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The Journal of Immunology, 2007, 178: 2429-2439.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fas-Associated Death Domain-Containing Protein-Mediated Antiviral Innate Immune Signaling Involves the Regulation of Irf71

Siddharth Balachandran2,*, Thiagarajan Venkataraman2,*, Paul B. Fisher{dagger} and Glen N. Barber3,*

* Department of Microbiology and Immunology and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL 33136; and {dagger} Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The induction of type I ({alpha}beta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-{alpha}4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-{alpha} subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-{alpha}s and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-{alpha} production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd–/– murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD’s antiviral activity involves regulation of IRF-7-dependent production of IFN-{alpha} subtypes and consequent induction of secondary antiviral genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The recognition of viral infection and subsequent triggering of antiviral innate immune responses has become the subject of intense research over the past few years. Particular attention has recently focused on the role of the TLRs, which have emerged as key transmembrane proteins responsible for recognizing conserved components of pathogenic microorganisms (referred to as pathogen-associated molecular patterns) (1, 2, 3). In mammalian cells, there appear to be at least 10 TLR family members, each of which respond to different pathogen-associated molecular patterns such as extracellular viral dsRNA (TLR3), ssRNA (TLR7), and unmethylated CpG DNA (TLR9) (1). Stimulation of these TLRs by their cognate ligands culminates in the transcriptional activation of numerous genes, such as the type I IFNs, that directly induce antiviral responses as well as facilitate innate and adaptive immune responses (1, 4, 5, 6, 7).

Although TLRs 3, 7, and 8 mediate recognition of extracellular and/or endosomal viral RNA species, distinct pathways for the recognition of cytosolic dsRNA also exist. Such mechanisms include those initiated by 2',5'-oligoadenylate synthase and the dsRNA-activated protein kinase R (PKR),4 which, following interaction with viral dsRNA, predominantly function to inhibit protein synthesis (8, 9). We have also recently demonstrated the existence of a TLR-independent cytosolic dsRNA recognition pathway involving Fas-associated death domain-containing protein (FADD) that may be related to the immune deficiency (IMD) innate immune pathway in Drosophila (10, 11). In addition, other molecules critical for the detection of virally produced cytosolic dsRNA include retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene (MDA)-5 (5, 12, 13, 14). RIG-I is a DExD/H box RNA helicase with an amino terminal caspase recruitment domain (CARD) and significant homology to another RNA helicase MDA-5 (15, 16). Although RIG-I appears to activate type I IFN through tank binding kinase (TBK)1/I{kappa}B kinase (IKK){epsilon}, little is known regarding the intermediate steps in this pathway. Recently, a CARD containing mitochondrial molecule called MAVS/VISA/Cardif/IPS-1 (hereafter referred to as IFN-beta promoter stimulator (IPS)-1) with homology to the CARDs of MDA-5 and RIG-I has been shown to transduce signals generated following activation of these helicases (17, 18, 19, 20). Although the mechanisms by which IPS-1 induces type I IFN remain to be clarified, data indicate that this molecule may activate IFN-beta via IFN regulatory factor (IRF)-3, NF-{kappa}B, and TNFR-associated factor (TRAF)3 (17, 18, 19, 20, 21).

In this study, we confirm that FADD is required for optimal antiviral signaling and RIG-I, MDA-5, and IPS-1 activity (10, 17, 22, 23). Importantly, our results implicate FADD in the production of secondary IFN-{alpha} genes following dsRNA stimulation or virus infection. In an effort to identify a mechanistic basis for this observation, we noticed that Fadd–/– murine embryonic fibroblasts (MEFs) are markedly impaired in the induction of Irf7 by virus infection or dsRNA stimulation. Indeed, reconstitution of Fadd–/– MEFs with IRF-7 was able to significantly rescue the IFN-{alpha} induction defect in these cells, and a constitutively active version of IRF-7 (but not RIG-I, MDA-5, or IPS-1) could activate a dsRNA-responsive promoter in Fadd–/– MEFs. Finally, we show that MC159, a FADD-interacting viral protein encoded by the poxvirus molluscum contagiosum, can efficiently block RIG-I, MDA-5, and IPS-1-mediated activation of a dsRNA-responsive promoter upstream of IRF-7. Thus, FADD is required for robust RIG-I, MDA-5, and IPS-1-mediated dsRNA-signaling, an event involving the control of Irf7.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

