The NEMO adaptor bridges the nuclear factor-jB andinterferon regulatory factor signaling pathways

Tiejun Zhao1, Long Yang1,3, Qiang Sun1, Meztli Arguello1,2, Dean W Ballard4, John Hiscott1–3 &Rongtuan Lin1,3

Intracellular detection of RNA virus infection is mediated by the RNA helicase RIG-I, which is recruited to mitochondria by the

adaptor protein MAVS and triggers activation of the transcription factors NF-jB, IRF3 and IRF7. Here we demonstrate that virus-

induced activation of IRF3 and IRF7 depended on the NF-jB modulator NEMO, which acted ‘upstream’ of the kinases TBK1

and IKKe. IRF3 phosphorylation, formation of IRF3 dimers and DNA binding, as well as IRF3-dependent gene expression, were

abrogated in NEMO-deficient cells. IRF3 phosphorylation and interferon production were restored by ectopic expression of NEMO.

Thus, NEMO, like MAVS, acts as an adaptor protein that allows RIG-I to activate both the NF-jB and IRF signaling pathways.

The success of innate host defense against viral infection depends onthe capacity of innate immune cells to detect the invading pathogen.Toll-like receptors and other innate immune receptors recognizepathogen-associated molecular patterns such as double-stranded(dsRNA) and/or 5¢-triphosphate RNA, which are common byproductsof viral replication, and trigger the activation of signaling cascadesthat culminate in the production of cytokines and chemokinesthat disrupt viral replication and initiate innate and adaptiveimmune responses1–7. Rapid induction of type I interferon is a centralevent in the establishment of the antiviral response and is tightlyregulated by extracellular and intracellular signals that activate thenuclear factor-kB (NF-kB), activator protein 1 and interferon-regulatory factor (IRF) transcription factors8–10. The synergistic effectof these regulatory proteins on the promoters of genes encoding type Iinterferon proteins triggers the immediate-early interferon response,characterized by the rapid release of interferon-b (IFN-b) and IFN-a1(ref. 1). Secreted interferon acts in a paracrine way on neighboringcells by engaging interferon receptors and activating the Jak and STATsignaling proteins11.

The central participant in NF-kB activation is the inhibitor ofNF-kB (IkB) kinase (IKK) complex, a trimeric holoenzyme composedof the kinases IKKa and IKKb and the regulatory subunit NEMO (alsocalled IKKg)12. NF-kB dimers are kept inactive in the cytoplasm byphysical association with the IkB family of inhibitory molecules, thebest characterized of which is IkBa12. After stimulation, the activatedIKK complex phosphorylates two serine residues (Ser32 and Ser36) inIkBa; this phosphorylation results in polyubiquitination and protea-somal degradation of IkBa12. Free NF-kB dimers can then translocateinto the nucleus and stimulate the transcription of target genesencoding inflammatory and immunoregulatory molecules.

Two IKK-related kinases, TANK (TRAF family member–associatedNF-kB activator)–binding kinase 1 (TBK1) and IKKe, regulateIRF3 and IRF7 activation13,14. Stimuli such as viral infection, dsRNAor lipopolysaccharide activate TBK1 and IKKe, which phosphorylateserine residues in IRF3 and IRF7; this elicits a conformational changethat allows the formation of IRF dimers, translocation of IRF into thenucleus and transactivation of target genes13,14. Despite their struc-tural and functional similarities, it is becoming apparent that neitherTBK1 nor IKKe or the canonical IKK proteins are redundant infunction but instead form part of a complex network whose moleculardetails are only now beginning to emerge10.

The cytoplasmic RNA helicase RIG-I is essential in the detection ofintracellular RNA virus infection7. RIG-I senses dsRNA and/or5¢-triphosphate RNA by means of its helicase domain and relayssignals ‘downstream’ through its amino-terminal (N-terminal) caspase-recruitment domain to the canonical IKKs and the IKK-related kinasesIKKe and TBK1, culminating in the activation of NF-kB and IRF3(refs. 5–7). The mitochondrial antiviral signaling adaptor MAVS (alsocalled IPS-1, VISA or Cardif), which links RIG-I to ‘downstream’signaling pathways, contains an N-terminal caspase-recruitmentdomain and a carboxy-terminal (C-terminal) mitochondrial trans-membrane domain that allows localization to the outer mitochondrialmembrane15–18. MAVS seems to constitute the ‘branching point’ ofRIG-I signals toward NF-kB and IRF3, as MAVS can interact with andactivate both the canonical IKK complex as well as TBK1 and IKKe.

Studies have shown that crosstalk between the canonical IKKs andIKK-related kinases influences NF-kB activation. TBK1 was firstdescribed as a kinase interacting with the adaptor protein TANK,which is able to activate NF-kB19. However, whether the canonicalIKKs influence IRF3 and IRF7 activation has not been investigated.

Received 2 November 2006; accepted 11 April 2007; published online 29 April 2007; doi:10.1038/ni1465

1Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, 2Department of Microbiology & Immunology, and 3Department of Medicine, McGillUniversity, Montreal H3T 1E2, Canada. 4Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.Correspondence should be addressed to R.L. (rongtuan.lin@mcgill.ca)

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Here we demonstrate that NEMO is essential for virus-inducedactivation of IRF3 and IRF7, NEMO acts ‘upstream’ of IKKe andTBK1 but ‘downstream’ of MAVS and RIG-I, and NEMO physicallyinteracts with TANK and mediates the recruitment of TBK1 and IKKeto the RIG-I–MAVS complex.

