Two Distinct Phases of Virus-induced Nuclear Factor kappa B Regulation Enhance Tumor Necrosis Factor-related Apoptosis-inducing Ligand-mediated Apoptosis in Virus-infected Cells*

Penny ClarkeDagger, Suzanne M. Meintzer, Lisa A. Moffitt, and Kenneth L. Tyler§

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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular transcription factors are often utilized by infecting viruses to promote viral growth and influence cell fate. We have previously shown that nuclear factor kappa B (NF-kappa 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-kappa B regulation. We show that at later times post-infection NF-kappa B activation is blocked in reovirus-infected cells. This results in the termination of virus-induced NF-kappa B activity and the inhibition of tumor necrosis factor alpha  and etoposide-induced NF-kappa B activation in infected cells. Reovirus-induced inhibition of NF-kappa B activation occurs by a mechanism that prevents Ikappa Balpha 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 Ikappa BDelta N2, which blocks NF-kappa B activation. These results indicate that the ability of reovirus to inhibit NF-kappa 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-kappa B regulation are required to efficiently activate host cell apoptotic responses to reovirus infection.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa B-regulated genes may determine cell fate (12). The prototypical form of NF-kappa B exists as a heterodimer of proteins p50 and p65 (RelA) (13, 14). NF-kappa B is normally sequestered in the cytoplasm by its binding to a family of inhibitor proteins, collectively known as Ikappa B (15, 16). In response to a variety of stimuli, Ikappa B is phosphorylated, resulting in its ubiquitination and subsequent degradation (17-20). This allows the release of NF-kappa 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-kappa 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-kappa B, and is required for reovirus-induced apoptosis (26). In HeLa cells reovirus-induced NF-kappa 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).

The experiments described below investigate the role of NF-kappa 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-kappa B activation that is required for reovirus-induced apoptosis in these cells. This is followed by a later phase of virus-induced NF-kappa B inhibition. Reovirus-induced inhibition of NF-kappa B activation is associated with impaired degradation of Ikappa 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 Ikappa BDelta N2, which blocks the activation of NF-kappa B. This suggests that the ability of reovirus to block NF-kappa 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-kappa B regulation are required to efficiently activate host cell apoptotic responses to reovirus infection represents a novel mechanism of viral-induced apoptosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 Ikappa BDelta N2, a strong dominant negative Ikappa B mutant lacking the NH2-terminal phosphorylation sites that regulate Ikappa B degradation and the consequent activation of NF-kappa 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, TNFalpha was obtained from Invitrogen, and etoposide and ribavirin were obtained from Sigma. Ribavirin was used at a concentration of 200 µM.

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 -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.

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-kappa B activation by EMSA using a 32P-labeled oligonucleotide consisting of the NF-kappa 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-kappa 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.).

Luciferase Gene Reporter Assays-- The NF-kappa B-dependent luciferase reporter construct was a gift from Dr. B. Sugden. The construct contains four NF-kappa 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-beta -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 beta -galactosidase activity using standard procedures (32) to normalize for transfection efficiency.

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 beta -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 beta -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 Ikappa Balpha (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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reovirus Induces the Activation of NF-kappa 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.

We have previously shown that reovirus activates NF-kappa 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-kappa B in reovirus-induced apoptosis in TRAIL-resistant cells we first investigated whether NF-kappa 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-kappa 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-kappa B to the probe sequences (Fig. 2A). Activated NF-kappa 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-kappa 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-kappa B-probe complexes (Fig. 2B).


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Fig. 2.   Activation of NF-kappa 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-kappa B consensus binding sequence. Incubation mixtures were resolved by acrylamide gel electrophoresis, dried, and exposed to film. Reovirus-induced NF-kappa B·DNA complexes are indicated. B, specificity of reovirus-induced NF-kappa 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-kappa B consensus binding sequence. The gel shows NF-kappa 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-kappa B (lane 4) consensus binding sequences. C, NF-kappa B-dependent luciferase expression in reovirus-infected HEK293 cells. HEK293 cells (1.5 × 105) expressing wild type (WT) Ikappa B or Ikappa BDelta N2 were transfected with 1 µg of a luciferase reporter construct containing NF-kappa 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.

Luciferase reporter gene assays were also used to show that NF-kappa 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-kappa 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 Ikappa BDelta N2, which lacks the sites necessary for Ikappa B phosphorylation. The subsequent ubiquitination and degradation of Ikappa B, which is necessary for NF-kappa B activation, is thus blocked in these cells. These results indicate that NF-kappa B is activated in a transient manner after reovirus-infection of HEK293 cells.