MEFs were obtained from the following sources: Fadd+/– and Fadd–/–, Tbk1+/+ and Tbk1–/–, Traf2+/+, and Traf2–/–, Traf6+/+ and Traf6–/– (provided by W.-C. Yeh, University of Toronto, Toronto, Canada); Trif+/+ and Trif–/– (provided by S. Akira, Osaka University, Osaka, Japan); and Stat1+/+ and Stat1–/– MEFs (provided by J. Durbin, Columbus Children’s Research Institute, Columbus, OH). All other cell lines were obtained from the American Type Culture Collection. Poly(I:C) (Amersham) was reconstituted in PBS at 2 mg/ml, denatured at 55°C for 30 min, and allowed to anneal to room temperature before use. MEFs were transfected with 6 µg of poly(I:C) in 8 µl of Lipofectamine 2000 per milliliter of medium to stimulate promoter activation and IFN production. Murine IFN-{alpha} and -beta ELISA Kits were acquired from PBL. NF-{kappa}B p65 EZ-Detect transcription factor ELISA Kits were purchased from Pierce. Anti-hemagglutinin (HA), anti-p50, and anti-p65 Abs were purchased from Santa Cruz Biotechnology. All other reagents were from Sigma-Aldrich, unless mentioned otherwise.

Plasmids

Expression vectors (pcDNA3-Neo; Invitrogen Life Technologies) encoding FLAG-tagged versions of human RIG-I, MDA-5, and LGP2, FADD, IPS-1, TBK-1, Toll/IL-1R domain-containing adaptor-inducing IFN-beta (TRIF), and their various mutants were generated by PCR. Other plasmids were obtained as follows: IFN-beta-Luc (provided by J. Hiscott, McGill University, Montreal, Canada), PRD-III-I-Luc (provided by T. Maniatis, Harvard University, Cambridge, MA), RL-1-Luc (provided by P. Pitha-Rowe, Johns Hopkins University, Baltimore, MD), NF-{kappa}B-Luc (Stratagene), expression vectors for MC159 (provided by J. Shisler, University of Illinois, Urbana, IL), TLR3, IL-1R-associated kinase (IRAK)1, TRAF6, IRF-7, and IRF-7 (superactive; SA) (InvivoGen).

DNA microarray analysis

Total RNA was extracted from MEFs stimulated with or without poly(I:C) (6 µg/ml in Lipofectamine 2000) or murine IFN-beta (200 U/ml) at the indicated time points. Preparation of cDNA and microarray analysis was performed at the W.M. Keck Foundation Biotechnology Research Laboratory DNA microarray facility at Yale University (New Haven, CT). The Mouse Genome 430 2.0 Array (Affymetrix) was used. Data analysis was performed with Microarray Suite software (version 5.0; Affymetrix) and GeneSpring software (Silicon Genetics).

Real-time PCR

Total RNA was isolated from cells using the RNeasy RNA extraction kit (Qiagen), and cDNA synthesis was performed using 1 µg of total RNA (Roche). Fluorescence real-time PCR analysis was performed using a LightCycler 2.0 instrument (Roche Molecular Biochemicals) and TaqMan Gene Expression Assays (Applied Biosystems). Relative amounts of mRNA were normalized to the 18S ribosomal RNA levels in each sample.

EMSA

Cells (5 x 106) washed two times with cold PBS were suspended in 400 µl of hypotonic buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.5 mM PMSF, and protease inhibitor mixture). After 15 min of incubation on ice, 25 µl of 10% Nonidet P-40 was added, and the sample was vigorously vortexed. The nuclei were collected by centrifugation at 3000 x g for 5 min at 4°C and washed once with buffer A, and the nuclear pellet was suspended in 75 µl of buffer B (20 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 5% glycerol, and protease inhibitor mixture) and incubated for 30 min at 4°C with brief mixing. The mixture was centrifuged at 10,000 x g for 5 min at 4°C. Protein concentrations were measured using the Bradford assay (Bio-Rad).

