tradd protein is an essential component of the rig-like helicase antiviral pathway

Immunity

Article

TRADD Protein Is an Essential Componentof the RIG-like Helicase Antiviral PathwayMarie-Cecile Michallet,1 Etienne Meylan,1,7 Maria A. Ermolaeva,2 Jessica Vazquez,1 Manuele Rebsamen,1

Joseph Curran,3 Hendrik Poeck,4 Michael Bscheider,5 Gunther Hartmann,4 Martin Konig,6 Ulrich Kalinke,6

Manolis Pasparakis,2 and Jurg Tschopp1,*1Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland2Institute for Genetics, University of Cologne, Zulpicher Strasse 47, 50674 Cologne, Germany3Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1 rue Michel Servet,

CH-1211 Geneva 4, Switzerland4Institute of Clinical Biochemistry and Pharmacology, University of Bonn, 53127 Bonn, Germany5Division of Clinical Pharmacology, Department of Internal Medicine, University of Munich, 80336 Munich, Germany6Paul-Ehrlich-Institut, Federal Agency for Sera and Vaccines, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany7Present address: Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E17-517,

Cambridge, MA 02139, USA.*Correspondence: [email protected]

DOI 10.1016/j.immuni.2008.03.013

SUMMARY

Upon detection of viral RNA, the helicases RIG-I and/or MDA5 trigger, via their adaptor Cardif (also knownas IPS-1, MAVS, or VISA), the activation of the tran-scription factors NF-kB and IRF3, which collaborateto induce an antiviral type I interferon (IFN) response.FADD and RIP1, known as mediators of death-recep-tor signaling, are implicated in this antiviral pathway;however, the link between death-receptor and antivi-ral signaling is not known. Here we showed thatTRADD, a crucial adaptor of tumor necrosis factorreceptor (TNFRI), was important in RIG-like helicase(RLH)-mediated signal transduction. TRADD is re-cruited to Cardif and orchestrated complex forma-tion with the E3 ubiquitin ligase TRAF3 and TANKand with FADD and RIP1, leading to the activationof IRF3 and NF-kB. Loss of TRADD preventedCardif-dependent activation of IFN-b, reduced theproduction of IFN-b in response to RNA viruses,and enhanced vesicular stomatitis virus replication.Thus, TRADD is not only an essential component ofproinflammatory TNFRI signaling, but is also re-quired for RLH-Cardif-dependent antiviral immuneresponses.

INTRODUCTION

Viruses are the most abundant and diverse pathogens that

depend on host cellular machinery for survival and replication.

A key aspect of the antiviral innate immune response is the rapid

synthesis and secretion of type I interferons (IFN-a and IFN-b)

(Isaacs and Lindenmann, 1957). It is now well established that

rapid immune recognition depends on highly conserved,

invariant structures of microbes that are distinct from the host

(Janeway, 1989). Such pathogen-associated molecular patterns

(PAMPs) include a range of components of bacteria, fungi, and

viruses including nucleic acids. Hence, virus-associated mole-

cules such as genomic DNA and RNA or double-stranded RNA

(dsRNA) can be recognized by host pattern-recognition recep-

tors (PRRs) expressed in innate immune cells such as dendritic

cells (DCs) (Iwasaki and Medzhitov, 2004; Theofilopoulos et al.,

2005). The sensing of viral PAMPs by host PRRs triggers the

initiation of signaling cascades that most importantly regulate

the synthesis of IFNs, which exhibit potent antiviral, antiprolifer-

ative, and immunomodulatory functions (Honda et al., 2005).

In mammals, the recognition of PAMPs by Toll-like receptors

(TLRs) is reasonably well understood. Among the characterized

TLRs, four members (TLR3, TLR7, TLR8, and TLR9) reside in

endosomes and are specialized in the detection of nucleic acids

(Akira et al., 2006). TLR3 recognizes dsRNA that is produced by

many viruses during replication (Alexopoulou et al., 2001),

whereas TLR7, TLR8, and TLR9 are activated by single-stranded

RNA (ssRNA) species and unmethylated CpG DNA motifs,

respectively (Diebold et al., 2004; Heil et al., 2004; Hemmi

et al., 2000). Although TLR pathways operate mainly within key

sentinel cells of the innate system (for instance, macrophages

and plasmacytoid DCs [pDCs]) to detect viral DNA and RNA,

TLRs are largely dispensable for effective antiviral defense in

most other cell types that rely on cytoplasmic virus sensors

(Creagh and O’Neill, 2006; Meylan and Tschopp, 2006). Two

such receptors, the RIG-like helicase (RLH) retinoic-acid-induc-

ible gene I (RIG-I) and melanoma differentiation antigen 5

(MDA5), were identified as important sensors of cytoplasmic viral

RNA (Gitlin et al., 2006; Kato et al., 2005; Yoneyama et al., 2004),

establishing an alternative pathway of virus detection. Mice lack-

ing RIG-I and MDA5 show dramatic phenotypes when chal-

lenged with RNA viruses in vitro and in vivo and highlight the

specificity of each helicase for different virus strains (Gitlin

et al., 2006; Kato et al., 2005; Kato et al., 2006). Recently, the

precise nature of the viral RNA structure recognized by RIG-I

was identified as 50-triphosphate RNA (Hornung et al., 2006;

Pichlmair et al., 2006), whereas the ligand for MDA5 remains

Immunity 28, 651–661, May 2008 ª2008 Elsevier Inc. 651

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Antiviral Role of TRADD Protein

uncharacterized. Given that RIG-I and MDA5 contain caspase

recruitment domain (CARD)-like structures, a CARD-containing

adaptor protein, Cardif (also called IPS-1, MAVS, and VISA)

was found to transmit the antiviral response downstream of

RIG-I and MDA5 (Kawai et al., 2005; Meylan et al., 2005; Seth

et al., 2005; Xu et al., 2005). RIG-I and MDA5 recruit Cardif

through CARD homotypic interactions, leading to the activation

of NF-kB and interferon regulating factor (IRF) 3 transcription

factors, which are both critical for cellular antiviral responses.

