b kinase subunits and are required for activation of nf- b...

JOURNAL OF VIROLOGY, Feb. 2007, p. 1360–1371 Vol. 81, No. 70022-538X/07/$08.00�0 doi:10.1128/JVI.01860-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

I�B Kinase Subunits � and � Are Required for Activation of NF-�Band Induction of Apoptosis by Mammalian Reovirus�

Mark W. Hansberger,1,2 Jacquelyn A. Campbell,1,2 Pranav Danthi,2,3 Pia Arrate,1Kevin N. Pennington,1 Kenneth B. Marcu,4,5 Dean W. Ballard,1

and Terence S. Dermody1,2,3*Departments of Microbiology and Immunology1 and Pediatrics,3 and Elizabeth B. Lamb Center for Pediatric Research,2

Vanderbilt University School of Medicine, Nashville, Tennessee 37232; Department of Biochemistry andCell Biology, Institute for Cell and Developmental Biology, Stony Brook University,

Stony Brook, New York 117944; and Centro Ricerca Biomedica Applicata, PAD23,St. Orsola University Hospital, University of Bologna, Bologna, Italy 401385

Received 25 August 2006/Accepted 10 November 2006

Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in thepathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is depen-dent on the activation of transcription factor NF-�B and downstream cellular genes. To better understand themechanism of NF-�B activation by reovirus, NF-�B signaling intermediates under reovirus control wereinvestigated at the level of Rel, I�B, and I�B kinase (IKK) proteins. We found that reovirus infection leadsinitially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. Thisbiphasic pattern of Rel protein activation is associated with the degradation of the NF-�B inhibitor I�B� butnot the structurally related inhibitors I�B� or I�B�. Using IKK subunit-specific small interfering RNAs andcells deficient in individual IKK subunits, we demonstrate that IKK� but not IKK� is required for reovirus-induced NF-�B activation and apoptosis. Despite the preferential usage of IKK�, both NF-�B activation andapoptosis were attenuated in cells lacking IKK�/Nemo, an essential regulatory subunit of IKK�. Moreover,deletion of the gene encoding NF-�B-inducing kinase, which is known to modulate IKK� function, had noinhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novelpathway of NF-�B/Rel activation involving IKK� and IKK�/Nemo, which together mediate the expression ofdownstream proapoptotic genes in reovirus-infected cells.

Mammalian reoviruses are nonenveloped viruses that con-tain a genome of 10 segments of double-stranded RNA (52).Following infection of newborn mice, reovirus disseminatessystemically, causing injury to the central nervous system(CNS), heart, and liver (76). Apoptosis induced by reovirusappears to be the primary mechanism for virus-induced en-cephalitis (53, 54, 59) and myocarditis (22, 23, 54). Disassemblyof internalized virus in the endocytic pathway provides theinitial viral trigger for stimulating the signaling pathways thatelicit an apoptotic response (19, 21).

Transcription factor NF-�B plays an important regulatoryrole in apoptosis evoked by reovirus in cultured cells (20) andin vivo (54). Inducible members of the NF-�B family are se-questered in the cytoplasm by inhibitory I�B proteins, includ-ing I�B�, I�B�, I�Bε, and p100/NF-�B2 (3, 31, 68, 79, 82). Inresponse to a wide variety of NF-�B inducers, I�B proteins arephosphorylated at specific serine residues, earmarking thesemolecules for destruction by the ubiquitin-proteasome path-way (7, 12, 31, 56, 75, 82). Phosphorylation of I�B proteins ismediated by cytokine-inducible I�B kinases (IKKs) IKK� andIKK� (47, 78), which can form higher-order complexes con-taining a regulatory subunit called IKK�/Nemo (26, 50, 62, 88,

91). A primary function of IKK� is to modulate the inhibitoryinteraction of I�B� with the prototypical form of NF-�B con-taining p50/RelA dimers (25, 26, 50, 58, 69). This regulatorycircuit, termed the classical pathway of NF-�B activation, isstrictly dependent on the presence of IKK�/Nemo (64, 65, 88).In contrast, IKK� functions in an alternative IKK�-indepen-dent pathway of NF-�B activation that leads to the proteolyticprocessing of p100 and the production of a functional p52 Relsubunit (16, 66, 71). Unlike the classical IKK�-directed path-way of NF-�B activation, the alternative pathway involvingIKK� is dependent on its prior phosphorylation by NF-�B-inducing kinase (NIK) (43, 66, 85). In addition to the cytoplas-mic function of IKK�, a nuclear role for IKK� in the tran-scriptional activation of NF-�B-responsive genes has beensuggested by in vitro studies (1, 32, 33, 41, 48, 69, 87).

To better understand the mechanism of NF-�B activation byreovirus, we conducted experiments to define the NF-�B/Rel,I�B, and IKK proteins that are under reovirus control. Thesestudies revealed that NF-�B/Rel proteins are mobilized to thenuclear compartment with biphasic kinetics following reovirusinfection. Reovirus-induced activation of NF-�B/Rel proteinsis accompanied by selective degradation of I�B�, suggesting arole for IKK�. However, additional studies with IKK-specificsmall interfering RNAs (siRNAs) and IKK-deficient cellsclearly demonstrate that IKK� rather than IKK� plays anessential role in the mechanism by which reovirus activatesNF-�B and downstream apoptotic genes. We also assembledevidence indicating that the reovirus/IKK� axis is intact in cells

* Corresponding author. Mailing address: Lamb Center for Pediat-ric Research, D7235 MCN, Vanderbilt University School of Medicine,Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615) 343-9723.E-mail: [email protected].

� Published ahead of print on 22 November 2006.