NF-kappa B Activation Is Required for Reovirus-induced Apoptosis-- After having shown that NF-kappa B is activated after reovirus infection of TRAIL-resistant cells we next wished to determine whether NF-kappa 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 Ikappa BDelta 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 Ikappa BDelta N2 (Fig. 3B). These results indicate that NF-kappa B activation is required for reovirus-induced activation of caspase 3 and apoptosis.


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Fig. 3.   NF-kappa B activation is required for reovirus-induced apoptosis and caspase 3 activity. HEK293 cells expressing wild type (WT) Ikappa B or Ikappa BDelta 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.

Reovirus Prevents the Activation of NF-kappa B by TNFalpha and Etoposide-- Although reovirus induces NF-kappa B after infection of HEK293 cells, this activation is transient in nature. We next investigated whether the transient nature of reovirus-induced NF-kappa B activation resulted from a block in NF-kappa B activation at later times pi. Both TNFalpha (100 ng/ml) and etoposide (100 µM) are classic inducers of NF-kappa B and cause a rapid and robust activation of NF-kappa 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-kappa B binding was seen as early as 1 h after treatment with TNFalpha and etoposide and was persistent. Prior infection of cells with reovirus blocked the appearance of the TNFalpha 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-kappa B-probe complex after etoposide or TNFalpha treatment in either mock or reovirus-infected cells. These results indicate that reovirus infection blocks the activation of NF-kappa B after treatment of HEK293 cells with TNFalpha or etoposide, indicating that reovirus infection both induces and then inhibits the activation of NF-kappa B.


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Fig. 4.   Reovirus prevents TNFalpha and etoposide-induced activation of NF-kappa B. A, time course of NF-kappa B activation after treatment with TNFalpha and etoposide. HEK293 cells were treated with TNFalpha (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-kappa B binding sequences. Shifted bands, corresponding to activated NF-kappa B·DNA complexes, are indicated. B, prior infection with reovirus prevents TNFalpha - and etoposide-induced NF-kappa B activation. HEK293 cells were infected with reovirus or were mock-infected. 12 h pi cells were treated with TNFalpha (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-kappa B binding sequences. Stimulus-induced NF-kappa B·DNA complexes are indicated.

Reovirus Blocks the Degradation of Ikappa B after Treatment of Cells with Etoposide and TNF-- Activation of NF-kappa B results from the stimulus-induced degradation of the inhibitor family of proteins, collectively known as Ikappa B. Treatment of HEK293 cells with the NF-kappa B-inducing stimuli etoposide (100 µM) and TNF (100 ng/ml) thus causes the degradation of Ikappa B as detected by Western blot analysis using an antibody directed against Ikappa Balpha (Fig. 5A). Degradation of Ikappa Balpha 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 Ikappa Balpha were detected after reovirus infection (Fig. 5B). We next determined whether reovirus blocked etoposide- and TNF-induced activation of NF-kappa B by inhibiting Ikappa Balpha 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 Ikappa Balpha degradation, cells were harvested and assayed for the presence of Ikappa Balpha by Western blot analysis. When etoposide was added 2 h after reovirus infection etoposide induced the degradation of Ikappa Balpha as expected. However, by 4 h pi the ability of etoposide to induce the degradation of Ikappa Balpha was inhibited, and at 12 h pi there was no degradation of Ikappa Balpha 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-kappa B activation at later times pi involves inhibition of Ikappa B degradation in reovirus-infected cells.


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Fig. 5.   Reovirus prevents etoposide-induced degradation of Ikappa Balpha . A, time course of etoposide- and TNF-induced Ikappa Balpha 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-Ikappa Balpha antibody. B, time course of levels of Ikappa Balpha 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-Ikappa Balpha antibody. C, reovirus prevents etoposide- and TNF-induced degradation of Ikappa Balpha . 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 Ikappa Balpha degradation, cells were harvested for Western blot analysis. After SDS-PAGE, blots were probed with an anti-Ikappa Balpha antibody. Also shown are the results from densitometric analysis.