IRF3 activation requires phosphorylation by two kinases belong-

ing to the IKK family, IKK3 and TBK1, which results in its dimer-

ization and nuclear translocation (Fitzgerald et al., 2003; Sharma

et al., 2003). Activated, nuclear IRF3 and NF-kB transcription

factors then trigger the expression of type I IFNs, which are

subsequently secreted to bind their respective receptors on

virus-infected and neighboring noninfected cells.

Fas-associated death domain (FADD) and receptor-interact-

ing protein (RIP) 1 are two death domain (DD)-containing

proteins originally described to have crucial roles in proinflam-

matory and proapoptotic signaling events because of their

interaction with tumor necrosis factor receptor (TNFR) family

members (Chinnaiyan et al., 1996; Hsu et al., 1996a; Kischkel

et al., 2000; Stanger et al., 1995). Recently, however, cells lack-

ing FADD were also shown to be defective in intracellular

dsRNA-activated gene expression, including production of

type I IFNs, resulting in increased susceptibility to viral infection

(Balachandran et al., 2004; Kumar et al., 2006). Similar defects in

dsRNA responses were observed in RIP1-deficient cells

(Balachandran et al., 2004), suggesting that FADD and RIP1

form a complex that is essential in dsRNA-dependent signaling.

Indeed, the first evidence of their role in the RLH signaling was

the demonstration that Cardif interacted with FADD and RIP1

via its non-CARD region to facilitate downstream NF-kB activa-

tion (Kawai et al., 2005). Furthermore, cleavage of initiator

caspases interacting with FADD, caspase-8, and caspase-10

has been reported during dsRNA stimulation, suggesting a role

of these two proteases in antiviral immune responses in addition

to their function in apoptosis (Takahashi et al., 2006). However,

despite observations that FADD and RIP1 complex with Cardif

and that caspases are cleaved in response to dsRNA, the mech-

anism of recruitment and the role of these proteins in RIG-I- or

MDA5-mediated antiviral signaling remain ill defined.

Here we investigated the role of TNFR-associated death

domain (TRADD) protein in RLH-dependent antiviral responses,

which, similar to FADD and RIP1, is a crucial signaling adaptor

mediating intracellular responses from TNFRI (Hsu et al.,

1996b; Hsu et al., 1995). We found that TRADD interacts with

Cardif, recruits the E3 ubiquitin ligase TRAF3 and the adaptor

protein TANK, and forms a complex with FADD and RIP1, lead-

ing to the activation of IRF3 and NF-kB transcription factors.

Silencing of TRADD expression resulted in a reduction of RIG-

I-mediated antiviral responses as well as an enhanced vesicular

stomatitis virus (VSV) replication. Moreover, deficiency of

TRADD prevented Cardif-dependent activation of IFN-b in fibro-

blasts and reduced the production of IFN-b in response to VSV

and Sendai viruses in vitro. Finally, virally infected TRADD-defi-

cient mice produced less IFN-b. Thus, TRADD serves not only

TNF-dependent inflammatory but also RLH-mediated antiviral

functions.

652 Immunity 28, 651–661, May 2008 ª2008 Elsevier Inc.

RESULTS

TRADD Activates IRF3 and NF-kB Promotersand Interacts with CardifAs a first approach to investigate whether TRADD may play a role

in the innate antiviral response, we overexpressed TRADD in

human embryonic kidney (HEK) 293T cells and determined

type I IFN responses. TRADD overexpression activated an IFN-

b-responsive promoter in a dose-dependent manner (Figure 1A)

as well as an IRF3-dependent interferon-stimulated regulatory

element (ISRE of ISG54; Figure 1B). As expected, overexpressed

TRADD also activated a NF-kB-dependent promoter (Figure 1C)

(Hsu et al., 1995). Finally, we observed a synergistic effect on

ISRE and NF-kB responses when TRADD was overexpressed

with Cardif, MDA5 activated by poly(I:C) (a synthetic dsRNA

that is commonly used to mimic viral infection, or RIG-I in the

presence of poly(I:C) or its specific ligand 3pRNA (Figures 1D

and 1E, and Figure S1 available online). This synergistic effect

was also detected when TRADD was cotransfected with

poly(I:C) alone and with IRF3 or IRF7 (Figure S2). Because

TLR3 is not involved in cytoplasmic poly(I:C) recognition, it was

likely that TRADD was involved in the RIG-I- or MDA5-mediated,

Cardif-dependent signaling pathway. To substantiate this possi-

bility, we coexpressed TRADD and Cardif and observed after

immunoprecipitation an interaction between both proteins

(Figure 1F). To map the TRADD-Cardif interaction, we used a

series of Cardif deletion constructs previously described (Meylan

et al., 2005). TRADD strongly interacted with a Cardif construct

lacking the N-terminal CARD domain (DCARD) and more weakly

with a construct lacking the C-terminal transmembrane region

(DTM) that is known to target Cardif to the mitochondria

(Figure 1F). No interaction was detected with a construct con-

taining only the CARD domain (Figure 1F), indicating that TRADD

associates with the intermediate region of Cardif. We also gener-

ated a TRADD construct lacking the DD, which interacted with

Cardif to a similar extent as the full-length molecule (Figure 1G).

These results show that neither the DD of TRADD nor the CARD

of Cardif is implicated in the Cardif-TRADD interaction. We next

investigated whether the interaction between TRADD and Cardif

also occurs at the endogenous level. As shown in Figure 1H,

Cardif DCARD was able to recruit endogenous TRADD protein.