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lacking NIK, an upstream activator of IKK�, but not in cellslacking the IKK� regulatory subunit IKK�/Nemo. Taken to-gether, these data suggest that reovirus activates the IKK�pathway of the NF-�B signaling apparatus downstream ofNIK, perhaps via direct interactions with the regulatory sub-unit IKK�/Nemo.

MATERIALS AND METHODS

Cells, viruses, and reagents. HeLa cells, 293T cells, and murine embryo fibro-blasts (MEFs) were maintained in Dulbecco modified Eagle medium containing10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml of penicillin, 100 �g/mlstreptomycin, and 25 ng/ml of amphotericin B (Invitrogen, Carlsbad, CA). MEFsdeficient in IKK� (34), IKK� (42), IKK�/Nemo (46), and NIK (89) have beendescribed previously. HeLa cells stably expressing IKK subunit-specific shorthairpin RNAs (shRNAs) were generated by stable transduction with ampho-typed Moloney murine retroviruses produced in Phoenix A packaging cellsprovided by Garry Nolan (Stanford University, Palo Alto, CA) (K. B. Marcu,unpublished data). The retroviral vectors were engineered to coexpress greenfluorescent protein (GFP) and blasticidin-resistance genes with or withoutshRNAs specific for human IKK� or IKK� inserted into Mir-30 cassettes linkedto GFP (55).

Reovirus type 3 Dearing (T3D) is a laboratory stock. Purified reovirus virionswere generated by using second- or third-passage L-cell lysate stocks of twice-plaque-purified reovirus as described previously (27). Viral particles were Freonextracted from infected cell lysates, layered onto 1.2- to 1.4-g/cm3 CsCl gradients,and centrifuged at 62,000 � g for 18 h. Bands corresponding to virions (1.36g/cm3) were collected and dialyzed in virion storage buffer (150 mM NaCl, 15mM MgCl2, 10 mM Tris-HCl [pH 7.4]). Concentrations of reovirus virions inpurified preparations were determined from an equivalence of one optical den-sity unit at 260 nm equaling 2.1 � 1012 virions (70). Viral titer was determinedby plaque assay using L cells (80). Particle-to-PFU ratios of these preparationsvaried from 10:1 to 100:1.

Antisera specific for I�B�, I�B�, I�Bε, p50, p65/RelA, RelB, c-Rel, IKK�/Nemo, and �-actin were purchased from Santa Cruz Biotechnology (Santa Cruz,CA). Antiserum specific for p100/p52 was purchased from Upstate Biotechnol-ogy (Lake Placid, NY). The agonistic lymphotoxin-� receptor antiserum waspurchased from BD Biosciences (San Jose, CA). A low-molecular-weight IKKinhibitor, Compound A (92), was obtained from Karl Ziegelbauer (Bayer HealthCare AG, Leverkusen, Germany).

EMSA. Cells (3 � 106) grown in 100-mm tissue-culture dishes (Costar, Cam-bridge, MA) were treated with 20 ng/ml of tumor necrosis factor alpha (TNF-�;Sigma-Aldrich, St. Louis, MO), adsorbed with T3D at a multiplicity of infection(MOI) of 100 PFU/cell, or treated with gel saline (mock infection). After incu-bation at 37°C for various intervals, nuclear extracts (10 �g total protein) wereassayed for NF-�B activation by electrophoretic mobility shift assay (EMSA)using a 32P-labeled oligonucleotide consisting of the NF-�B consensus-bindingsequence (Santa Cruz Biotechnology) as described previously (20). For super-shift assays, 2 �g of rabbit polyclonal antiserum specific for p50, p52, RelA, RelB,or c-Rel was added to the binding reaction mixtures and incubated at 4°C for 30min prior to the addition of radiolabeled oligonucleotide. Nucleoprotein com-plexes were subjected to electrophoresis in native 5% polyacrylamide gels at 180V for 90 min, dried in a vacuum, and exposed to Biomax MR film (Kodak,Rochester, NY). Band intensity was quantified by determining photostimulusluminescence (PSL) units using a Fuji2000 phosphorimager and Multi Gaugesoftware (Fuji Medical Systems, Inc., Stamford, CT).

Immunoblot assay. Cells (8 � 105) were treated with 20 ng/ml of TNF-�,adsorbed with T3D at an MOI of 100 PFU/cell, or mock infected. Whole-cellextracts were generated by incubation with lysis buffer (10 mM HEPES [pH 7.4],10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.1% Igepal,1 mM Na4O7P2, 1 mM NaF, 1 mM NaVO3, 1 �M microcystin). Extracts (10 to50 �g total protein) were resolved by electrophoresis in 10% polyacrylamide gelsand transferred to nitrocellulose membranes. Membranes were blocked at 4°Covernight in blocking buffer (phosphate-buffered saline [PBS] containing 0.1%Tween 20 and 5% bovine serum albumin). Immunoblots were performed byincubating membranes with primary antibodies diluted 1:500 to 1:2,000 in block-ing buffer at room temperature for 1 h. Membranes were washed three times for10 min each with washing buffer (PBS containing 0.1% Tween 20) and incubatedwith horseradish peroxidase-conjugated goat anti-rabbit (Amersham Bio-sciences, Piscataway, NJ) and bovine anti-goat antibodies (Santa Cruz Biotech-nology) diluted 1:2,000 and 1:3,000, respectively. After three washes, membraneswere incubated for 1 min with chemiluminescent peroxidase substrate (Amer-

sham Biosciences) and exposed to film. Band intensity was quantified by usingthe Image J program (NIH, Bethesda, MD).