NF-{kappa}B consensus oligonucleotides (Santa Cruz Biotechnology) were end-labeled with ATP using T4 polynucleotide kinase. Before the addition of oligonucleotide probe, 10 µg of nuclear protein was incubated with binding buffer (2 µg poly(dI-dC), 12% glycerol, 20 mM HEPES (pH 7.0), 1 mM DTT, 1 mM EDTA, and 50 mM NaCl) for 10 min at room temperature. Radiolabeled oligonucleotide was incubated with reaction mixture for 15 min at room temperature and subjected to 5% nondenaturing PAGE in 0.5x Tris-borate-EDTA buffer. The gels were dried and analyzed by autoradiography. Supershift was performed by adding Abs to the incubation mixture of nuclear extract and incubating for 15 min on ice before the addition of radiolabeled probe.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Requirement for FADD in RIG-I, MDA-5, and IPS-1-mediated induction of IFN-beta

We and others have recently demonstrated the existence of a TLR-independent cytosolic dsRNA recognition pathway involving FADD (10, 17, 22, 23). It has been also been shown that the RNA helicases RIG-I and MDA-5 associate with intracellular dsRNA to activate IFN-beta (16, 23), whereas a CARD-less RIG-I/MDA-5 homologue, LGP2, may act as a dominant-negative inhibitor of RIG-I and MDA-5 signaling (23, 24). Recent data further show a role for IPS-1 in TBK1/IKK{epsilon}-mediated activation of IFN-beta by RIG-I and MDA-5 (25, 26). Given that FADD also governs dsRNA-triggered activation of IFN-beta, we investigated the role of FADD in RIG-I/MDA-5/IPS-1-mediated dsRNA signaling. To this end, Fadd+/– or Fadd–/– MEFs were transfected in the presence or absence of dsRNA with expression vectors encoding RIG-I, MDA-5, and their respective CARD-containing modules, LGP2, IPS-1, and TRIF. As expected, we found that overexpressed RIG-I and MDA-5 robustly induced the IFN-beta promoter in the presence of dsRNA (>100-fold) in Fadd+/– MEFs (Fig. 1). The CARDs of RIG-I and MDA-5 alone was found to induce the IFN-beta promoter >100- to 300-fold compared with controls, even in the absence of dsRNA. In contrast, overexpression of LGP2 inhibited activation of the IFN-beta promoter in response to dsRNA (Fig. 1). When RIG-I or MDA-5 or their corresponding CARDs alone were transfected into Fadd–/– MEFs, there was a significant reduction in activation of the IFN-beta promoter following dsRNA stimulation (Fig. 1). Importantly, these studies also demonstrated a clear role for FADD in optimal IPS-1 signaling. Whereas IPS-1 overexpression induced the IFN-beta promoter >200-fold in Fadd+/– MEFs, it caused only a ~10-fold activation of this promoter in Fadd–/– MEFs (Fig. 1A). TRIF signaling was unimpaired in FADD-deficient cells, indicating that, at least in MEFs, FADD is predominantly involved in TLR-independent signaling, similar to its role in Drosophila innate immune responses (27, 28) (Fig. 1).


Figure 1
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  		FIGURE 1. FADD is required for optimal RIG-I, MDA-5, and IPS-1 signaling. The indicated expression vectors, together with the IFN-beta-Luc firefly luciferase reporter plasmid were transfected into Fadd+/– and Fadd–/– MEFs. Twenty-four hours later, these cells were either left untreated, or were transfected with poly(I:C) for a further 6 h, and IFN-beta promoter-driven firefly luciferase activity was measured at that time. Values are normalized to an internal Renilla luciferase control. Error bars, ±SD.