Importantly, specific formation of the TRADD- and Cardif-con-

taining complex was dependent on the activation of RIG-I and/

or MDA5 by poly(I:C) (Figure 1I), but never detected upon TNFRI

activation (Figure S3). Finally, DAI (DLM-1 or ZBP1) has been

recently identified as a cytoplasmic DNA sensor leading to the

production of type I IFN (Takaoka et al., 2007). We therefore

investigated whether TRADD may also play a role in the DAI-

dependent pathway. However, TRADD siRNA had no effect on

poly(A:T)-induced ISRE promoter activity (Figure S4). Thus,

TRADD is recruited to Cardif upon stimulation of RIG-I or MDA5.

TRADD Knockdown Inhibits RIG-I-InducedAntiviral ResponsesTo examine the functional significance of the TRADD-Cardif in-

teraction, we determined whether TRADD siRNA could inhibit

RIG-I- or Cardif-induced IFN responses. TRADD knockdown

with short-interfering RNA (siRNA) attenuated RIG-I-induced

ISRE promoter activation (Figure 2A, Figure S5). Trif is an adaptor

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protein that is essential for TLR3- and TLR4-dependent IFN

responses. ISRE promoter activation by Trif activation was nor-

mal in the absence of TRADD, demonstrating a specific effect of

TRADD on RIG-I IFN responses, but not TLR3 and TLR4 IFN

responses.

Similar results were observed with FADD siRNA, in agreement

with previously published results (Takahashi et al., 2006), and in

the presence of RIP1 siRNA (Figure 2A, Figure S5). We next

investigated whether RIG-I-dependent NF-kB responses may

also be affected by TRADD deficiency. The presence of TRADD

Figure 1. TRADD Interacts with Cardif and

Induces NF-kB and IRF3 Promoter Activa-

tion

(A–C) HEK293T cells were transfected with an IFN-

b, ISRE, or NF-kB reporter plasmid, together with

an empty plasmid (Mock) or increasing amounts

of TRADD constructs, and analyzed 24 hr later

for (A) IFN-b-, (B) ISRE-, or (C) NF-kB-dependent

luciferase activity, respectively.

(D and E) HEK293T cells were transfected with an

(D) ISRE or (E) NF-kB reporter plasmid, together

with an empty plasmid, TRADD, Cardif, RIG-I,

and poly(I:C) at 50 ng/ml where indicated. Data

are mean values ± standard deviation (SD) from

one experiment representative of at least three in-

dependent experiments.

(F–H) HEK293T cells were transfected with the

indicated VSV, Flag, or myc constructs, and the

immunoprecipitates (IP) and cell extracts were

analyzed by immunoblot (IB). Ig(H)* denotes immu-

noglobulinheavychain,and ) indicatesnonspecific

band.

(I) HEK293T cells were transfected with an empty

plasmid (�) or TBK1 + poly(I:C) 50 ng/ml (TBK1

overexpression is needed to induce RLH expres-

sion), and the immunoprecipitates and cell extracts

were analyzed by immunoblot. The anti-TRADD

used for immunoprecipitation is a mouse IgG1

antibody, and an anti-mouse IgG1 was used as

a control. The anti-TRADD used for immunoblot

analysis of endogenous TRADD in (H) and (I) is

a rabbit polyclonal IgG antibody. Data are repre-

sentative of at least two independent experiments.

siRNA but not control siRNA resulted in

a clear reduction of RIG-I-induced

NF-kB activation (Figure 2B, Figure S5).

A consistent reduction of Cardif- or RIG-

I-induced NF-kB responses was also

observed in HEK293T cells stably ex-

pressing a TRADD-specific short-hairpin

RNA (shRNA) construct (Figure 2C).

Interestingly, RIP1 siRNA had the same

inhibitory effect as TRADD siRNA, dem-

onstrating that RIP1, which is a binding

partner of TRADD in the TNF signaling

complex, is also essential for RIG-I-de-

pendent NF-kB responses (Figure 2B).

siRNA knockdown of both TRADD and

RIP1 produced an additive effect, reduc-

ing RIG-I-induced NF-kB activation in

a manner comparable to that observed after FADD deficiency

(Kawai et al., 2005; Takahashi et al., 2006) (Figure 2B). Finally,

to corroborate the role of TRADD in antiviral signaling, we deter-

mined Cardif-induced IFN responses in TRADD-deficient mouse

embryonic fibroblasts (MEFs). As shown in Figures 2D and 2E,

Cardif-induced ISRE and IFN-b responses were strongly de-

creased in MEFs from Tradd�/� mice. Moreover, IFN-b produc-

tion as well as phosphorylation of the transcription factor IRF3

induced by Cardif overexpression were totally impaired in

TRADD-deficient MEFs (Figure 2F). All of these results show that


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Figure 2. RIG-I- and Cardif-Induced NF-kB

and IFN Responses Are TRADD Dependent

(A and B) HEK293T cells were transfected with 20

nM of indicated siRNAs or a control (scramble) and

24 hr later were retransfected with an (A) ISRE or

(B) NF-kB reporter plasmid, together with an

empty plasmid (Mock), Trif, or RIG-I [in combina-

tion with 50 ng/ml poly(I:C)]. Luciferase activity

was measured 24 hr after the second transfection.

Effectiveness of siRNAs is shown in Figure S5.

(C) HEK293T cells stably expressing shRNA con-

struct directed against Tradd (same construct

used in the experiment for Figure 5B) or the gene

that encodes lamin (control) were transfected

with an NF-kB reporter plasmid, together with Car-

dif, RIG-I [in combination with 50 ng/ml poly(I:C)],

or an empty plasmid (Mock) and analyzed for

NF-kB-dependent luciferase activity. All data are

mean values ± SD from one experiment represen-

tative of at least three independent experiments.