Kinase assay. Cells (8 � 105) were treated with 20 ng/ml of TNF-�, adsorbedwith T3D at an MOI of 100 PFU/cell, or mock infected. Whole-cell extracts wereincubated with an IKK�-specific antiserum in the presence of ELB buffer (50mM HEPES [pH 7.4], 250 mM NaCl, 5 mM EDTA, 0.1% Igepal). Immunopre-cipitates were equilibrated in kinase buffer (10 mM HEPES [pH 7.4], 0.5 mMdithiothreitol, 5 mM MgCl2, 1 mM MnCl2, 12.5 mM �-glycerophosphate, 50 �MNa3VO4, 2 mM NaF) and incubated with 10 �M ATP, 5 �Ci of [�-32P]ATP(PerkinElmer), and 1 �g of recombinant glutathione S-transferase (GST) pro-tein fused to amino acids 1 to 54 of I�B� (GST-I�B�) at 30°C for 30 min (13).Kinase reactions were terminated by heat denaturation in the presence of 1%sodium dodecyl sulfate (SDS). Radiolabeled products were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose mem-branes, and visualized by autoradiography.

RNA interference and NF-�B reporter assays. 293T cells (105) grown in24-well tissue-culture plates (Costar) were transfected with plasmids encodingshRNAs specific for IKK�, IKK�, or IKK�/Nemo or a negative-control shRNA(Millipore Corp., Billerica, MA) using FuGENE 6 transfection reagent (Roche,Basel, Switzerland) according to the manufacturer’s instructions. Cells wereincubated at 37°C for 48 h before a second transfection with plasmids encodingthe appropriate shRNA along with 0.15 �g/well of NF-�B-reporter plasmidpNF-�B-Luc, which expresses firefly luciferase under NF-�B control (9), and0.05 �g/well of pRenilla-Luc, which expresses Renilla luciferase constitutively(9), by using FuGENE 6. After an additional 24 h of incubation at 37°C, cellswere treated with 10 ng/ml of TNF-�, adsorbed with T3D at an MOI of 100PFU/cell, or mock infected. Luciferase activity was assessed by using a Dual-Luciferase assay kit (Promega Corp., Madison, WI) according to the manufac-turer’s instructions.

Caspase 3/7 activity assay. Cells (3 � 103) grown in clear-bottom, black-walled, 96-well tissue culture plates (Costar) were inoculated with 10 ng/ml ofTNF-�, a combination of 10 ng/ml of TNF-� and 10 �g/ml of cycloheximide(Sigma-Aldrich), or T3D at an MOI of 1,000 PFU/cell or were mock infected.Following incubation at 37°C for either 12 (TNF-�) or 24 (T3D) h, caspase 3/7activity was quantified by using a Caspase-Glo 3/7 assay (Promega). Lumines-cence was detected by using a Topcount NXT luminometer (Packard BiosciencesCo., Meriden, CT). The treatment of cells with cycloheximide blocks proteinsynthesis and enhances apoptosis induced by TNF-� (83).

Trypan blue exclusion assay. Cells (4 � 104) grown in six-well tissue cultureplates were inoculated with 10 ng/ml of TNF-�, a combination of 10 ng/ml ofTNF-� and 10 �g/ml of cycloheximide, or T3D at an MOI of 1,000 PFU/cell orwere mock infected. Following incubation at 37°C for either 12 (TNF-�) or 24(T3D) h, cells were collected and washed with PBS. The cell pellet was resus-pended in 50 �l of PBS and stained using 100 �l of a solution containing 0.4%trypan blue (Kodak) in PBS. For each experiment, 200 to 300 cells were counted,and the percentage of cell death was determined by light microscopy (Axiovert200; Zeiss, Oberkochen, Germany).

Statistical analysis. Mean values obtained by EMSAs, immunoblotting, andapoptosis assays were compared by using unpaired Student’s t test as applied withMicrosoft Excel software (Microsoft, Redmond, WA). P values of less than 0.05were considered to be statistically significant.

RESULTS

Reovirus infection results in the biphasic activation of NF-�B/Rel DNA-binding proteins. In prior studies, we found thatreovirus activates the functional expression of p50/RelA com-plexes, suggesting the involvement of classical NF-�B signaling(20). However, it remained unclear whether NF-�B/Rel pro-teins linked to the alternative pathway of the NF-�B pathwayare activated during reovirus infection. To more completelydefine the composition of NF-�B complexes activated by reo-virus, we used nuclear extracts from reovirus-infected HeLacells and Rel-specific antibodies to monitor the composition ofDNA-binding complexes formed in EMSAs. NF-�B/Rel DNA-binding activity was readily detected over background levels(mock treatment) within 2 h after infection with reovirus strainT3D (Fig. 1A), which potently induces apoptosis in culturedcells (20, 60, 77) and the murine CNS (53). Peak levels of

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NF-�B/Rel DNA-binding activity were observed at 4 to 8 hpostinfection. Supershift analysis of extracts obtained at 4, 6,and 8 h postinfection revealed the presence of DNA/proteincomplexes containing p50 and RelA but neither p52 nor RelB(Fig. 1B), suggesting preferential usage of the classical versusalternative NF-�B pathway by reovirus. Complexes containingc-Rel were apparent in supershift assays only after 8 h ofinfection (Fig. 1B). Thus, reovirus induces a biphasic pattern ofNF-�B/Rel activation featuring the initial nuclear transloca-tion of complexes consisting of p50 and RelA, followed bythose containing p50, RelA, and c-Rel. Given that the cellulargene encoding c-Rel contains functional NF-�B-binding sites(29), this expression pattern may reflect de novo synthesis ofc-Rel rather than its mobilization from a latent cytoplasmicpool.