RESULTS

NEMO in virus-induced interferon production

To examine the physiological function of NEMO in virus-mediatedinduction of interferon and interferon-inducible gene expression, wemeasured the expression of interferon and interferon-inducible genesin immortalized mouse embryonic fibroblasts (MEFs) derived frommice lacking NEMO (Ikbkg–/–). We reconstituted Ikbkg–/– MEFswith the ‘pBabe’ retroviral vector or with pBabe containing cDNAencoding human NEMO, and then infected the cells with Sendai virus(SV) or vesicular stomatitis virus (VSV). Virus-induced expression ofIfnb1 and other IRF3-responsive genes was abrogated in Ikbkg–/–

MEFs but was restored by transduction with the NEMO-expressingretrovirus (Fig. 1a,b).

Next we measured SV-induced production of IFN-a and IFN-b byenzyme-linked immunosorbent assay (ELISA). The first wave of IFN-aproduction depends mainly on the action of IRF3 and IRF7, whereasIFN-b production is regulated by NF-kB as well as IRF3 and IRF7.Ikbkg–/– and Irf3–/– MEFs produced much less IFN-a and IFN-b thandid wild-type MEFs; retroviral expression of wild-type NEMO

restored virus-induced IFN-a and IFN-b production by Ikbkg–/– MEFs(Fig. 1c). IFN-b production was approximately 25% lower in Ikbkb–/–

(IKKb-deficient) MEFs. These data indicate that NEMO but not thecanonical IKKb is essential for virus-induced interferon production.

Interferon subsequently stimulates the Jak-STAT signaling pathway,thereby amplifying interferon responses11,20. SV infection inducedSTAT1 expression and phosphorylation in wild-type and Ikbkb–/–

MEFs but not in Ikbkg–/– or Irf3–/– MEFs (Fig. 1d). These resultswere consistent with the lack of virus-induced interferon productiondetected by ELISA (Fig. 1c). Expression of wild-type NEMO partiallyrestored virus-induced STAT1 expression (Fig. 1e) and production ofIFN-a and IFN-b (Fig. 1c) in Ikbkg–/– MEFs. This partial recovery mayhave been due to higher expression of retrovirus-encoded NEMO thanits endogenous counterpart (Fig. 1e). Indeed, when transiently over-expressed in wild-type MEFs, NEMO attenuated virus-induced acti-vation of an interferon-stimulated response element (ISRE)–drivenreporter gene (data not shown). Consistent with our results, NEMOalso exerts dose-dependent effects on other signaling pathways21.

To investigate the physiological functions of NEMO in other cells,we used a small interfering RNA (siRNA) specific for Ikbkg to reduceendogenous NEMO expression in the mouse leukemic monocytemacrophage cell line RAW264.7. ‘Knockdown’ of NEMO expressionmediated by siRNA inhibited SV-induced STAT1 expression (Fig. 1f).Thus, the function of NEMO in the induction of the interferonresponse extends beyond NF-kB activation.

++ ––

IkbkgControlsiRNA

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+ Vec

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–/– Ikbkg–/–

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++++– – – –SV++++++ ––––––SVIr f3–/

– Irf9–/

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0 6 16 24 0 6 16 24

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1.41.21.00.80.60.40.2

0

02.04.0

IFN

-α (

ng/m

l)IF

N-β

(ng

/ml)

6.08.0

10.0

a

d e

f

b c

Figure 1 Responses to RNA virus infection in NEMO-deficient MEFs. (a,b) RT-PCR of transcripts encoding NEMO (Ikbkg), ISG15 (a ubiquitin-like modifier;

Isg15), IFIT1 (Ifit1), IFN-b (Ifnb1), IRF7 (Irf7), IKKe (Ikke), TBK1 (Tbk1) or GAPDH (encoding glyceraldehyde phosphate dehydrogenase (loading control);

Gapdh) in Ikbkg–/– MEFs transduced with an empty retroviral vector (+ vector) or a retroviral vector encoding wild-type NEMO (+ WT) and infected (time,

above lanes) with SV (a) or VSV (b). (c) ELISA of IFN-a and IFN-b in wild-type, Ikbkg–/–, Ikbkb–/– or Irf3–/– MEFs left untransduced or transduced with

retroviral vectors as described in a,b (horizontal axis) and then ‘mock infected’ (NI) or infected with SV or VSV for 14 h. (d) Immunoblot analysis of

whole-cell lysates of wild-type, Ikbkg–/–, Ikbkb–/–, Irf3–/– or Irf3–/–Irf9–/– MEFs left untransduced or transduced with retroviral vectors as described in a,b

(above lanes) and then ‘mock infected’ (–) or infected with SV (+) for 14 h. p-, phosphorylated. (e) Immunoblot analysis of whole-cell lysates of wild-type

and Ikbkg–/– MEFs left untransduced or transduced with retroviral vectors as described in a,b and then left uninfected or infected with SV for 14 h.(f) Immunoblot analysis of whole-cell lysates of RAW264.7 cells transfected with control or Ikbkg-specific siRNA and infected with SV for 16 h. Actin (d–f),

loading control. Data are representative of three experiments (a,b,d) or represent the mean ± s.d. of three experiments (c).

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NEMO in RIG-I-mediated interferon responses

To examine the involvement of NEMO in RIG-I-mediated activationof IRF3 and IRF7, we measured the expression of luciferase reportergenes driven by tandem ISRE elements (ISRE-Luc), the IFNA1promoter (IFNA1-Luc) or the IFNA4 promoter (IFNA4-Luc) inIkbkg–/– MEFs stably reconstituted with vector alone or with vectorencoding wild-type NEMO. SV infection or ectopic expression ofMAVS, the adaptor protein TRIF (Toll–interleukin 1 receptordomain–containing adaptor inducing IFN-b) or the constitutivelyactive form of RIG-I (DRIG-I) resulted in strong induction of ISRE-Luc reporter activity only in Ikbkg–/– MEFs infected with the retrovirusexpressing NEMO (Fig. 2a). Activation of the IFNA1 and IFNA4promoters, which depends heavily on IRF7 function13,14, also requiredNEMO expression (Fig. 2b,c). In contrast, ectopic expression of TBK1promoted activation of the ISRE, IFNA1 and IFNA4 reporter geneconstructs independently of NEMO (Fig. 2a–c), suggesting thatNEMO acts ‘upstream’ of TBK1 and IKKe.