(D and F) Wild-type (WT) or Tradd�/� primary

MEFs were transfected with an empty plasmid

(Mock) or mouse Cardif (mCardif) with the Amaxa

Nucleofection technology. An ISRE reporter plas-

mid was added in (D), and luciferase activity was

measured 24 hr after electroporation. (F) Superna-

tants and cell extracts were collected 24 hr after

electroporation to measure IFN-b production by

ELISA and phosphorylation of IRF3 by immuno-

blot, respectively.

(E) Wild-type (WT) or Tradd�/� transformed MEFs

were transfected with an IFN-b reporter plasmid,

together with an empty plasmid (Mock) or mCardif,

through the use of lipofectamine 2000. Luciferase

activity was measured 24 hr after transfection. LF

denotes lipofectamine alone. Data are mean

values ± SD from one experiment representative

of at least two independent experiments.

TRADD plays a critical role in RIG-I-induced innate immune

signaling that leads to IRF3 and NF-kB activation.

TRADD Interacts with TRAF3 and TANK, Leadingto IRF3 ActivationBecause the interaction between TRADD and Cardif has a func-

tional impact on signaling pathways implicated in antiviral innate

responses, we reasoned that TRADD might influence the forma-

tion of a complex containing other components present in the

Cardif-signaling pathway. Because Cardif is recruited by RIG-I

and MDA5, we first looked at whether TRADD interacted with

these two helicases, which was not the case (Figure S6). Cardif

was suggested to interact with several TRAF molecules (Saha

et al., 2006; Seth et al., 2005; Xu et al., 2005); hence, we per-

formed immunoprecipitation experiments between TRADD and

the seven TRAF family members. As expected from previous

reports, the specific binding of TRADD to TRAF1 and TRAF2

(Figure 3A) was observed, a complex operating in TNFRI-signal-

ing cascades (Hsu et al., 1996b). An interaction between TRADD

and TRAF3 was also seen (Figure 3A). This observation was

interesting because TRAF3 is known to interact with Cardif and

to be necessary for Cardif-induced type I IFN production (Saha

et al., 2006). We therefore investigated whether TRADD inter-

acted with other effectors that mediate the activation of IRF


transcription factors downstream of Cardif, such as the nonclas-

sical IKKs, TBK1, and IKK3 (Fitzgerald et al., 2003; Sharma et al.,

2003). In contrast to TRAF members, no binding of TRADD to

these kinases was detected (Figure 3A, Figure S7), suggesting

that TRADD acts more upstream of these kinases in the Cardif

signaling pathway. To validate this hypothesis, we investigated

whether TRADD-induced IRF3 responses were affected by the

absence of TRAF3 or Cardif. Overexpression of TRADD in

HEK293T cells led to an increase of ISRE reporter activity, which

was strongly inhibited by TRAF3 siRNA (Figure 3B). In contrast,

Cardif siRNA had little effect (Figure 3B), confirming that TRADD

most likely acts upstream of TRAF3 but downstream of Cardif.

Finally, we determined whether TRADD may be involved in the

formation of a complex containing TRAF3 and TANK (also called

I-TRAF [Cheng and Baltimore, 1996; Rothe et al., 1996]), a protein

shown to interact with TRAF3 and TBK1 (Li et al., 2002; Pomer-

antz and Baltimore, 1999). In immunoprecipitation experiments,

TRADD interacted specifically with TANK (Figure 3C) in a com-

plex that included TRAF3 (Figure 3C). We observed that overex-

pressed TRADD recruited endogenous TANK and TRAF3

molecules (Figure 3D). Taken together, these results show that

RIG-I-mediated IRF responses are dependent on a molecular

complex that contains at least TRADD, TRAF3, and TANK, which

act downstream of Cardif but upstream of TBK1 and IKK3.

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Figure 3. TRADD Interacts with TRAF3 and TANK

(A) HEK293T cells were transfected with myc-TRADD together with the indicated Flag constructs where indicated, and the immunoprecipitates (IP) and cell ex-

tracts were analyzed by immunoblot (IB). ) indicates nonspecific band.

(B) HEK293T cells were transfected with 20 nM of indicated siRNAs or a control (scramble) and 24 hr later were retransfected with an ISRE reporter plasmid,

together with a TRADD construct or an empty plasmid (Mock). Luciferase activity was measured 24 hr after second transfection. Data are mean values ± SD

from one experiment representative of at least three independent experiments.

(C) HEK293T cells were transfected with the indicated VSV or Flag constructs together with myc-TRADD where indicated, and the immunoprecipitates and cell

extracts were analyzed by immunoblot. The anti-TRADD used for immunoblot analysis is the rabbit polyclonal IgG antibody.

(D) HEK293T cells were transfected with myc-TRADD, and the immunoprecipitates and cell extracts were analyzed by immunoblot. The anti-TRADD used for

immunoblot analysis is the mouse IgG1 antibody. ) indicates nonspecific band.

TRADD Forms a Multiprotein Complex with FADDand RIP1 to Activate NF-kBFADD and RIP1 have been linked to dsRNA-induced antiviral

responses (Balachandran et al., 2004) and also to Cardif-mediated

NF-kB activation (Kawai et al., 2005). To determine the role of

TRADD in Cardif-induced NF-kB responses, we performed immu-

noprecipitation experiments involving Cardif, TRADD, and RIP1.