These initial experiments conducted over an 8-h time courseprovided no evidence for the capacity of reovirus to stimulatethe nuclear expression of p52, a signature Rel protein involvedin the alternative pathway of NF-�B signaling. To further in-vestigate whether reovirus interfaces with the alternativeNF-�B pathway, we extended the time course of T3D infectionto 24 h and monitored extracts for the processing of p100 top52 (66). Levels of p100 were significantly reduced between 16and 24 h postinfection (Fig. 2). Consistent with a precursor/product relationship, diminution of p100 protein levels was

FIG. 1. Biphasic activation of NF-�B/Rel proteins in reovirus-in-fected cells. (A) Nuclear extracts were prepared from uninfected HeLacells (0 h), mock-infected cells (Mock), or cells infected with T3D at anMOI of 100 PFU/cell for the times shown. Cells also were treated with20 ng/ml of TNF-� for 30 min as a positive control. Extracts wereincubated with a radiolabeled NF-�B consensus oligonucleotide, andthe resulting protein-oligonucleotide complexes were resolved by poly-acrylamide gel electrophoresis, dried, and exposed to film. (B) Nuclearextracts prepared at 4, 6, and 8 h postinfection were incubated withantisera specific for p50, p52, RelA, RelB, or c-Rel prior to the addi-tion of a radiolabeled NF-�B consensus oligonucleotide. NF-�B-con-taining complexes are indicated.

FIG. 2. Processing of p100 to p52 during reovirus infection.(A) Whole-cell extracts were prepared from uninfected HeLa cells (0h), mock-infected cells (Mock), or cells infected with reovirus T3D atan MOI of 100 PFU/cell for the times shown. Cells also were treatedwith 2 �g/ml of an agonistic lymphotoxin-� receptor antiserum for 8 has a positive control. Extracts were resolved by SDS-PAGE, trans-ferred to nitrocellulose membranes, and immunoblotted by using anantiserum specific for p100/p52. Band intensity was quantified by usingthe Image J program. The results are expressed as the mean ratio of(B) p100/actin or (C) p52/p100 for three independent experiments.Error bars indicate standard deviations. *, P � 0.05 as determined byStudent’s t test in comparison to untreated cells (0 h).

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accompanied by a significant increase in the steady-state levelsof p52 (Fig. 2A and C). RelB nuclear localization also wasobserved approximately 16 to 24 h following reovirus infection(data not shown). Taken together with the NF-�B/Rel profilingdata shown in Fig. 1B, these findings suggest that reovirusengages not only the classical pathway of NF-�B signaling butalso the alternative pathway, albeit at much later times ofinfection.

Reovirus infection leads to the selective degradation ofI�B�. Activation of the classical NF-�B pathway by physio-logic agonists is dependent primarily on the degradation ofI�B� (reviewed in references 4, 28, and 30), an inhibitor thatsequesters p50/RelA complexes in the cytoplasmic compart-ment (3). We have shown that degradation-resistant forms ofI�B� attenuate reovirus-induced apoptosis, which is criticallydependent on NF-�B activation (20). However, mammalian

FIG. 3. Reovirus infection leads to degradation of I�B� but not I�B� or I�Bε. Cytoplasmic extracts were prepared from uninfected HeLa cells(0 h), mock-infected cells (Mock), or cells infected with reovirus T3D at an MOI of 100 PFU/cell for the times shown. Cells also were treated with20 ng/ml of TNF-� for 10 min as a positive control. Extracts were resolved by SDS-PAGE, transferred to nitrocellulose membranes, andimmunoblotted by using antisera specific for (A) I�B�, (C) I�B�, or (E) I�Bε. An actin-specific antiserum was used to detect levels of actin asa loading control. Band intensity corresponding to levels of (B) I�B�, (D) I�B�, and (F) I�Bε was quantified by using the Image J program. Theresults are expressed as the mean ratio of I�B/actin for three independent experiments. Error bars indicate standard deviations. *, P � 0.05 asdetermined by Student’s t test in comparison to untreated cells (0 h).



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cells express other labile inhibitors that are structurally similarto I�B�, such as I�B� (44) and I�Bε (82). Indeed, prior studiessuggest a potential role for signal-dependent degradation ofI�B� (74) and I�Bε (82) in the inducible nuclear entry ofc-Rel. To determine whether any of these inhibitors is underreovirus control, we monitored their levels in T3D-infectedcells in immunoblot studies using I�B-specific antibodies. Thecellular pool of I�B� was significantly reduced within 4 h afterinfection with T3D (Fig. 3A and B). In contrast, levels of I�B�and I�Bε were maintained under the same stimulatory condi-tions over the entire 8-h time course (Fig. 2C to F). We con-clude that I�B� is a primary cellular target of reovirus, which

is fully consistent with its capacity to stimulate nuclear trans-location of NF-�B p50/RelA.

IKK� and IKK�/Nemo are required for reovirus-inducedNF-�B activation. We next investigated the mechanism bywhich reovirus destabilizes I�B proteins. Cytokine-induceddegradation of NF-�B inhibitors is dependent on their phos-phorylation at specific serine residues by IKKs such as IKK�and IKK� (7, 26, 91). These structurally related enzymes caninteract and form higher-order complexes with other cellularproteins (reviewed in references 28 and 30). Integration of theregulatory protein IKK�/Nemo into such complexes is re-quired for the activation of IKK� (64, 65, 88) but not IKK�

FIG. 4. Involvement of IKKs in reovirus-induced NF-�B activation. (A) Whole-cell extracts were prepared from uninfected HeLa cells (0 h),mock-infected cells (Mock), or cells infected with reovirus T3D at an MOI of 100 PFU/cell for the times shown. Cells also were treated with 20ng/ml of TNF-� for the times shown as a positive control. The IKK complex was immunoprecipitated by using an IKK�-specific antiserum priorto incubation with a GST-I�B� substrate in the presence of [�-32P]ATP. Kinase reactions were resolved by SDS-PAGE, transferred to nitrocel-lulose, and visualized by autoradiography. (B) HeLa cells were pretreated with IKK inhibitor Compound A for 1 h at the concentrations shownand then uninfected (Untreated), mock infected (Mock), or infected with reovirus T3D at an MOI of 100 PFU/cell for the times shown. Nuclearextracts were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted by using a RelA-specific antiserum. (C) Band intensity wasquantified relative to uninfected cells by using the Image J program. The results are expressed as the mean RelA band intensity for threeindependent experiments. Error bars indicate standard deviations. *, P � 0.05 as determined by Student’s t test in comparison to untreated cells(0 �M). (D) Nuclear extracts from the experiment shown in panel B were incubated with a radiolabeled NF-�B consensus oligonucleotide, andthe resulting protein-oligonucleotide complexes were resolved by polyacrylamide gel electrophoresis, dried, and exposed to film. (E) Band intensitywas quantified by determining PSL units relative to uninfected cells for four independent experiments. Error bars indicate standard deviations. *,P � 0.05 as determined by Student’s t test in comparison to untreated cells (0 �M).



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(16, 24, 66). The best-characterized substrate of IKK� is I�B�(35, 50), whereas IKK� catalyzes phosphorylation of p100/NF-�B2 (66, 85).

To determine whether either IKK� or IKK� is required forthe reovirus-induced activation of NF-�B, cellular IKK com-plexes were immunopurified from HeLa cells either before orafter infection with T3D and monitored for the capacity tophosphorylate I�B� in vitro. In keeping with the kinetics ofI�B� degradation (Fig. 3A) and NF-�B activation (Fig. 1A),I�B kinase activity exceeding basal levels in uninfected cellswas readily detected within 4 h after exposure to T3D andsustained for at least an additional 4 h (Fig. 4A). These datasuggest that IKKs are critically involved in the mechanism bywhich reovirus diminishes the cellular pool of I�B� (Fig. 3A).

To determine whether IKK activation is required for the nu-clear translocation of NF-�B by reovirus, cells were treated withescalating doses of Compound A, a low-molecular-weight inhib-

itor of IKK (92), prior to infection with T3D. Importantly, Com-pound A inhibits IKK� more efficiently than IKK� (92). As dem-onstrated by immunoblots measuring p65/RelA nuclear localization,treatment of cells with Compound A suppressed NF-�B traffick-ing induced by reovirus (Fig. 4B and C). Concordantly, EMSAsperformed with nuclear extracts from the same panel of infectedcells indicated that Compound A had a marked inhibitory effecton reovirus-induced NF-�B DNA-binding activity (Fig. 4D andE), although this inhibition was incomplete, perhaps reflectingresidual IKK� activity. These pharmacological data suggest thatreovirus activates NF-�B via a mechanism involving IKK�, IKK�,or both of these IKKs.

To identify the IKK subunits responsible for NF-�B activa-tion by reovirus, NF-�B-dependent gene expression was as-sessed by using 293T cells transiently transfected with shRNA-encoding plasmids engineered to diminish expression of IKK�,IKK�, or the IKK� regulatory subunit IKK�/Nemo (Fig. 5A).

FIG. 5. IKK� and IKK�/Nemo are required for reovirus-induced activation of NF-�B. (A) 293T cells were transfected with plasmids encodingshRNAs specific for IKK�, IKK�, or IKK�/Nemo or a negative control shRNA (Neg. Con.). After incubation at 37°C for 48 h, cells werecotransfected with plasmids encoding IKK subunit-specific shRNAs, pNF-�B-Luc, and pRenilla-Luc. Cells were treated with 10 ng/ml of TNF-�and adsorbed with T3D at an MOI of 100 PFU/ml or mock infected prior to assessments of luciferase activity in cell-culture lysates. The resultsare expressed as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Errorbars indicate standard deviations. (B) Wild-type MEFs or MEFs deficient in IKK�, IKK�, or IKK�/Nemo were uninfected (0 h), mock infected(Mock), infected with reovirus T3D at an MOI of 100 PFU/cell for 8 h, or treated with 20 ng/ml of TNF-� for 1 h. Nuclear extracts were resolvedby SDS-PAGE, transferred to nitrocellulose, and immunoblotted by using a RelA-specific antiserum. (C) Nuclear extracts from the experimentshown in panel B were incubated with a radiolabeled NF-�B consensus oligonucleotide. Resulting protein-oligonucleotide complexes wereresolved by polyacrylamide gel electrophoresis, dried, and exposed to film. NF-�B-containing complexes are indicated. (D) Band intensity wasquantified by determining the number of PSL units relative to that in uninfected cells for three independent experiments. Error bars indicatestandard deviations. *, P � 0.05 as determined by Student’s t test in comparison to mock-treated cells (0 h).