 
Defective induction of primary antiviral genes in the absence of FADD

To confirm a role for FADD in dsRNA- and virus-triggered production of type I IFN, Fadd+/– and Fadd–/– MEFs were transfected with poly(I:C), or infected with vesicular stomatitis virus (VSV) or Sendai virus (SeV) at multiplicities of infection (m.o.i.) of 1 or 10, and examined for type I IFN protein production at 6, 12, or 24 h posttreatment. As before, and in agreement with previous results (10, 22, 23), we found that Fadd–/– MEFs exhibited a significant defect in the production of IFN-beta following poly(I:C) transfection or VSV infection (Fig. 2A, left and middle panels). Indeed, Fadd–/– MEFs produced only 30–50% the amount of IFN-beta of similarly treated Fadd+/– MEFs. When these cells were infected with SeV, however, Fadd–/– MEFs produced approximately normal levels of IFN-beta, albeit with delayed kinetics, compared with controls (Fig. 2A, right panel). To determine whether ectopic overexpression of RIG-I and MDA-5 was able to overcome the IFN-beta production defects seen following transfection with poly(I:C), we transfected Fadd+/+ and Fadd–/– MEFs with expression vectors encoding full-length RIG-I and MDA-5, and stimulated such cells with poly(I:C) for 24 h before examining supernatants for IFN-beta by ELISA. Overexpressed RIG-I or MDA-5 were unable to stimulate effective IFN-beta production in the absence of FADD, confirming a pivotal role for FADD downstream of these helicases in the induction of IFN-beta (Fig. 2B). To determine whether defective induction of IFN-beta extended to other primary antiviral genes, we compared the gene induction profile of Stat1-independent (i.e., "primary") genes in Fadd–/– MEFs to their heterozygous counterparts. For the purpose of this analysis, all genes that were induced by dsRNA treatment at least 2-fold in Stat1+/+ MEFs and were also induced at least 2-fold or more in the Stat1–/– MEFs were classified as primary dsRNA-responsive antiviral genes. After ensuring that these subsets were also induced at least 2-fold in the Tbk1+/+ and Fadd+/– MEFs, their behavior was examined in Tbk1–/– and Fadd–/– MEFs. This analysis revealed that, whereas the majority of primary genes were induced in Fadd–/– MEFs to the same or greater extent than they were in Fadd+/– MEFs, the induction of a subset of primary genes (such as Ifit1, Ifit2, and Icam1) was found to be significantly impaired in the absence of FADD (Fig. 2C). Thus, FADD appears to be required for the optimal induction of some, but not all, STAT1-independent genes.


Figure 2
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  		FIGURE 2. FADD participates in the regulation of a subset of primary dsRNA-responsive genes. A, ELISA was performed to detect levels of IFN-beta in the supernatants of Fadd+/– and Fadd–/– MEFs treated with poly(I:C), VSV, or SeV at the m.o.i. and times indicated. Error bars, ±SD. *, Not detectable. B, Expression vectors encoding RIG-I or MDA-5 were transfected into Fadd+/– and Fadd–/– MEFs. Twenty-four hours posttransfection, these cells were treated with poly(I:C) for a further 24 h, and levels of IFN-beta were measured in the supernatants at that time. Error bars, ±SD. C, Fadd+/– and Fadd–/– MEFs in tandem with Tbk1+/+, Tbk1–/–, Stat1+/+, and Stat1–/– MEFs, were either left untreated or were transfected with poly(I:C) for 3 h, and DNA microarray analysis of dsRNA-induced primary gene expression was performed as described in Materials and Methods and Results.

 
Global defects in secondary dsRNA-responsive gene induction in the absence of FADD

To determine whether defective IFN-beta secretion in the absence of FADD resulted in compromised induction of secondary (IFN-beta-dependent) gene induction, including that of the other type I IFNs (IFN-{alpha}s), we next examined the production of IFN-{alpha} in Fadd+/– and Fadd–/– MEFs following dsRNA stimulation or virus infection. Accordingly, Fadd+/– and Fadd–/– MEFs were either transfected with poly(I:C) or infected with VSV or SeV at different m.o.i. (1 and 10), and supernatants from these cells were examined for secreted IFN-{alpha} production. As we had reported previously, Fadd–/– MEFs produced profoundly reduced levels of IFN-{alpha} compared with Fadd+/– MEFs following dsRNA treatment (Fig. 3A, left panel) (10). More importantly, we noticed that virus infection of Fadd–/– MEFs also resulted in significantly reduced IFN-{alpha} production. In fact, IFN-{alpha} was undetectable in the supernatant of VSV-infected Fadd–/– MEFs at all m.o.i.s and time points tested (Fig. 3A, middle panel). Notably, and unlike the case with IFN-beta, IFN-{alpha} production was also significantly defective (reduced by ~80%, compared with controls) in Fadd–/– MEFs after SeV infection (Fig. 3A, right panel). Along with our previous observation that IFN-{alpha} production is important for protection against virus infection (10), these results provide an explanation for the remarkable susceptibility of early passage Fadd–/– MEFs to viral infection.