Interestingly, although TRADD and RIP1 coimmunoprecipitated

with Cardif (Figure 4A), RIP1 overexpression enhanced the

TRADD-Cardif interaction, supporting the notion that Cardif,

TRADD, and RIP1 can form a complex (Figure 4A). This result

was confirmed because endogenous TRADD and RIP1 were spe-

cifically recruited toCardif uponRIG-I activationorwhen its expres-

sion was doxycycline induced (Figures 4B and 4C). In experiments

that employed DDD constructs of TRADD and RIP1, the formation

of the complex was unaffected (Figure 4A), indicating that TRADD

and RIP1 interact with Cardif independently of their DDs.

We have previously shown that TRADD and RIP1 associate

with FADD to form the so-called cytoplasmic complex II in the

TNFRI-mediated signaling pathway (Micheau and Tschopp,

2003). We hypothezised that a similar complex may exist in the

Cardif-induced NF-kB pathway. Immunoprecipitation experi-

ments were performed, which demonstrated FADD recruitment

to Cardif only in the presence of RIP1 and TRADD (Figure 4D,

Figure S8). In contrast, no direct interaction between FADD

and any Cardif construct could be detected (Figure S9). These

results suggest that TRADD and RIP1 are adaptor proteins nec-

essary for FADD to be recruited to Cardif. Furthermore, FADD

recruitment was mediated through a DD homotypic interaction

with TRADD and RIP1, because FADD was not precipitated

with DD-deleted TRADD and RIP1 constructs (Figure 4D).

Finally, we examined whether TRADD was involved in c-Jun

N-terminal kinase (JNK) activation, given the evidence that

Cardif mediates JNK phosphorylation (Seth et al., 2005). TRADD


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Figure 4. TRADD Interacts with FADD and RIP1 Downstream of

Cardif

(A) HEK293T cells were transfected with the indicated VSV or myc constructs

together with Flag-Cardif where indicated, and the immunoprecipitates (IP)

and cell extracts were analyzed by immunoblot (IB). Ig(H)* denotes immuno-

globulin heavy chain. ) indicates nonspecific band.

(B) HEK293T cells inducibly expressing Flag-Cardif were treated with doxycy-

cline (200 ng/ml) for 24 hr where indicated, and the immunoprecipitates and

cell extracts were analyzed by immunoblot.

(C) HEK293T cells were transfected with an empty plasmid (�) or RIG-I 2CARD

(a constitutively active form of RIG-I), and the immunoprecipitates and cell ex-

tracts were analyzed by immunoblot.


knockdown with siRNA had no effect on Cardif-induced JNK

phosphorylation, as determined by immunoblotting with a phos-

pho-specific JNK antibody (Figure S10). These results show that

TRADD effects downstream of RIG-I and Cardif are restricted to

IRF and NF-kB responses.

TRADD Knockdown Enhances VSV Replicationand Impairs IFN-b Responses to Sendai VirusThe prior data strongly suggest that TRADD and its binding part-

ners play a crucial role in type I IFN and NF-kB responses medi-

ated by RIG-I and Cardif. Thus, it was expected that the absence

of TRADD would have an effect on a viral infection. The amount

of TRADD protein was modulated via shRNA or cDNA overex-

pression in cells infected with VSV, which is known to be sensed

by RIG-I in most cell types (Kato et al., 2006). TRADD and Cardif

overexpression comparably impaired VSV replication, as mea-

sured by a viral-plaque assay in HeLa cells, but also in Vero cells,

which do not respond to IFN-b (Figure 5A, Figure S11). Consis-

tent with this observation, TRADD knockdown or deficiency

resulted in a 2- to 4-fold increase in virus titer compared to con-

trols (Figures 5B and 5C). Importantly, when TRADD-deficient

mice (M.A.E., M.-C.M., N. Papadopoulou, O. Utermohlen, K.

Kranidioti, G. Kollias, J.T. and M.P., unpublished data) were

infected with VSV, the amounts of IFN-b 24 hr infection were sub-

stantially decreased (Figure 5D). In addition, TRADD-deficient

MEFs infected with another RNA virus, Sendai virus (SeV),

showed a partial reduction of antiviral IFN-b levels (Figure 5E).

IFN-b production was totally abolished in MEFs from Cardif �/�

mice (Figure 5F, Figure S12). Altogether, these in vitro and in

vivo findings indicate that TRADD is an important mediator of

RLH-dependent responses.

DISCUSSION

The DD protein TRADD was originally identified as an adaptor in

the TNFRI-signaling pathway because of its specific interaction

with the DD of this death receptor (Hsu et al., 1995). Overexpres-

sion of TRADD leads to two major TNF-induced responses,

apoptosis and activation of NF-kB (Hsu et al., 1995), which are

dependent on its specific interaction with FADD and TRAF2,

respectively (Hsu et al., 1996b). In the present study, we reveal

an unexpected additional role for TRADD as a central mediator

of RLH-induced antiviral immune responses that lead to the pro-

duction of type I IFNs.

Activation of the NF-kB transcriptional program is a fundamen-

tal early step of the innate antiviral immune response, because it

is one of the signaling pathways required for the synthesis of

antiviral type-I IFNs. As a consequence, it represents a prime

candidate for viral interference (Hiscott et al., 2006). In this con-

text, the hepatitis C virus (HCV) is particularly active. The HCV

serine protease NS3-4A, which is required to cleave the HCV

polyprotein during virus replication, cleaves Cardif, causing

(D) HEK293T cells were transfected with Flag-Cardif and various FADD, RIP1,

and TRADD constructs, and the Flag-immunoprecipitates and cell extracts

were analyzed by immunoblot. The anti-TRADD used for immunoprecipitation

is the mouse IgG1 antibody, and an anti-mouse IgG1 was used as a control.

The anti-TRADD used for immunoblot analysis of endogenous TRADD in (B)

and (C) is the rabbit polyclonal IgG antibody. ) indicates nonspecific band.