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NF-�B reporter gene activity was dramatically reduced in cellsexpressing shRNA specific for either IKK� or IKK�/Nemo incomparison to cells expressing IKK�-specific shRNA or neg-ative control shRNA, suggesting that IKK� and IKK�/Nemoare required for reovirus-induced NF-�B activation. To con-firm a requirement for IKK� and IKK�/Nemo in the activationof NF-�B in response to reovirus, RelA nuclear localizationwas assessed with MEFs deficient in IKK�, IKK�, or IKK�/Nemo. Nuclear extracts from wild-type and IKK-deficientMEFs were probed by immunoblotting for RelA followinginfection with T3D for 8 h, which corresponds to peak levels ofNF-�B activation (Fig. 1A). Reovirus stimulated nuclear local-ization of RelA in wild-type MEFs, as did the cytokine TNF-�(Fig. 5B), a potent IKK agonist (26, 91). Similar results wereobtained with nuclear extracts from reovirus-infected MEFslacking IKK� (Fig. 5B). However, this response was dimin-ished in MEFs lacking IKK� (Fig. 5B), indicating preferentialusage of IKK� relative to IKK�. MEFs lacking the regulatorysubunit IKK�/Nemo also were incapable of reovirus-mediatedRelA nuclear localization (Fig. 5B). EMSA studies using nu-clear extracts from the same panel of infected cells confirmedthese results (Fig. 5C and D). NF-�B DNA-binding activitywas detected in wild-type MEFs and IKK�-deficient MEFs butnot in MEFs lacking IKK� or IKK�/Nemo. Differences inreovirus-mediated signal transduction in IKK-deficient MEFscould not be attributed to differences in viral infection orgrowth (data not shown). Thus, these findings provide strongevidence that IKK� and IKK�/Nemo are required for reovirus-induced NF-�B activation.

The protein kinase NIK phosphorylates and activates IKK�following cellular stimulation with agonists of the alternativeNF-�B pathway, such as lymphotoxin-� (43, 49). To determinewhether NIK is required for NF-�B activation by reovirus, wemonitored this response in NIK-deficient MEFs by immuno-blotting and EMSA (Fig. 6). Reovirus infection resulted inNF-�B activation in both wild-type and NIK-deficient MEFs,indicating that NIK is dispensable for reovirus-induced NF-�Bactivation. Taken together, these data confirm a requirementfor endogenous IKK in the mechanism by which reovirus ac-tivates NF-�B and demonstrate that this virus selectively uti-lizes IKK� and not IKK� to drive the canonical branch of theNF-�B signaling pathway. Surprisingly, IKK�/Nemo, which isknown to regulate IKK� rather than IKK�, is also required forNF-�B activation by reovirus, but NIK is dispensable.

IKK� and IKK�/Nemo are required for reovirus-inducedapoptosis. Since IKK� and IKK�/Nemo mediate NF-�B acti-vation following reovirus infection (Fig. 5), we examinedwhether IKK stimulation by reovirus is required for apoptoticcell death. IKK-deficient MEFs were infected with reovirusT3D, and apoptosis was assessed by quantitation of caspase 3/7activity (Fig. 7A). Levels of activated caspase 3/7 followinginfection of wild-type and IKK�-deficient MEFs were substan-tially greater than those following infection of MEFs deficientin either IKK� or IKK�/Nemo. To corroborate these results,we tested wild-type and IKK subunit-deficient MEFs for via-bility following infection with T3D (Fig. 7B). A significantlygreater percentage of IKK�- and IKK�-deficient MEFs thanwild-type and IKK�-deficient MEFs remained viable duringthe time course of reovirus infection.

To determine whether NIK is required for apoptosis induc-

tion following reovirus infection, NIK-deficient MEFs wereinfected with reovirus T3D, and apoptosis was assessed byquantification of caspase 3/7 activity (Fig. 7C). Levels ofcaspase 3/7 activity in MEFs deficient in NIK were comparableto those in wild-type cells following infection with T3D. Inparallel with these results, we observed no remarkable differ-ence in the viability of wild-type and of NIK-deficient MEFsfollowing T3D infection (Fig. 7D), suggesting that NIK is notrequired for reovirus-induced apoptosis.

To confirm a requirement for IKK� in reovirus-inducedapoptosis using an independent approach, we examinedwhether HeLa cells stably expressing retrovirus-transducedshRNAs for either IKK� or IKK� were capable of undergoingapoptosis in response to reovirus. Steady-state levels of IKK�and IKK� in the respective shRNA-expressing cells were sig-nificantly reduced in comparison to levels of those proteins incells expressing an empty retrovirus vector control (Fig. 8A).IKK� knockdown resulted in a marked decrease in reovirus-induced apoptosis assessed by both caspase 3/7 activity (Fig.8B) and cell viability (Fig. 8C) in comparison to levels ofapoptosis in cells transduced with an IKK�-specific shRNA orthe empty retrovirus vector control. Together, these functionaldata with IKK-specific siRNA-mediated knockdown and IKK-and NIK-deficient MEFs strongly correlate with the capacity ofreovirus to modulate I�B� and NF-�B during infection (Fig. 1to 6). Our findings indicate that both IKK� and IKK�/Nemo,

FIG. 6. Reovirus-induced activation of NF-�B in NIK-deficientcells. (A) Wild-type MEFs or NIK-deficient MEFs were uninfected (0h), mock infected (Mock), infected with reovirus T3D at an MOI of1,000 PFU/cell for 8 h, or treated with 20 ng/ml of TNF-� for 30 min.Nuclear extracts were resolved by SDS-PAGE, transferred to nitrocel-lulose, and immunoblotted by using a RelA-specific antiserum.(B) Nuclear extracts from the experiment shown in panel A wereincubated with a radiolabeled NF-�B consensus oligonucleotide. Theresulting protein-oligonucleotide complexes were resolved by polyacryl-amide gel electrophoresis, dried, and exposed to film. NF-�B-contain-ing complexes are indicated.



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but neither IKK� nor NIK, are required for the activation ofNF-�B and induction of apoptosis in response to reovirus.

