (56), bovine herpesvirus 4 (57),
adenovirus (58) and HIV 1 (59, 60). Similarly, apoptosis induced by
hepatitis B (61), HIV-1 (62), bovine herpesvirus 4 (57), and parvovirus H-1 (63) may involve the TNF receptor signaling pathway. NF-
B regulation is, thus, likely to have implications for apoptosis and
disease resulting from a variety of viral infections.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institute of Health Public Health Service Grant 1RO1AG14071, Merit and Research Enhancement Award Program (REAP) grants from the Department of Veterans Affairs, U. S. Army Medical Research and Material Command Grant DAMD17-98-1-8614, and by the Reuler-Lewin Family Professorship of Neurology.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 the Ovarian Cancer Research Fund.
¶ To whom correspondence should be addressed: Dept. of Neurology (B 182), University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver CO 80262. Tel.: 303-393-2874; Fax: 303-393-4686; E-mail: Ken.Tyler@uchsc.edu.
Published, JBC Papers in Press, March 13, 2003, DOI 10.1074/jbc.M300265200
2 P. Clarke, S. M. Meintzer, L. Moffitt, and K. L. Tyler, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
TRAIL, TNF-related apoptosis-inducing ligand;
NF-B, nuclear
factor
B;
I
B, inhibitor
B;
pi, post-infection;
EMSA, electrophoretic mobility shift assay;
m.o.i., multiplicity of
infection;
HIV-1, human immunodeficiency virus type.
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