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a potent inhibition of NF-kB signaling (Meylan et al., 2005). Apart

from NF-kB, viruses encode proteins that modulate TNF and

other death-receptor signaling pathways. For example, HCV

via its core protein is able to disrupt the binding of TRADD to

TNFRI (Zhu et al., 2001). In addition, the HCV NS5A protein inter-

acts specifically with TRADD, inhibiting its association with

FADD- and TNF-mediated apoptosis and resulting in persistent

infection (Majumder et al., 2002; Miyasaka et al., 2003). This

dual targeting of TRADD by HCV suggested to us that TRADD

might be implicated in an antiviral pathway in addition to its

known role in TNFRI signaling. On the basis of the data pre-

sented here, we propose that TRADD indeed plays an important

role in both NF-kB and IRF3 transcription factor activation by

RIG-I and MDA5. Thus, by targeting TRADD, the production of

type I IFN by the chief cytoplasmic viral sensors RIG-I and

MDA5 is predicted to be considerably impaired. Moreover, by

Figure 5. TRADD Inhibits Vesicular Stomati-

tis Virus Replication and Is Required for

Antiviral Responses and Impaired IFN-

b Production to Sendai Virus in TRADD-

Deficient Cells

(A) HEK293T cells were transfected with different

amounts of TRADD, Cardif, or an empty plasmid

(Mock) and 24 hr later infected with VSV as de-

scribed in the Experimental Procedures. HeLa

cells were used to determine VSV titer. Data are

duplicates from one experiment representative of

three independent experiments. PFU denotes

plaque-forming units.

(B) HEK293T cells stably expressing shRNA con-

structs directed against the Tradd gene (two differ-

ent constructs) or the gene that encodes lamin

(control) were infected with VSV as described in

the Experimental Procedures. HeLa cells were

used to determine VSV titer. Data are duplicates

from one experiment representative of three inde-

pendent experiments.

(C) As (B), but TRADD WT or Tradd�/�MEFs were

infected with VSV at a MOI of 0.1.

(D) Wild-type and Tradd�/� mice were infected

with VSV (2 3 106 PFU per mouse) via tail-vein in-

jection. Sera were collected 24 hr after infection to

measure IFN-b production by ELISA. Data are

mean values ± SD from one representative ex-

periment.

(E and F) Primary TRADD-deficient MEFs (E) and

primary Cardif-deficient MEFs (F) were infected

with SeV as described in the Experimental Proce-

dures. Cell supernatants were recovered 24 hr

after infection to measure IFN-b production by

ELISA. ND denotes not detectable. Data are

mean values ± SD from one experiment represen-

tative of at least two independent experiments.

manipulating TRADD expression or

function, a virus will be able to modulate

TNF-induced proinflammatory cytokine

production, TNF-dependent apoptosis,

and host antiviral immunity.

Our data also show that FADD and

RIP1, in addition to TRADD, play a crucial

role in RIG-I-induced NF-kB activation.

This is in line with previous results where FADD- and RIP1-defi-

cient cells were shown to be very susceptible to viral infection

(Balachandran et al., 2004). Moreover, Kawai et al. (2005) dem-

onstrated that overexpression of FADD and RIP1 stimulates

NF-kB promoter activity, and that FADD and RIP1 interact with

Cardif. Our data confirm the Cardif-RIP1 interaction but clearly

reveal that FADD recruitment to Cardif is mediated by TRADD

and RIP1. We failed to observe a direct interaction between

FADD and Cardif. It has been suggested that FADD and RIP1

may function preferentially on the NF-kB axis, because their

overexpression did not activate IFN-b promoter, and because

overexpression of a mutant FADD-death-effector domain had

no effect on IRF-dependent promoters (Kawai et al., 2005). How-

ever, our data show that FADD and RIP1 are likely to be impli-

cated in IRF3 activation too, because FADD and RIP1 siRNAs

strongly inhibited RIG-I-induced ISRE promoter activity.


Immunity


It appears that signaling by proinflammatory TNFRI and antivi-

ral RIG-I is very similar. We propose that TRADD, RIP1, and

FADD form a central molecular signaling unit that stands at the

apex of both signaling pathways (Figure 6). We previously

showed this trimolecular complex to function in the TNFRI-medi-

ated signaling pathway and called it complex II (Micheau and

Tschopp, 2003). In the context of TNFRI, complex II (we propose

to call it TRADDosome) is derived from complex I, which con-

tains TRADD and RIP1 and initially forms around the DD present

in the cytoplasmic portion of TNFRI upon TNF addition. This is

followed by the dissociation of TRADD-RIP1, which permits

the association of FADD to the liberated DD of TRADD and

RIP1, thus forming the TRADDosome. Within the TRADDosome,

RIP1-bound TRAF2 can act as an E3 ligase to ubiquitinate RIP1

at Lys63. This modification induces NEMO recruitment, which

acts as a scaffold for IKKa and IKKb, leading to NF-kB activation.

The TNF-derived TRADDosome also contains TRAF2-associ-

ated TANK that recruits TBK1, thereby potentiating NF-kB acti-

vation (Pomerantz and Baltimore, 1999). Finally, the third

member of the TRADDosome, FADD, engages caspase-8 and

its inhibitor FLIP and thus regulates NF-kB activity. In the

absence of FLIP, caspase-8 becomes proapoptotic and can

kill the cell.