Reovirus-induced Inhibition of Stimulus-induced Ikappa Balpha Degradation Requires Viral RNA Synthesis-- The ability of reovirus to inhibit stimulus-induced Ikappa Balpha degradation and subsequent NF-kappa 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 Ikappa 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 Ikappa B antibody. Fig. 6A shows that etoposide treatment of mock-infected cells in the presence or absence of ribavirin, results in the degradation of Ikappa B. As expected, in reovirus-infected cells the ability of etoposide to induce the degradation of Ikappa B is blocked. However, etoposide does induce Ikappa B degradation in cells treated with both reovirus and ribavirin, indicating that viral RNA synthesis is required for reovirus-induced inhibition of stimulus-induced Ikappa B degradation.


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Fig. 6.   In HEK293 cells reovirus replication is required for reovirus-induced inhibition of NF-kappa 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 Ikappa Balpha 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-Ikappa Balpha 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.

Reovirus-induced Apoptosis in HEK293 Cells Requires Viral RNA Synthesis-- Having shown that reovirus-induced inhibition of Ikappa 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).

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-kappa B Activation Sensitizes Cells to Apoptosis Induced by TRAIL and TNFalpha -- 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-kappa B activation in infected cells at later times pi we wished to determine whether inhibition of NF-kappa B activation was the mechanism by which reovirus sensitizes TRAIL-resistant cells to TRAIL-induced apoptosis. HEK293 cells expressing Ikappa BDelta 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 Ikappa BDelta 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 Ikappa BDelta 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 Ikappa BDelta N2 compared with cells expressing vector alone, and at 200 ng/ml TRAIL induced a 20-fold increase. The expression of Ikappa BDelta N2, thus, sensitizes HEK293 cells to TRAIL-induced apoptosis, suggesting that reovirus-induced inhibition of NF-kappa B activation is the mechanism by which reovirus sensitizes of cells to TRAIL.


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Fig. 7.   Expression of Ikappa BDelta N2 sensitizes cells to TRAIL and TNFalpha -induced apoptosis. HEK293 cells expressing WT Ikappa B or Ikappa BDelta N2 were treated with the indicated concentrations of TRAIL (A), TNFalpha (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.

Fig. 4 shows that TNFalpha and etoposide induce the activation of NF-kappa B in HEK293 cells. TRAIL also induces NF-kappa B activation in these cells (31). Having shown that the expression of Ikappa BDelta N2 sensitizes cells to TRAIL-induced apoptosis, we next determined whether the expression of Ikappa BDelta N2 would also sensitize HEK293 cells to TNF and etoposide-induced apoptosis. Neither TNFalpha (200 ng/ml) nor etoposide (100 µM) induced apoptosis in HEK293 cells. However, whereas levels of TNFalpha as low as 2 ng/ml induced significant apoptosis in cells expressing Ikappa BDelta N2 (Fig. 7B), the expression of Ikappa BDelta 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 TNFalpha (20 and 200 ng/ml) treatment of cells expressing Ikappa BDelta N2 but not after TNFalpha treatment of cells expressing vector alone (Fig. 7B). Again, caspase-3 activation was not enhanced in etoposide-treated cells expressing Ikappa BDelta N2 compared with cells expressing vector alone (Fig. 7C). These results indicate that the expression of Ikappa BDelta N2 sensitizes HEK293 cells to TRAIL and TNFalpha but not etoposide-induced apoptosis.

Virus Infection Sensitizes HEK293 Cells to Apoptosis Induced by TNFalpha and TRAIL-- We have previously shown that reovirus infection sensitizes HEK293 cells to TRAIL-induced apoptosis. Results described above show that blocking NF-kappa B activation sensitizes cells to both TRAIL- and TNFalpha -induced apoptosis. Because reovirus-induced inhibition of NF-kappa B activation is the mechanism by which reovirus sensitizes cells to TRAIL-induced apoptosis, we wanted to determine whether the inhibition of NF-kappa B activation after reovirus infection would also sensitize these cells to TNFalpha -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 TNFalpha (20 ng/ml). Apoptosis was then determined after a further 24 h. Fig. 8 shows that TRAIL and TNFalpha 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 TNFalpha 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 TNFalpha and reovirus when these agents are used alone, indicating that reovirus infection acts synergistically with TRAIL and TNFalpha to induce apoptosis. In contrast, reovirus did not sensitize cells to etoposide-induced apoptosis, in agreement with the observation that the expression of Ikappa BDelta N2 also does not sensitize these cells to etoposide.