Next we determined whether the defective interferon promoteractivity of NEMO-deficient MEFs was due to a requirement forNF-kB activity. Activation of the ISRE promoter mediated by SV,intracellular dsRNA and MAVS was impaired in the absence of NEMO

but not in the absence of IKKb (Fig. 2d). Furthermore, SV-mediatedactivation of the ISRE promoter was maintained even in MEFs lackingboth IKKa and IKKb (Fig. 2e). Ectopic expression of NEMO, but notof IKKa, IKKb or the NK-kB subunit RelA, restored SV-mediatedactivation of the ISRE promoter in NEMO-deficient MEFs, whereascoexpression of a dominant negative form of IkBa with S32A andS36A substitutions, which is an efficient inhibitor of NF-kB signaling,had no effect on SV-mediated activation of the ISRE promoter inNEMO-reconstituted Ikbkg–/– MEFs (Fig. 2f). However, RelA, IKKaand IKKb activated an NF-kB promoter–driven luciferase reporterconstruct in Ikbkg–/– MEFs reconstituted with wild-type NEMO; in thesame cell type, the dominant negative IkBa blocked VSV-mediatedactivation of NF-kB (Fig. 2g). Thus, NEMO but not NF-kB activity isessential for virus-induced interferon production. Furthermore,NEMO expression is required for the activation of IRF7 and IRF3.These results collectively demonstrate that NEMO acts ‘upstream’ ofTBK1 and IKKe in the activation of IRF3 and IRF7 in response toRIG-I stimulation.

Ser396 in the C-terminal cluster of serine and threonine residues inIRF3, as well as Ser477 and Ser479 in IRF7, are targeted forphosphorylation in vivo after virus infection and are essential in IRF

IκBα D

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b c

Figure 2 NEMO is required for the activation of ISRE and IFNA promoters. Luciferase activity of MEFs transfected with the plasmids described below and,

8 h after transfection, left untreated or treated for an additional 16 h with SV (a–f), dsRNA (d) or VSV (g); luciferase activity was measured 24 h after

transfection. (a) Ikbkg–/– MEFs transduced with empty retroviral vector (+ vector) or retroviral vector encoding wild-type NEMO (+ WT) and then transiently

transfected with an ISRE–luciferase reporter plasmid and either a control plasmid (Vector) or plasmids encoding various signaling molecules (horizontal axis).

(b,c) Analysis as described in a, but in cells transfected with a plasmid encoding IRF7 and with luciferase reporters driven by IFNA1 (b) or IFNA4 (c)

promoters. (d) Ikbkg–/– or Ikbkb–/– MEFs transduced with retroviral vectors (horizontal axis) as described in a and transiently transfected with an ISRE–

luciferase reporter plasmid, followed by transfection with either a control plasmid (–) or a plasmid encoding MAVS or followed by transfection with the

control plasmid and then treatment with either SV or dsRNA. (e) Wild-type and Ikbka–/–Ikbkb–/– MEFs transfected with an ISRE–luciferase reporter plasmid

and then left untreated (–) or treated with SV. (f) Ikbkg–/– MEFs transduced with retroviral vectors as described in a (key) and then transiently transfected

with an ISRE–luciferase reporter plasmid and either control plasmid (Vector) or plasmids encoding various signaling molecules (horizontal axis). The cells

were then infected (+) or not (�) with SV. (g) Ikbkg–/– MEFs transduced with a retroviral vector encoding wild-type NEMO, then transiently transfected with

an ISRE–luciferase reporter plasmid and either control plasmid (Vector) or plasmids encoding various signaling molecules (horizontal axis). The cells were

then infected or not (�) with VSV. DN, dominant negative. Luciferase activity is expressed as ‘fold induction’ relative to basal activity. Data represent themean ± s.d. of three experiments.

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activation22. To investigate the phosphorylation states of IRF3 andIRF7 after SV infection, we expressed human IRF3 or IRF7 in Ikbkg–/–

MEFs. Serine phosphorylation was completely abolished in SV-infected Ikbkg–/– MEFs but was easily detectable in Ikbkg–/– MEFsreconstituted with wild-type NEMO (Fig. 3a). Similarly, expression ofMAVS or the constitutively active RIG-I induced IRF3 and IRF7phosphorylation only in Ikbkg–/– MEFs infected with the NEMO-expressing retrovirus (Fig. 3a). In contrast, expression of TBK1induced serine phosphorylation regardless of the presence or absenceof NEMO (Fig. 3a).

To determine whether NEMO influences the activity of endogenousmouse IRF3, we assessed VSV-induced IRF3 phosphorylation, form-ation of dimers, and DNA-binding activity in Ikbkg+/+ and Ikbkg–/–

MEFs. VSV induced the expression of a form of IRF3 with slowermigration, which corresponds to phosphorylated IRF313,23, in Ikbkg+/+

but not Ikbkg–/– MEFs (Fig. 3b). Similarly, we detected the formationof IRF3 dimers by native gel electrophoresis in Ikbkg+/+ but notIkbkg–/– MEFs (Fig. 3b). Furthermore, electophoretic mobility-shiftassay (EMSA) demonstrated that VSV infection induced the forma-tion of an IRF3 protein–DNA complex that was supershifted afterthe addition of IRF3-specific antibody in Ikbkg+/+ but not Ikbkg–/–

MEFs (Fig. 3c). Finally, STAT1 expression was induced in VSV-infected Ikbkg+/+ but not Ikbkg–/– MEFs (Fig. 3b). These resultsdemonstrate that NEMO is crucial for virus-induced activation ofendogenous IRF3.