An almost identical signaling pathway appears to be triggered

upon viral infection. Instead of the cytoplasmic portion of TNFRI,

the TRADDosome assembles on the intermediary region of

Cardif at the mitochondrial outer membrane, when the RNA

helicases RIG-I and MDA5 detect viral nucleic acids and conse-

quently interact with Cardif. The Cardif-bound TRADDosome in-

duces the activation of IKKa and IKKb upon RIP1 ubiquitination

and NEMO recruitment, resulting in the phosphorylation and

degradation of I-kB. Cardif also directly recruits TRAF3, which

is required for RIP1 ubiquitination, and TANK, which is interact-

ing with TBK1. Recruited NEMO also serves the activation of

IKK3 and TBK1, which in turn cause the phosphorylation, dimer-

ization, and nuclear translocation of IRF3. As with TNFRI, the

Cardif-bound TRADDosome recruits via FADD caspase-8 (and/

or -10), which is obligatory for NF-kB and IRF responses.

Although the proposed model is compatible with most of the

published data, there are still several open questions. For exam-

ple, if the TRADDosome is utilized by the RLH and the TNFRI

Figure 6. A Schematic Overview of the Sig-

naling Pathways Triggered by RLH-Cardif

and TNFRI

Viral RNA binds to RIG-I or MDA5, which transmit

the downstream signals via Cardif to activate IRF3

and NF-kB. A central signaling unit is the TRADDo-

some, which consists of TRADD-RIP1 and FADD

and is also crucial for TNFRI-mediated proinflam-

matory responses. See text for details.

pathway, NEMO recruitment, and IKKs,

why does potent IRF3 activation occur

only in the virally activated pathway?

There are several possible explanations.

The TNFRI and RLH pathways are clearly

distinct with respect to the site of assem-

bly. Although the TNFRI complex is

formed at the cell membrane, Cardif needs to be anchored in

the outer mitochondrial membrane to be active (Li et al., 2005;

Seth et al., 2005), suggesting that proteins required for IRF acti-

vation, but not NF-kB activation, are present on the mitochon-

drial membrane. In support of this hypothesis, we observed

that binding of Cardif to TRAF3, which is essential for IRF3

activation but not NF-kB activation, is dependent on Cardif’s

mitochondrial localization (data not shown).

Another open question remains as to the role of caspase-8 or

-10 in the NF-kB signaling pathway. In the NF-kB signaling path-

way emanating from the T cell receptor, caspase-8 was shown to

be required for the processing of FLIP, which, in its processed

form, recruits TRAF2 (Budd et al., 2006). A TRADDosome-

TRAF modulating role of processed FLIP may therefore also be

envisaged.

Viral infection remains a considerable health threat. Although

antiviral drugs inhibiting replication of viruses have been devel-

oped, many viruses such as the human immunodeficiency virus

(HIV) mutate and produce drug-resistant strains. In this context,

a better knowledge of antiviral signaling pathways exerted by

host immune systems may lead to the development of new strat-

egies aimed at combating viral infection. The identification of

TRADD as a crucial player in the host antiviral response may

contribute to the development of new therapeutic strategies for

enhancing the immune response against viral diseases.

EXPERIMENTAL PROCEDURES

Expression Vectors

pCR3-RIP1-Flag, pCR3-Trif-Flag, Flag-tagged constructs of Cardif, and lucif-

erase reporter constructs were previously described (Meylan et al., 2004; Mey-

lan et al., 2005). shRNA constructs directed against the tradd gene in pLKO.1

vectors were obtained from Openbiosystems. Ha-IRF3 and pcDNA3-IRF7

were kindly provided by K. Fitzgerald and G.P. Dotto, respectively.

Flag-RIG-I, Flag-TBK1, Flag-IKK3, Flag-TRAF7, Flag-JNK, ha-TRAF3, and

VSV-FADD were gifts from T. Fujita, M. Nakanishi, T. Maniatis, H.B. Shu, C.

Widmann, S. Rothenberger, and K. Burns, respectively. Flag-TRAF1–6 were

previously described (Thome et al., 1999). pCR3-TRADD-myc was obtained

by restriction of the pRK5-TRADD (provided by D.V. Goeddel, Tularik) and sub-

sequently cloned into a derivative of pCR3 (Invitrogen), in frame with an N-ter-

minal myc tag. Human TANK, human MDA5, and mouse Cardif were amplified

from EST clones by standard PCR with Pfx polymerase (Invitrogen) and were

subsequently cloned into a derivative of pCR3, in frame with an N-terminal VSV


Immunity


or Flag tag. The RIP1 delta death domain (DDD) (amino acids 1–568) and the

TRADDDDD (amino acids 1–168) constructs were obtained by standard

PCR of the pCR3-RIP1-VSV and appropriate restriction of the pCR3-

TRADD-myc, respectively. The RIG-I 2CARD (amino acids 1–200), a constitu-

tively active form of RIG-I, was amplified from pCR3-RIG-I-VSV by standard

PCR. RIP1DDD, TRADDDDD, and RIG-I 2CARD were subsequently cloned

into a derivative of pCR3 (Invitrogen) in frame with an N-terminal VSV tag.

The fidelity of all PCR amplifications was confirmed by sequencing.

Mice, Cells, and Culture Conditions

The generation of Cardif �/� mice is described in the Supplemental Data

(Figure S12). Tradd�/� mice were generated and provided by M. Paspara-

kis. Mouse embryonic fibroblasts (MEFs) from wild-type and mutant mice

were prepared from day 13.5 embryos and cultured in Dulbecco’s modified

Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% heat-inacti-

vated FCS and 100 mM b-mercaptoethanol. The HEK 293T, HeLa cells,

Vero cells, fibrosarcoma HT1080 cells, HEK293T cells inducibly expressing

Flag-Cardif upon doxycycline treatment (200 ng/ml), and HEK293T cells

stably expressing shRNA constructs directed against the tradd gene were

grown in DMEM supplemented with 10% heat-inactivated FCS. The

Flag-tagged TNF-a used for TNFRI complex analysis was obtained from

Apotech.