Figure 3
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  		FIGURE 3. Global defects in secondary gene induction by dsRNA in the absence of FADD. A, ELISA was performed to detect levels of IFN-{alpha} in the supernatants of Fadd+/– and Fadd–/– MEFs treated with poly(I:C), VSV, or SeV at the m.o.i. and times indicated. Error bars, ±SD. *, Not detectable. B, Fadd+/– and Fadd–/– MEFs together with Tbk1+/+, Tbk1–/–, Stat1+/+, and Stat1–/– MEFs, were either left untreated or were transfected with poly(I:C) for 12 h, and DNA microarray analysis of dsRNA-induced secondary gene expression was performed as described in Materials and Methods and Results.

 
Given that type I IFN induction is severely impaired in the absence of FADD, we next directly addressed the expression profile of secondary (type I IFN/STAT1-dependent) antiviral genes in Fadd+/– and Fadd–/– MEFs. For this experiment, all genes that were induced at least 2-fold in Stat1+/+ MEFs, but were not induced (i.e., 1-fold or lower) in Stat1–/– MEFs, were classified as secondary dsRNA-responsive genes (Fig. 2B). The induction profiles of such genes were then examined in Tbk1–/– and Fadd–/– MEFs, after ensuring that they were induced at least 2-fold in Tbk1+/+ and Fadd+/– MEFs. This study revealed a significant, global impairment of the induction of a spectrum of secondary responsive genes in the absence of FADD (Fig. 2A). For example, genes such as Casp7, Tlr3, eif2ak2 (PKR), Rnasel, Ddx58 (RIG-I), and Ube11 were poorly activated in Fadd–/– MEFs (Fig. 3B). This analysis also confirmed a severe impairment in the induction of the genes encoding secondary IFNs (such as Ifnab, Ifna2, Ifna5, Ifna6, Ifna7, and Ifna9) 12 h after dsRNA treatment of Fadd–/– MEFs. Interestingly, whereas inducibility of the majority of secondary dsRNA-responsive genes was compromised in Fadd–/– MEFs, a subset of such genes (including Myd88 and B2m) were induced normally in these cells (Fig. 3B). These results indicate that, whereas FADD appears essential for robust secondary gene induction following dsRNA treatment, FADD-independent mechanisms for the induction of these also likely exist. Nevertheless, taken together with the fact that RIG-I and MDA-5-dependent IFN-{alpha} and IFN-beta production is significantly compromised in the absence of FADD (Fig. 3B), our findings suggest an indispensable role for FADD in autocrine production of type I IFN by cytosolic dsRNA.