Because TBK1 is key in IRF3 phosphorylation, we investigatedwhether NEMO is involved in the activation of the catalytic activity of

TBK1. VSV infection induced TBK1 kinase activity in Ikbkg+/+ but notIkbkg–/– MEFs, as demonstrated by specific TBK1-mediated in vitrophosphorylation of the C-terminal glutathione S-transferase–IRF3(amino acids 380–427) peptide substrate (Fig. 3d). Similar quantitiesof TBK1 were immunoprecipitated in each reaction.

Ikbkg–/– MEFs produce more virus

To determine if NEMO is involved in the establishment of theinterferon-mediated antiviral state, we infected Ikbkg+/+ and Ikbkg–/–

MEFs with VSV or SV and measured viral protein expression atvarious times after infection. We detected VSV proteins (nucleocapsid,surface glycoprotein and matrix) at 9 h after infection in Ikbkg–/–

MEFs; in contrast, we detected small amounts of viral proteinsbeginning at 12 h after infection in Ikbkg+/+ MEFs (Fig. 4a). Weobtained similar results with SV protein expression (data not shown).

Next we used recombinant green fluorescent protein–taggedVSV (GFP-VSV) to directly visualize viral replication. Ikbkg–/– MEFs

a

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0.800.730.750.91.12.42.91.0

161290161290VSV (h)

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Figure 3 Impaired IRF3 and IRF7 activation in Ikbkg–/– MEFs.

(a) Immunoblot analysis of whole-cell extracts of Ikbkg–/– MEFs transduced

an empty retroviral vector (KO) or a retroviral vector encoding wild-type

NEMO (WT) and then transfected with plasmids encoding IRF3 and

IRF7 together with empty vector (Vector) or plasmids expressing Myc-tagged

MAVS, DRIG-I or TBK1; at 10 h after transfection, some cells (+ SV) were

infected for 14 h with SV. (b) Immunoblot analysis (top row and bottom three)

with various antibodies (left margin) and native gel electrophoresis (secondfrom top) of whole-cell extracts of Ikbkg+/+ and Ikbkg�/� MEFs infected with

VSV for various periods of time (above lanes). p-IRF3, phosphorylated IRF3;

VSV G, surface glycoprotein; VSV N, nucleocapsid protein. (c) EMSA of

whole-cell extracts from b incubated with a 32P-labeled probe corresponding

to the ISRE motif from the promoter of the gene encoding the ubiquitin-like

modifier ISG15. Far right lane, anti-IRF3 added to a wild-type sample. Arrow

indicates DNA binding of IRF3. (d) Kinase activity (top) and immunoblot

analysis (IB; middle) of TBK1 immunoprecipitated from whole-cell extracts

of Ikbkg+/+ and Ikbkg–/– MEFs infected with VSV for various periods of time

(above lanes). Kinase activity of immunoprecipitated TBK1 was measured

with glutathione S-transferase–IRF-3 (amino acids 380–427) as a substrate.

Immunoblot analysis is of TBK1 immunoprecipitates, using anti-TBK1.

Bottom, Coomassie staining of whole-cell lysates; numbers below lanes

indicate amount of IRF3 in each lane relative to basal amount (set as 1.0).

Data are representative of two (a,d) or three (b,c) experiments.

1815129630Time after infection (h)

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cFigure 4 Defective antiviral response in Ikbkg–/– MEFs. (a) Immunoblot

of whole-cell extracts of Ikbkg+/+ and Ikbkg–/– MEFs infected with VSV for

various periods of time (above lanes), analyzed with VSV-specific antisera.

Left margin, VSV N, G and M (matrix) proteins. (b) Phase-contrast

microscopy (top) and fluorescence microscopy (bottom) of Ikbkg+/+ and

Ikbkg–/– MEFs infected for 16 h with recombinant GFP-VSV virus. Original

magnification, �50. (c) Standard plaque assay of VSV replication assessed

in supernatants of Ikbkg+/+ and Ikbkg–/– MEFs transduced with emptyretroviral vector (circles) or retroviral vector encoding wild-type NEMO

(triangles) and infected with VSV for various periods of time (horizontal

axis); titers are presented as plaque-forming units per ml (PFU/ml).

Data are representative of three (a) or two (b,c) experiments.

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were more permissive to viral infection and replication than wereIkbkg+/+ MEFs (as assessed by GFP fluorescence; Fig. 4b). Quantifica-tion of virus replication by plaque assay showed that the yield ofinfectious virus in Ikbkg–/– MEFs was 1 to 2 logs higher than that inIkbkg–/–MEFs reconstituted with NEMO (Fig. 4c). Thus, NEMO isrequired for the effective generation of host interferon-mediatedantiviral responses.

NEMO interacts with TBK1 and IKKe through TANK

To elucidate the mechanism by which NEMO regulates IRF3 and IRF7activation, we examined the possibility that NEMO could interactwith TBK1 and/or IKKe. We expressed Flag-tagged NEMO togetherwith GFP-tagged IKKe (Fig. 5a, left) or GFP-tagged TBK1 (Fig. 5a,right), with or without Myc-tagged TANK, in HEK293 cells. GFP-tagged IKKe and GFP-tagged TBK1 immunoprecipitated togetherwith NEMO only in the presence of TANK (Fig. 5a). This observationis consistent with published studies demonstrating that TANK inter-acts with IKKe and/or TBK1 to activate the IKK complex24,25. Notably,a NEMO mutant with internal deletion of residues 196–250, whichremoves the TANK-binding domain24, failed to associate with TBK1or IKKe in cells coexpressing TANK (Fig. 5a). These results suggestthat TANK is involved in the formation of tripartite TBK1-TANK-NEMO or IKKe-TANK-NEMO complexes.