Transfections

Transient transfection of HEK293T cells was performed as previously de-

scribed (Thome et al., 1998). siRNAs and poly(I:C) (Invivogen) were also trans-

fected into HEK293T cells via the calcium-phosphate precipitation method, at

final concentrations of 20 nM and 50 ng/ml, respectively. siRNA oligonucleo-

tides against RIP1 (50-GGCGAAGAUGAUGAACAGA-30), TRADD (50-GGAGGA

UGCGCUGCGAAAUUU-30) (Zheng et al., 2006), FADD (50-GAAGACCUGUGU

GCAGCAUUU-30 ), TRAF3 (50-GGAGGUUACAAGGAAAAGUTT-30) and Cardif

(50-CCCACAGGGUCAGUUGUAUTT-30) were obtained from Ambion. The

synthetic DNA poly(dA-dT) (Sigma) was used at a concentration of 1.5 mg/ml

after complex formation with lipofectamine 2000 (Invitrogen) at a ratio of 1.0

ml lipofectamine to 1.0 mg DNA in Optimem (Invitrogen). Chemically synthe-

sized 50-triphosphate dsRNA (3pRNA) was generated and kindly provided by

H. Poeck. Transient transfection of primary and immortalized MEFs was per-

formed by electroporation with the Amaxa Nucleofection technology (Amaxa

Biosystems) and lipofectamine 2000 (Invitrogen), respectively, according to

the manufacturers’ instructions.

Immunoprecipitation and Immunoblot

For immunoblot analysis, transfected HEK293T cells were resuspended in

lysis buffer (50 mM Tris [pH 7.8], 150 mM NaCl, 0.1% Nonidet P-40, 5 mM

EDTA, and complete protease inhibitor cocktail [Roche]) for 15 min on ice,

and lysis was completed by three cycles of freeze and thaw. For coimmuno-

precipitation experiments, lysates were incubated overnight at 4�C on a rotat-

ing wheel with anti-Flag (M2) beads (Sigma) or anti-VSV (P5D4, Sigma) bound

to protein G. Beads were recovered and washed five times with lysis buffer be-

fore analysis by SDS-PAGE and immunoblotting. TNFRI complex analysis was

performed as previously described (Micheau and Tschopp, 2003). In brief,

HT1080 cells were stimulated for the indicated times with 1 mg/ml of Flag-

tagged TNF-a (Apotech) and lysed in lysis buffer (20 mM Tris-HCl [pH 7.4],

250 mM NaCl, 1% Nonidet P40, 10% glycerol, and complete protease inhibitor

cocktail) for 15 min on ice. Lysates were immunoprecipitated overnight with

M2-Flag beads (Sigma) at 4�C on a rotating wheel. Beads were recovered

and washed five times with lysis buffer before analysis by SDS-PAGE and im-

munoblotting. Antibodies used for immunoblot analysis were anti-RIP1 (clone

38, Transduction Labs), anti-TRADD (mouse IgG1, catalog number T50320,

Transduction Labs; rabbit polyclonal IgG, clone H-278, Santa Cruz), anti-

TRAF3 (clone H-122, Santa-Cruz), anti-TANK (rabbit polyclonal, catalog num-

ber sc-8314, Santa Cruz), anti-FADD (mouse IgG1, catalog number 610400,

BD PharMingen), anti-Cardif (mouse IgG2b, clone Adri-1, Alexis), anti-IkB

(clone C-21, Santa Cruz), anti-phospho-IkB (mouse IgG1, Cell Signaling),

anti-TNFRI (IgG2b, clone H5, Santa Cruz), anti-phospho-JNK (catalog number

9251, Cell Signaling), anti-phospho-IRF3 (4D4G rabbit, Cell Signaling),

anti-myc (Upstate), anti-Flag, and anti-VSV (Sigma).

Luciferase Reporter Assays

These assays were performed as previously described (Meylan et al., 2002).

Plaque Assay

Virus yield was measured from culture supernatants collected from cells stably

expressing cDNA or shRNA constructs infected for 6 hr with VSV at a multiplic-

ity of infection of 1 or 2, respectively. HeLa or Vero cells were infected in dupli-

cates with serial dilutions of recovered viruses for 1 hr and were overlaid with

DMEM containing 0.3% agarose 1% FCS low-melting agarose. Viral plaques

were counted after 24 hr of incubation.

Viral Infections

Cells (MEFs) were seeded onto 12-well microtiter plates. When 50%–60%

confluent, they were infected with the equivalent of 10 ml of a Sendai virus

(SeV) DIH4 stock prepared in the allantoic cavity of embryonic eggs. Infections

were performed in serum-free medium (1 ml per well) for 1 hr at 37�C. The in-

fecting medium was then removed and replaced with 1 ml of normal growth

medium. Cell supernatants were recovered 24 hr after infection and stored

at �70�C until the analysis. Wild-type and Tradd�/� mice were infected with

VSV (2 3 106 plaque-forming unit [PFU] per mouse) via tail-vein injection.

Sera were collected 24 hr after infection to measure IFN-b production by

ELISA. The ELISA kit for mouse IFN-b was purchased from PBL Biomedical

Laboratories.

SUPPLEMENTAL DATA

Twelve figures are available at http://www.immunity.com/cgi/content/full/

28/5/651/DC1/.

ACKNOWLEDGMENTS

We thank S. Lippens and D.A. Muruve for critical readings and discussions.

J.T. is supported by grants of the Swiss National Science Foundation. M.-

C.M. is a recipient of a fellowship from European Molecular Biology Organiza-

tion (EMBO). H.P. and M.B. are supported by Gradiertenkolleg 1202 of the

Deutsche Forschungsgemeinschaft.

Received: July 29, 2007

Revised: January 15, 2008

Accepted: March 4, 2008

Published online: April 24, 2008

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