DISCUSSION

Apoptosis is a genetically programmed form of cell deaththat plays an important regulatory role in a wide spectrum ofbiological processes. Many viruses are capable of inducingapoptosis of infected cells (63). In some cases, apoptosis trig-gered by viral infection may serve as a component of hostdefense to limit viral replication or spread. In other instances,apoptosis may enhance viral infection by facilitating viral dis-semination or allowing virus to evade host inflammatory re-sponses (5, 6, 63). Apoptosis plays an important role in thevarious patterns of disease caused by reovirus infection (22, 23,53, 54). Although we previously uncovered an essential func-

tion for NF-�B activation in reovirus-induced apoptosis (20), itwas not known how reovirus activates this signal-transductionmechanism. Results reported here identify constituents of theNF-�B signaling apparatus induced by reovirus and provideevidence that NF-�B activation during reovirus infection re-quires select components of the classical and alternativeNF-�B signaling pathways.

Using siRNA-mediated knockdown of individual IKK sub-units and IKK subunit-deficient MEFs, we demonstrate thatreovirus-infected cells lacking IKK� are impaired for NF-�Bactivation (Fig. 5) and apoptotic programming (Fig. 7 and 8),whereas both of these processes are operative in cells lackingIKK�. Despite its preferential usage of IKK�, reovirus retainsthe capacity to elicit both NF-�B activation and apoptosis inthe absence of NIK (Fig. 6 and 7), a known activator of IKK�

FIG. 7. IKK� and IKK�/Nemo are required for reovirus-induced apoptosis. Wild-type MEFs or (A) MEFs deficient in IKK�, IKK�,IKK�/Nemo, or (C) NIK were mock infected (Mock), infected with reovirus T3D at an MOI of 1,000 PFU/cell for 24 h (T3D), treated with 10ng/ml of TNF-� for 12 h (TNF�), or treated with 10 ng/ml of TNF-� and 10 �g/ml of cycloheximide for 12 h (TNF�/CHX). Caspase 3/7 activitywas quantified by using a luminescent substrate. The results are expressed as the mean caspase activity relative to that of mock-infected cells forthree independent experiments. Wild-type MEFs, (B) IKK-deficient MEFs, or (D) NIK-deficient MEFs were mock infected, infected with reovirusT3D at an MOI of 1,000 PFU/cell for 48 h, treated with 10 ng/ml of TNF-� for 24 h, or treated with 10 ng/ml of TNF-� and 10 �g/ml ofcycloheximide for 24 h. Cell viability was quantified by trypan blue exclusion. The results are expressed as the mean percentage of cell death forthree independent experiments. Error bars indicate standard deviations. *, P � 0.05 as determined by Student’s t test in comparison tomock-infected cells.



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in cytokine-treated cells (43, 66). Furthermore, degradation ofthe RNA or targeted disruption of the gene encoding IKK�/Nemo, which is dispensable for cytokine-induced activation ofIKK� (16, 24), significantly attenuates reovirus-inducedNF-�B activation and apoptosis (Fig. 5 and 7). Our findingswith NIK and IKK�/Nemo suggest that reovirus activates thecanonical NF-�B pathway by a novel mechanism involvingIKK� instead of IKK�. The simplest interpretation of theseresults is that reovirus accesses the cellular NF-�B machineryby directly interfacing with IKK�/IKK� complexes, with IKK�/Nemo serving as an adaptor that docks one or more reovirusgene products. In keeping with this possibility, IKK�/Nemotethers the HTLV1 Tax protein to IKK complexes, resulting inthe persistent activation of IKK� and NF-�B (9, 14). In whatmay be another related finding, IKK�/Nemo also is requiredfor Tax-induced activation of IKK� (84). Although dataemerging from studies of IKK�-deficient mice suggest thepresence of functional IKK�/IKK� complexes (40, 42, 61, 72),direct evidence for the existence of IKK�/IKK� complexes inwild-type animals has not been reported. Notwithstanding, ourresults clearly establish that reovirus activates NF-�B anddownstream proapoptotic genes via a mechanism involvingIKK� but not IKK�.

The principle in vivo substrate of IKK� is I�B� (26, 50, 58).This cytoplasmic inhibitor tightly controls the nuclear translo-cation of p50/RelA dimers (3), effectors of the classical NF-�Bpathway (reviewed in references 4, 28, and 30). The principlein vivo substrate of IKK� is p100/NF-�B2 (66), an integralinhibitor in the alternative NF-�B pathway that retains theRelB transactivator protein in the cytoplasm (66, 71). Follow-ing IKK�-mediated phosphorylation, p100 is processed to p52via a proteasome-dependent mechanism, permitting the nu-clear entry of p52/RelB complexes (66, 71). Given these dis-tinct mechanisms, our findings with reovirus-infected cells sug-gest an unconventional function for IKK� in substratetargeting. Specifically, we were unable to detect either p52 orRelB DNA-binding activity in nuclear extracts from cells fol-lowing 4 to 8 h of reovirus infection (Fig. 1B). Instead, at theseearly time points, the predominant Rel species detected werep50 and RelA (Fig. 1B), which are primarily under I�B� con-trol (2, 3). Consistent with this Rel profile, I�B� protein levelswere significantly reduced by 4 h postinfection (Fig. 3). Al-though p100 processing to p52 was observed at late time pointsduring reovirus infection (16 h), it seems unlikely that this verydelayed response contributes to the more rapidly developingsignals required for apoptosis (19, 20, 60). Accordingly, wepropose that IKK� rather than IKK� targets I�B� for proteo-lytic destruction and regulates the nuclear translocation ofp50/RelA complexes in reovirus-infected cells. This workingmodel is fully concordant with the phenotype of cell linesdeficient for either IKK�, p50, or p65/RelA, all of which areimpaired for reovirus-induced NF-�B activation and proapop-totic signaling (Fig. 5 and 7) (20). In agreement with thismodel, prior studies with recombinant proteins indicate thatIKK� can efficiently phosphorylate NF-�B-bound forms ofI�B� in vitro (39, 90). Additionally, RANKL (receptor activa-tor of NF-�B ligand)-mediated signaling of epithelial cells invitro requires IKK�-dependent phosphorylation of I�B� toactivate NF-�B (8).