Because TANK orchestrates the formation of NEMO-TBK1 andNEMO-IKKe complexes, we reasoned that TANK may be involved invirus-triggered signaling. To test that idea, we used short hairpin RNA(shRNA) constructs containing sequences complementary to humanTANK cDNA to inhibit endogenous TANK expression in HEK293 cells.Five TANK-specific shRNA constructs suppressed TANK expression tovarying degrees (Fig. 5b). To determine whether TANK is involved invirus-mediated ISRE activation, we assessed SV-induced transcriptionof an ISRE–luciferase reporter construct in HEK293 cells expressingTANK-specific shRNA. ‘Knockdown’ of TANK expression inhibitedSV-mediated ISRE activation (Fig. 5b). The extent of inhibitioncorrelated with the efficiency of the ‘knockdown’ of TANKexpression. Consistent with these data, ‘knockdown’ of TANKexpression also suppressed SV-mediated induction of the interferon-inducible gene encoding IFIT1 (also called ISG56) and the geneencoding RIG-I (Fig. 5b).

Mapping the functional domains of NEMO

NEMO contains many distinct domains, including a TANK-bindingdomain24, two coil-coiled domains (CC1 and CC2), a leucine zipperregion and a zinc finger domain26. To identify the functional domainsrequired for RIG-I- and MAVS-mediated induction of interferon, wegenerated a series of NEMO deletion mutants (Fig. 6a). We expressed

a b

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IP: α-Flag

IB: α-GFP

IB: α-Myc

IB: α-Flag

IB: α-GFP

IB: α-Myc

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Figure 5 TANK links IKKe and TBK1 to NEMO. (a) Immunoblot analysis

of HEK293 cells transfected with empty vector (–) or with vectors

encoding Flag-tagged wild-type NEMO or NEMO lacking the TANK-binding

domain (D196–250) together with GFP-tagged IKKe or TBK1 and

Myc-tagged TANK; at 24 h after transfection, Flag-tagged proteins were

immunoprecipitated (IP) for analysis, and whole-cell extracts (WCE) were

also prepared for analysis. a-, antibody to. (b) Luciferase activity and

immunoblot analysis of HEK293 cells transfected with plasmids encodingcontrol (Vector) or TANK-specific shRNA constructs (shRNA50, shRNA51,

shRNA52, shRNA53 and shRNA54); after selection in puromycin, cells

were transfected with an ISRE–luciferase reporter for an additional 8 h,

then were left uninfected (–) or were infected with SV (+) for 15 h (below

lanes). Top, luciferase activity, measured 24 h after the second

transfection, expressed as ‘fold induction’ relative to basal activation.

Bottom, immunoblot analysis of whole-cell lysates. Data are representative

of two (a) or three (b, bottom) experiments or the mean + s.d. of three

experiments (b, top).

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Figure 6 NEMO domains required for ISRE activation. (a) NEMO deletion mutants. Top (left), 419–amino acid human NEMO: CC1 and CC2, coil-coiled

domains 1 and 2; LZ, leucine zipper region; ZF, zinc finger domain. Below (left), deletion mutants: 1–395, 1–369 and 1–350, deletion of 24, 50 or

69 amino acids, respectively, from the C-terminal region; 1–306, deletion to amino acid 306; 92–419 and 141–419, deletion of 91 or 140 amino acids,respectively, from the N-terminal region; 151–419 and 251–419, further deletion to position 150 or 250, respectively; D196–250, D196–290 and

D262–290, internal deletion of residues 196–250 (TANK-binding domain), 196–290 (TANK-binding and CC2 domains) and 262–290 (almost the entire

CC2 domain), respectively. Right, immunoblot analysis of whole-cell extracts of Ikbkg–/– MEFs transiently transfected with plasmids encoding the NEMO

mutants at left. (b) Luciferase activity of Ikbkg–/– MEFs transfected with an ISRE–luciferase reporter plasmid and plasmids encoding the NEMO mutants in

a (horizontal axes) and a plasmid encoding MAVS (right) or treated with SV for 16 h (left). Luciferase activity, analyzed 24 h after the second transfection,

is expressed as ‘fold induction’ relative to basal activity. FL, full-length. Data represent the mean ± s.d. of three experiments.

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plasmids encoding each mutant in Ikbkg–/– MEFs and assessed theirability to transduce ISRE-activating signals. Deletion of 24, 50 or69 amino acids from the C-terminal region of NEMO only modestlyreduced SV-induced ISRE–luciferase reporter activity; in contrast,further deletion to position 306, which eliminated the leucine zipperdomain, or internal deletion of residues 196–250, which removed theTANK-binding domain, abrogated SV-triggered ISRE–luciferasereporter induction (Fig. 6b, left). Deletion of 91 or 140 amino acidsfrom the N-terminal region of NEMO moderately reducedSV-mediated ISRE-luciferase activity, whereas further deletion toposition 150 or 250 completely abolished SV-induced ISRE-luciferaseactivity (Fig. 6b, left). An internal deletion that removed almost theentire CC2 domain modestly reduced virus-induced ISRE promoteractivity. We obtained similar results with VSV (Supplementary Fig. 1online) or MAVS (Fig. 6b, right) as an ISRE promoter stimulus.

Next we determined whether NEMO is required for ISRE activationtriggered by cytosolic B-form DNA. NEMO constructs lacking thezinc finger domain (deletion of amino acids 1–395, 1–369 or 1–350),the CC2 domain (deletion of amino acids 262–290), internal aminoacids 196–250, or 91 or 140 N-terminal amino acids supportedcytosolic DNA-induced ISRE promoter activity (SupplementaryFig. 2 online). In contrast, further deletion that eliminated the leucinezipper region (amino acids 1–306) or deletion to residue 250 from theN-terminal region essentially abrogated cytosolic DNA-induced tran-scription of the ISRE–luciferase reporter.