Control of Irf7 by FADD

Secondary IFN-{alpha} gene expression requires members of the IRF family of transcription factors for their expression (29, 30, 31). During our earlier analyses, we noticed that Irf7 was among those genes whose induction by dsRNA was defective in the absence of FADD (10). We therefore further evaluated IRF expression levels in Fadd+/– and Fadd–/– MEFs before and after dsRNA treatment. Irf1, Irf2, Irf3, Irf5, Irf7, and Irf9 were present in detectable levels in unstimulated wild-type cells (Fig. 4A). In contrast, expression of Irf4, Irf6, and Irf8 were undetectable in fibroblasts either basally or following dsRNA or IFN stimulation, and were therefore not evaluated further (data not shown). Irf3 was present in equivalent levels in both Fadd+/– and Fadd–/– MEFs and was not inducible by dsRNA treatment (Fig. 4A). Following dsRNA stimulation, we noticed that Irf1, Irf2, and Irf9 were induced normally in Fadd–/– MEFs. Importantly, however, we observed a significant and marked impairment in the induction of Irf7 (and, to a lesser extent, Irf5) by dsRNA in Fadd–/– MEFs 3 h after dsRNA stimulation (Fig. 4A). Both IRF-5 and IRF-7 are virus-activated transcription factors implicated in antiviral host defense. However, IRF-7 has recently emerged as the master regulator of type I IFN-production following virus infection, whereas IRF-5 appears to participate primarily in inflammatory cytokine production (32, 33). Furthermore, induction by dsRNA of Irf7 was more severely compromised than Irf5 in Fadd–/– MEFs. For these reasons, we examined the regulation of Irf7 by FADD in greater detail. Defective induction of Irf7 by dsRNA in the absence of FADD was confirmed by real-time PCR analysis and persisted for at least 12 h poststimulation (Fig. 4, B and C). It is noteworthy that induction of Irf7 (or any of the other inducible IRFs) by type I IFN treatment per se is not defective in the absence of FADD, indicating that the FADD is required for the activation of these genes at a step before type I IFN-triggered Jak/STAT signaling (Fig. 4D). In support of this fact, DNA microarray analysis of Irf7 in Stat1+/+ and Stat1–/– MEFs showed that Irf7 cannot be induced by dsRNA in the absence of STAT1 and, by extension, type I IFN signaling (Fig. 4C). Taken together, these data indicate that the poor inducibility of Irf7 seen in Fadd–/– MEFs is not the result of defective IFN Jak/STAT signaling but rather suggests a role for FADD in the direct dsRNA-stimulated production of IRF-7 itself.


Figure 4
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  		FIGURE 4. Defective Irf7 induction by dsRNA in Fadd–/– MEFs. A, Tbk1+/+, Tbk1–/–, Fadd+/–, and Fadd–/– MEFs were either left untreated or were transfected with poly(I:C) for 3 h, and DNA microarray analysis of Irf gene expression was performed as described in Materials and Methods. B, RNA extracted from Fadd+/– and Fadd–/– MEFs treated with or without poly(I:C) for the indicated times was examined from Irf7 induction by real-time PCR. C, Fadd+/–, Fadd–/–, Stat1+/+, Stat1–/–, Tbk1+/+, and Tbk1–/– MEFs were either left untreated or were transfected with poly(I:C) for 12 h, and DNA microarray analysis of Irf7 gene expression was performed as described in Materials and Methods. D, Fadd+/– and Fadd–/– MEFs were either left untreated or were treated with murine IFN-beta (500 U/ml) for 8 h, and DNA microarray analysis of Irf gene expression was performed as described in Materials and Methods.

 
Ectopic overexpression of IRF-7 partially rescues IFN production defects in FADD–/– MEFs

Next, we performed experiments to examine whether reconstitution of Fadd–/– MEFs with wild-type IRF-7 can rescue defective dsRNA signaling seen in these cells. To this end, Fadd+/– and Fadd–/– MEFs were transfected with expression vectors encoding either wild-type IRF-7 or a constitutively SA mutant of IRF-7 (IRF-7 (SA)) together with the IFN-beta-Luc reporter plasmid. IRF-7 (SA) differs from wild-type human IRF-7 in that it contains a deletion of a negative regulatory region (aa 238–410) that allows it to activate IFN-beta without a requirement for prior phosphorylation by TBK1/IKK{epsilon} (34). Twenty-four hours posttransfection, these cells were stimulated with dsRNA and examined for reporter activity 6 h poststimulation. Fadd+/– MEFs showed robust activation of the IFN-beta promoter after dsRNA stimulation, whereas Fadd–/– MEFs showed almost no responsiveness to the same stimulus (~25-fold activation in Fadd+/– MEFs, compared with ~1.5-fold in Fadd–/– MEFs; Fig. 5A). Remarkably, however, Fadd–/– MEFs transfected with wild-type IRF-7 were now able to respond to dsRNA, albeit to a lesser extent (~50%) than the littermate control-derived Fadd+/– fibroblasts. In agreement with this finding, IRF-7 (SA) was able to activate the IFN-beta promoter equally well in the presence or absence of FADD (~200-fold in both cases; Fig. 5A). We next complemented these studies by directly examining whether IRF-7 overexpression can boost type I IFN production in Fadd–/– MEFs following dsRNA stimulation. Accordingly, Fadd+/– and Fadd–/– were transfected with an expression vector encoding IRF-7 and subsequently stimulated with dsRNA for 24 h. Supernatants from cells treated in this manner were examined by ELISA for either IFN-{alpha} or IFN-beta. Transfection of a plasmid encoding IRF-7 increased the ability of Fadd–/– MEFs to produce IFN-{alpha} by ~4-fold (from ~200 pg/ml to ~800 pg/ml after dsRNA treatment; p > 0.0001; Fig. 5B). IFN-beta production was also increased by ~2-fold, from ~2500 pg/ml to ~5000 pg/ml after IRF-7 overexpression (Fig. 5C; p > 0.001). These results demonstrate that IRF-7 can at least partially rescue the defects in induction of type I IFN seen in Fadd–/– MEFs.