Both IKK� and IKK� contain regulatory serine phosphoac-

FIG. 8. Knockdown of IKK� diminishes apoptosis in response toreovirus. (A) Extracts of HeLa cells transduced with retroviruses con-taining empty vector or encoding shRNAs specific for either IKK� orIKK� were resolved by SDS-PAGE, transferred to nitrocellulose, andimmunoblotted by using IKK�-, IKK�-, or tubulin-specific antiserum.Immunoblots were scanned and quantified by using Odyssey software.(B) HeLa cells stably expressing empty vector or shRNAs specific foreither IKK� or IKK� were mock infected (Mock), infected with reo-virus T3D at an MOI of 1,000 PFU/cell for 24 h (T3D), treated with 10ng/ml of TNF-� for 12 h (TNF�), or treated with 10 ng/ml of TNF-�and 10 �g/ml of cycloheximide for 12 h (TNF�/CHX). Caspase 3/7activity was quantified by using a luminescent substrate. The results areexpressed as the mean caspase activity relative to that of mock-infectedcells for three independent experiments. (C) HeLa cells stably express-ing empty vector or shRNAs specific for either IKK� or IKK� weremock infected, infected with reovirus T3D at an MOI of 1,000 PFU/cell for 48 h, treated with 10 ng/ml of TNF-� for 24 h, or treated with10 ng/ml of TNF-� and 10 �g/ml of cycloheximide for 24 h. Cellviability was quantified by trypan blue exclusion. The results are ex-pressed as the mean percentage of cell death for three independentexperiments. Error bars indicate standard deviations. *, P � 0.05 asdetermined by Student’s t test in comparison to mock-infected cells.



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ceptors in their so-called T-loop domains (25, 50). Signal-dependent phosphorylation of the T-loop serines in IKK� andIKK� is a prerequisite for their catalytic activation (25). Basedon in vitro studies with recombinant proteins, IKK� and IKK�can autophosphorylate at these T-loop serines (25, 65, 73).Physiologic agonists of the alternative NF-�B pathway stimu-late T-loop phosphorylation and activation of IKK� via theupstream kinase NIK (43, 66, 85). However, NIK is dispens-able in the mechanism by which reovirus induces NF-�B acti-vation and apoptosis (Fig. 6 and 7). Which signal transducerscouple reovirus to the IKKs? Experiments using pharmacolog-ical inhibitors suggest that NF-�B-dependent apoptotic signal-ing is triggered by viral replication steps that occur after dis-assembly but prior to RNA synthesis (19). Strain-specificdifferences in the capacity of reovirus to induce apoptosis seg-regate with the viral genes encoding the 1 and �1 proteins(18, 60, 77), which play important roles in viral attachment (38,81) and membrane penetration (10, 11, 45), respectively. Im-portantly, transient expression of �1 is sufficient to induceapoptosis in cell culture (17), implicating this protein in thereovirus/NF-�B signaling axis. We envision three potentialmechanisms by which �1, or perhaps another viral gene prod-uct, initiates NF-�B signal transduction during reovirus infec-tion. First, �1 may activate viral sensors that engage the adap-tor protein IPS-1 (beta interferon promoter stimulator 1),which mediates the activation of NF-�B in response to viralinfections by recruiting TNF-associated factor 6 to the signal-ing complex (37, 51, 67, 86). Second, �1 may activate a cellularkinase distinct from NIK that phosphorylates the T loop ofIKK�. Third, �1 may interact with IKK complexes directly,leading to conformational changes that stimulate oligomeriza-tion and selectively trigger IKK�-dependent autophosphoryla-tion of the T loop in IKK� rather than IKK� (36, 57). Studiesto test these models for reovirus-induced activation of IKK�are currently under way.

Reovirus infection leads to NF-�B activation in numerouscell types (15, 20, 54). However, the functional consequencesof NF-�B activation in vivo differ depending on the infectedtissue. NF-�B activation in the CNS leads to high levels ofneuronal apoptosis and encephalitis (54). In contrast, NF-�Bactivation in the heart leads to IFN-� production, which limitsviral replication and protects against apoptosis (54). In theabsence of NF-�B signaling, reovirus infection induces wide-spread apoptotic damage to cardiac myocytes, resulting inmyocarditis. Mechanisms underlying the divergent cellularfates following NF-�B activation by reovirus in vivo remainunclear. Considering the results presented in this report, thesemechanisms may involve tissue-specific activation of differentIKK subunits, which in turn may influence the composition ofnuclear NF-�B complexes. Experiments using tissue-specificIKK�- or IKK�-deficient mice should clarify the function ofIKK in reovirus-induced disease.

ACKNOWLEDGMENTS

We thank members of our laboratory for many helpful discussionsand Jim Chappell, Geoff Holm, and Denise Wetzel for careful reviewsof the manuscript. We thank Karl Ziegelbauer and Bayer Health CareAG for Compound A, Amgen for NIK-deficient cells, and RobertSchreiber for NIK wild-type cells. K.B.M. thanks Garry Nolan forPhoenix A packaging cells, Ross Dickins for the pLMP and pTMPretroviral vectors, and Dorothee Vicogne for technical assistance.

This research was supported by Public Health Service awards T32CA09385 (J.A.C.), R01 GM066882 (K.B.M.), R01 AI52379 andCA82556 (D.W.B.), and R01 AI50080 (T.S.D.) and the Elizabeth B.Lamb Center for Pediatric Research. Additional support was providedby Public Health Service awards CA68485 for the Vanderbilt-IngramCancer Center and DK20593 for the Vanderbilt Diabetes Researchand Training Center.

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