The results of the mutagenesis studies reported above indicated thatISRE activation mediated by SV, MAVS and cytosolic DNA requiresdistinct NEMO domains. Next we identified the NEMO domainsrequired for activation of NF-kB promoter–driven reporter constructs.C-terminal deletions (of amino acids 1–395, 1–369 or 1–350) orinternal deletions (of amino acids 196–250 or 262–290) resulted in lessVSV-induced transcription of an NF-kB luciferase reporter (Supple-mentary Fig. 1). Notably, deletion of the first 91, 140, 150 or 250amino acids from the N-terminal region of NEMO completelyabolished VSV-induced NF-kB promoter activity (SupplementaryFig. 1). These results further support the idea that different functionaldomains of NEMO are required for the transduction of differentintracellular signals.

NEMO substitutions impair ISRE activation

In humans, hypomorphic mutations in the gene encoding NEMO leadto ectodermal dysplasia with immunodeficiency and to increasedsusceptibility to bacterial and virus infections27,28. To determine

whether ectodermal dysplasia with immunodeficiency is in part dueto impaired NEMO-mediated transduction of signals leading tointerferon production, we generated constructs encoding some ofthe known NEMO substitutions associated with ectodermal dysplasiawith immunodeficiency (L80P, L153R, R175P, L227P, D311N, D406Vand C417R) and assessed their ability to stimulate ISRE–luciferasereporter activity in Ikbkg–/– MEFs (Fig. 7a). The L80P substitution hadessentially no effect on SV- or MAVS-mediated activation of the ISREpromoter, whereas the D406V and C417R substitutions slightlyreduced MAVS- but not SV-induced ISRE promoter activity(Fig. 7b). In contrast, substitutions located in the TANK-binding,CC1 and LZ domains (L227P, L153R, R175P and D311N) almostcompletely abolished MAVS-induced promoter activity and partiallyimpaired SV-induced ISRE promoter activity.

NEMO is a sensor of K63-linked polyubiquitination, and NEMOsubstitutions that prevent recognition of polyubiquitin chains byNEMO (Y308S, D311N and L329P) abolish IKK activation29,30. TheY308S, D311N, L329P and K285R substitutions also abrogated MAVS-induced ISRE promoter activity and substantially reduced SV-inducedISRE promoter activity (Fig. 7b). The simultaneous C396S and C400Ssubstitutions in the NEMO zinc finger domain also resulted inimpaired MAVS- and virus-mediated activation of ISRE promoter(Fig. 7b). Notably, the L80P substitution abolished VSV-inducedNF-kB activation but had no effect on VSV-mediated ISRE activation(Supplementary Fig. 3 online). These results indicate that mostmutations in the gene encoding NEMO that are associated withectodermal dysplasia with immunodeficiency also compromise inter-feron signaling.

DISCUSSION

Here we have documented an essential function for NEMO in RNAvirus–induced activation of IRF3 and IRF7 mediated by RIG-Iand MAVS and subsequent induction of interferon production.Virus-mediated production of endogenous interferon and interferon-inducible gene expression was severely impaired in Ikbkg–/– MEFs.Activation of STAT1 and the generation of an interferon-inducedantiviral state were also abolished in Ikbkg–/– MEFs. Expression ofMAVS or the constitutively active DRIG-I in Ikbkg–/– MEFs failed tostimulate phosphorylation and activation of IRF3 and IRF7 as well assubsequent ISRE reporter gene activation. TBK1 triggered ISREpromoter activity regardless of the presence or absence of NEMO,indicating that NEMO acts ‘upstream’ of TBK1. The association ofNEMO with TANK facilitated the recruitment of TBK1 and IKKe to

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SV MSVa b

Figure 7 NEMO point substitutions affecting activation of the ISRE promoter. (a) NEMO substitution mutants. Top (left), 419–amino acid human NEMO (as

described in Fig. 6a). Below (left), substitution mutants. Right, immunoblot analysis of whole-cell extracts of Ikbkg–/– MEFs transiently transfected with

plasmids encoding the NEMO mutants at left. (b) Luciferase activity of Ikbkg–/– MEFs, assessed as described in Figure 6b but using the NEMO substitution

mutants (horizontal axes). Data represent the mean ± s.d. of three experiments.

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the MAVS mitochondrial complex, and the TANK-binding, coiled-coiland leucine zipper domains of NEMO were required for virus-inducedISRE activation.

NEMO is an integral regulatory subunit of the canonical IKKcomplex. Targeted disruption of the gene encoding NEMO rendersIKK nonfunctional and consequently abolishes NF-kB activity in re-sponse to many stimuli, including tumor necrosis factor, interleukin 1and virus infection31,32. The abrogation of virus-mediated activationof IRF3 and IRF7 was not due to the lack of canonical NF-kB functionin Ikbkg–/– MEFs, as production of interferon and activation ofinterferon-inducible gene expression was normal in IKKb-deficientMEFs. The adaptor proteins TANK and NAP1 have been shown tointeract with TBK1 and IKKe19,33,34, and in fact TBK1 was initiallycharacterized as a TANK-binding kinase19; IKKe has also been shownto interact with TANK33. It has been suggested that the association ofTANK with these two kinases positively regulates NF-kB activity19,33.Notably, TANK has also been also identified as a NEMO-interactingprotein24. Exogenous TBK1 and IKKe did not immunoprecipitatetogether with NEMO; however, coexpression of TANK promotedinteraction between TBK1 and NEMO as well as between IKKe andNEMO24. Furthermore, NEMO mutants lacking the TANK-bindingdomain failed to interact with IKKe and TBK1 or to transduce virus-or MAVS-mediated signals culminating in interferon production andISRE reporter activity. ‘Knockdown’ of TANK expression also inhib-ited SV-mediated ISRE activity and expression of the genes encodingIFIT1 and RIG-I.