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Fig. 8.   Reovirus sensitizes HEK293 cells to TRAIL and TNFalpha -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, TNFalpha , 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

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-kappa B and that this activation is required for reovirus-induced apoptosis (26). The results presented in this report describe the role of NF-kappa B in reovirus-induced apoptosis in TRAIL-resistant (HEK293) cells. We show that reovirus-induced NF-kappa B activation is highly regulated in these cells. At early times pi (2-4 h) reovirus activates NF-kappa B, as demonstrated by the presence of NF-kappa B in the nucleus of reovirus-infected cells and by the ability of this NF-kappa B to bind to radiolabeled oligonucleotide probe sequences comprising NF-kappa B binding sites. Activation of NF-kappa B-responsive genes after reovirus infection of HEK293 cells is also demonstrated by luciferase reporter gene assays. NF-kappa 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 Ikappa BDelta 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-kappa B-regulated genes.

Although required for virus-induced apoptosis, NF-kappa B activation is transient in both reovirus-infected TRAIL-sensitive and TRAIL-resistant cells. The transient nature of NF-kappa B activation in HEK293 cells results from the inhibition of NF-kappa B activation at later times pi since reovirus can block the ability of both etoposide and TNFalpha to induce NF-kappa B activation. This inhibition of NF-kappa B activation results from the inhibition of stimulus-induced Ikappa 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 Ikappa Balpha . However, at later times post-infection (8-12 h) neither reagent induces Ikappa Balpha degradation. These results are consistent with the fact that reovirus only activates NF-kappa B at early times pi.

Although the inhibition of NF-kappa B activation in reovirus-infected cells might be expected to induce a concordant increase in levels of Ikappa Balpha at later times pi, we were unable to detect such a change. We predict that this is because of the low levels of NF-kappa B that are activated in reovirus-infected cells and the relative insensitivity of Western blotting compared with EMSA.

Reovirus-induced activation of NF-kappa 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-kappa 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 Ikappa B was completely blocked, indicating that viral RNA synthesis is required for this process.

NF-kappa B regulates genes with both pro- and anti-apoptotic effects. The ability of reovirus to block NF-kappa 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 Ikappa BDelta N2. This indicates that blocking NF-kappa B activation sensitizes cells to TRAIL-induced apoptosis. Second, ribavirin, which blocks the ability of reovirus to inhibit stimulus-induced Ikappa 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.

In neurons TNFalpha may be more important in mediating reovirus-induced apoptosis than TRAIL (5). The results presented here indicate that reovirus can also sensitize cells to TNFalpha -induced apoptosis by inhibiting NF-kappa 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 Ikappa BDelta 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 TNFalpha . Previous studies show that NF-kappa B activation is required for etoposide-induced apoptosis (32), supporting our observation that the expression of Ikappa BDelta N2 does not sensitize HEK293 cells to etoposide-induced apoptosis.

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-kappa B is also critical for virus-induced apoptosis. NF-kappa 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-kappa B-regulated genes. Both TRAIL and its receptors are regulated by NF-kappa B (32-34). It is, thus, likely that the pro-apoptotic effects of NF-kappa 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-kappa B in this process has yet to be established. At later times pi, reovirus inhibits the activation of NF-kappa B in infected cells. This has the effect of blocking stimulus-induced NF-kappa B activation. In uninfected HEK293 cells TRAIL induces the activation of NF-kappa B (31). Our results suggest that TRAIL-induced NF-kappa B activation has an inhibitory effect on TRAIL-induced apoptosis in these cells. Thus, the ability of reovirus to block TRAIL-induced NF-kappa 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-kappa B activation is in accordance with TRAIL release from reovirus-infected cells, which occurs at later times pi (7). Thus, it appears that NF-kappa B activation is turned off in reovirus-infected cells before TRAIL is released to facilitate reovirus-induced apoptosis in TRAIL-resistant cells.

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-kappa B activation. Other studies also indicate that blocking NF-kappa 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-kappa B.

The NF-kappa B pathway provides an attractive target to viral pathogens for modulating host cell events. NF-kappa 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-kappa 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-kappa 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-kappa 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-kappa B activity to enhance replication or contribute to viral pathogenicity.

The results demonstrated here indicate that reovirus both activates and then inhibits NF-kappa B activity to efficiently induce apoptosis in infected cells. This is the first time that two phases of NF-kappa 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-kappa B by reovirus is critical for TRAIL- and TNFalpha -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-kappa 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.

Dagger 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-kappa B, nuclear factor kappa B; Ikappa B, inhibitor kappa 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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DISCUSSION
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