We propose a model in which RIG-I senses virus-encoded5¢-triphosphate RNA through its helicase domain and relays ‘down-stream’ signals through its N-terminal caspase-recruitment domain tothe mitochondrial adaptor MAVS (Supplementary Fig. 4 online)35.MAVS then recruits TRAF3 and/or TRAF6 through a direct interactionbetween the TRAF domain of TRAF3 and/or TRAF6 and a TRAF-interaction motif in MAVS16,36. The association of NEMO withTANK facilitates the recruitment of TBK1 and IKKe to the MAVS-TRAF mitochondrial complex and results in the activation ofTBK1 and IKKe. Activated TBK1 and IKKe phosphorylate IRF3and IRF7, which elicit ISRE activation, interferon production andthe adoption of an antiviral state. MAVS-TRAF complexes canalso lead to NF-kB activation by means of the canonical NEMO-IKKa-IKKb complex, which phosphorylates the inhibitory subunitIkBa, resulting in the release of NF-kB and activation of proinflam-matory gene transcription.

NEMO senses K63-linked polyubiquitination, and binding ofNEMO to K63-linked polyubiquitin chains is required for activationof the canonical IKK complex29,30. Several single point substitutions,including L329P, D311N and Y308S, which impaired the ability ofNEMO to bind to K63-linked polyubiquitin chains, also abolish therecruitment of IKK to the tumor necrosis factor receptor and theactivation of NF-kB29,30. Notably, these substitutions also abrogatedthe ability of NEMO to transduce MAVS-mediated signals resulting inactivation of IRF3 and IRF7. Data indicate that IKKe is recruited tothe MAVS mitochondrial complex after virus infection37; whetherNEMO is involved in the recruitment of IKKe to the mitochondrialsignaling complex remains to be determined.

Mutations in IKBKG have been directly linked to rare humangenetic disorders, including incontinentia pigmenti and ectodermaldysplasia with immunodeficiency27,38. Most patients with incontinen-tia pigmenti have an identical genomic deletion from exon 4 to exon10 (loss of function) in IKBKG, whereas patients with the disorderectodermal dysplasia with immunodeficiency have hypomorphicmutations in IKBKG that impair but do not abolish NF-kB activation.

These mutations result in a combined, variable but profoundimmunodeficiency associated with increased susceptibility to bacterialand viral infection39. Here we have demonstrated that most mutantsresulting from mutations in IKBKG that are associated with ectoder-mal dysplasia with immunodeficiency failed to transduce signalsemanating from RIG-I. Our observations raise the possibility thatmutations in IKBKG that are associated with impaired RIG-I-inducedinterferon production contribute to the immunodeficiency found inpatients with ectodermal dysplasia with immunodeficiency.

METHODSPlasmid construction and site-directed mutagenesis. Plasmids encoding IRF7,

MAVS, DRIG-I, TRIF, IKKe, TBK1, ISRE-Luc, P2(2)-TK pGL3, IFNA1-pGL3,

IFNA4-pGL3, pRLTK, IKKa, IKKb, RelA and IkBa (S32,36A) have been

described13,40,41. TANK cDNA was amplified by PCR from a human T cell

cDNA library and was cloned into Myc-pcDNA3.1-Zeo. Plasmids encoding

human full-length NEMO, NEMO constructs of amino acids 1–395 or 251–419,

and the L329P NEMO substitution mutant have been described29. Other

NEMO deletion mutants were generated by standard PCR methods, and point

mutations were introduced with a Quickchange Kit according to the manu-

facturer’s instructions (Stratagene). DNA sequencing was done to confirm the

mutations. The cDNA encoding human NEMO and IKKb42 was cloned into the

pBabePuro retroviral vector carrying the puromycin-resistance gene43.

Cell culture, transfection and luciferase assays. Irf3–/– or Irf3–/–Irf9–/– MEFs44,

Ikbkb–/–, Ikbkg–/– and wild-type MEFs32,45 and MEFs derived from mice lacking

IKKa and IKKb46 have been described. MEFs were grown in DMEM media

(Wisent) supplemented with 10% (vol/vol) FBS, glutamine and antibiotics.

Retroviral stocks were generated as described47. After retroviral infection of

Ikbkb–/–, Ikbkg–/– MEFs47, stably transduced bulk cultures were selected in

media containing puromycin (2 mg/ml; Sigma), and individual populations

expressing each construct were isolated. RAW264.7 cells were grown in RPMI

1640 media (Wisent) supplemented with 10% (vol/vol) FBS, sodium pyruvate

and antibiotics. For luciferase assays, MEFs grown to subconfluency in DMEM

(Wisent) supplemented with 10% (vol/vol) FBS, glutamine and antibiotics

were transfected with 200 ng of pRLTK reporter (renilla luciferase; internal

control), 200 ng of ISRE-pGL3, IFNA1-pGL3 or IFNA4-pGL3 luciferase

reporter (firefly luciferase; experimental reporter), 100 ng IRF7 expression

plasmid, 200 ng of MAVS, DRIG-I, TRIF, IKKe or TBK1 expression plasmid,

and 20 ng NEMO expression plasmid using Lipofectamine 2000 according to

the manufacturer’s instructions (Invitrogen). At 24 h after transfection,

luciferase activity was measured with a Dual-Luciferase Reporter Assay System

according to the manufacturer’s instructions (Promega). Some cells were

treated with SV (40 hemagglutination units per ml) or were transfected for

15 h with dsRNA (4 mg/ml of poly(I:C); Sigma) or poly(dA:dT) (10 mg/ml; GE

Biosciences) using Lipofectamine 2000.

Immunoblot analysis. Whole-cell extracts (20–100 mg) were separated by 8%

SDS-PAGE. After electrophoresis, proteins were transferred for 1 h at 4 1C to

Hybond-C transfer membranes (Amersham) in a buffer containing 30 mM Tris,

200 mM glycine and 20% (vol/vol) methanol. Membranes were blocked for 1 h

at 25 1C in 5% (wt/vol) dried milk in PBS and 0.1% (vol/vol) Tween-20 (PBST)

and then were probed with antibody to Flag (anti-Flag (M2); Sigma), VSV

whole virus antisera, SV antisera, anti-IKKa (H744; Santa Cruz Biotechnology),

anti-IKKb (2C8; Cell Signaling), anti-NEMO (DA10-12; Cell Signaling), anti-

IRF3 (ZM3; Zymed), anti-IRF3 (FL-425; Santa Cruz Biotechnology), antibody

to IRF3 phosphorylated at Ser396 (ref. 22), antibody to IRF7 phosphorylated at

Ser 477 and Ser479 (ref. 48), anti-STAT1 (C-111; Santa Cruz Biotechnology),

anti-TANK (C-20; Santa Cruz Biotechnology), antibody to STAT1 phosphory-

lated at Tyr701 (58D6; Cell Signaling) or anti-actin (MAB1501; Chemicon) at a

dilution of 1 mg/ml in 5% (wt/vol) milk in PBS. After four 10-minute washes

with 5% (wt/vol) dried milk in PBS and 0.1% (vol/vol) Tween-20, membranes

were incubated for 1 h with horseradish peroxidase–conjugated goat anti-rabbit

or anti-mouse (Amersham) at a dilution of 1:3,000 in blocking solution. The

reaction was then visualized with an enhanced chemiluminescence detection

system as recommended by the manufacturer.

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Analysis of protein-protein interactions. HEK293 cells were transfected

with expression plasmids. Whole-cell extracts (300 mg) were prepared from

transfected cells and were incubated for 1 h at 4 1C with 1 mg anti-Myc (9E10;

Sigma-Aldrich) or anti-Flag (M2; Sigma-Aldrich) crosslinked to 100 ml protein

A/G PLUS-Agarose (Santa Cruz Biotechnology)49. Precipitates were washed five

times with lysis buffer and proteins were eluted by boiling of the beads for

3 min in 1� SDS sample buffer. Eluted proteins or 5% of the input whole cell

extracts were separated by 10% SDS-PAGE. After electrophoresis, proteins were

transferred for 1 h to Hybond transfer membranes (Amersham) in a buffer

containing 30 mM Tris, 200 mM glycine and 20% (vol/vol) methanol.

Membranes were blocked by incubation for 1 h in PBS containing 5% (wt/vol)

dried milk and then were probed with anti-GFP (A11122; Invitrogen), anti-

hemagglutinin (H7; Sigma-Aldrich), anti-Flag (M2; Sigma-Aldrich) or anti-Myc

(9E10; Sigma-Aldrich), each at a concentration of 1 mg/ml. Immunocomplexes

were detected with a chemiluminescence-based system (ECL; Amersham).

Virus production, quantification and infection. Wild-type VSV (Indiana

serotype), AV1 VSV and GFP-VSV were propagated in Vero cells as described50.

Viruses were obtained from cell-free supernatants and were titrated on Vero cells

by standard plaque assay50. Virus yields are expressed as plaque-forming units

(PFU) of released virus per milliliter. Cells were infected with VSV virus as

described50. For GFP-VSV, virus replication was analyzed with inverted micro-

scopy equipped with an ultraviolet lamp (Axiovert 25; Zeiss).

RNA interference, RT-PCR, in vitro kinase assay, formation of IRF3 dimers,

ELISA and EMSA. These procedures are described in the Supplementary

Methods online, and primers used in RT-PCR are listed in Supplementary

Table 1 online.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank M. Schmidt-Supprian, S.C. Sun, S. Yamaoka, G. Sen and T. Dermodyfor reagents used in this study; D. Goubau and M. Solis for isolation of humanprimary macrophages; and members of the Molecular Oncology Group at theLady Davis Institute for discussions. We thank J.D. Ashwell (National Institutesof Health) for plasmids encoding human full-length NEMO, NEMO constructsof amino acids 1–395 or 251–419, and the L329P NEMO substitution mutant;K. Mossman (McMaster University) for Irf3–/– or Irf3–/–Irf9–/– MEFs; M. Karin(University of California, San Diego) for Ikbkb–/–, Ikbkg–/– and wild-type MEFs;I. Verma (The Salk Institute) for MEFs derived from mice lacking IKKa andIKKb; J. Bell (Ottawa Cancer Centre) for VSV whole virus antisera; andI. Julkunen (National Public Health Institute and University of Helsinki)for SV antisera. Supported by the Cancer Research Society (R.L.), CanadianInstitutes of Health Research (R.L. and J.H.), the National Cancer Institute ofCanada with support from the Canadian Cancer Society (J.H.), the NationalInstitutes of Health (AI052379 and CA082556 to D.B.W.), Fonds de laRecherche en Sante Quebec (R.L.) and the Canadian Institutes of HealthResearch (J.H.).

AUTHOR CONTRIBUTIONSR.L. designed the research, did experiments, analyzed data, supervised allexperiments and wrote the paper; T.Z. did RT-PCR, ELISA, RNA interference andfluorescence microscopy assays; L.Y. did EMSAs, plaque assays and some of theimmunoblots; Q.S. did the in vitro kinase assays; J.H. and M.A. wrote the papertogether; and D.W.B. provided new reagents and contributed to discussions.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

Reprints and permissions information is available online at http://npg.nature.com/

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