Figure 5
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  		FIGURE 5. Ectopic overexpression of IRF-7 in FADD–/– MEFs partially rescues dsRNA signaling defects. A, Fadd+/– and Fadd–/– MEFs were transfected with either wild-type IRF-7 or a constitutively active version of IRF-7 (IRF-7 (SA)), together with the IFN-beta-Luc firefly reporter plasmid for 24 h, and subsequently treated with or without poly(I:C) for a further 6 h. IFN-beta promoter-driven firefly luciferase activity was then measured and normalized to an internal Renilla luciferase control. Error bars, ±SD. B, An expression vector encoding wild-type IRF-7 was transfected into Fadd+/– and Fadd–/– MEFs. Twenty-four hours posttransfection, these cells were treated with poly(I:C) for a further 24 h, and levels of IFN-beta or (C) IFN-{alpha} were measured in the supernatant by ELISA. Error bars, ±SD. **, p < 0.001; ***, p < 0.0001.

 
Impaired activation of NF-{kappa}B in the absence of FADD

In Drosophila, FADD participates in an innate immune signaling cascade that culminates in NF-{kappa}B-mediated activation of antimicrobial genes (11). Because mammalian FADD is also capable of activating NF-{kappa}B (17, 35), and because several important antiviral genes (including IFN-beta and Irf7) have functional NF-{kappa}B binding sites, we examined the role of FADD in activation of NF-{kappa}B following dsRNA stimulation (36, 37). We initially overexpressed FADD (in tandem with IPS-1 and TRIF as controls) together with either an IFN-beta promoter reporter plasmid (IFN-beta-Luc), or reporter plasmids responsive to either IRF3/7 (PRD III-I-Luc) or NF-{kappa}B (NF-{kappa}B-Luc). We found that FADD was clearly capable of robust NF-{kappa}B activation (150-fold), in agreement with previous results (Fig. 6A) (17, 35). However, we also observed that FADD could modestly stimulate IFN-beta-Luc and the IRF3/7 responsive PRD III-I promoter (~8-fold and ~5-fold, respectively; Fig. 6A).


Figure 6
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  		FIGURE 6. Partial requirement for FADD in activation of NF-{kappa}B by dsRNA. A, Expression vectors encoding FADD, IPS-1, or TRIF were transfected into 293T cells, together with the indicated firefly luciferase reporter plasmids. Forty-eight hours posttransfection, firefly luciferase activity was measured, and normalized to an internal Renilla luciferase control. Error bars, ±SD. B, Nuclear extracts prepared from Fadd+/– and Fadd–/– MEFs stimulated for the indicated times with transfected dsRNA were analyzed by EMSA for NF-{kappa}B activity as described in Materials and Methods. C, Nuclear extracts prepared from Fadd+/– and Fadd–/– MEFs stimulated for the indicated times with transfected dsRNA were analyzed by p65-specific ELISA for NF-{kappa}B activity per the manufacturer’s instructions (EZ-Detectp65 Transcription Factor Kit; Pierce). Competition with mutant and wild-type NF-{kappa}B consensus sequences shows specificity. TNF-{alpha}-treated HeLa nuclear lysate was used as a positive control. The x-ray exposure of duplicate samples is shown. D, An expression vector encoding FADD was transfected into 293T cells, together with a firefly luciferase reporter plasmid driven by the full-length human Irf7 promoter (RL-1-Luc). Firefly luciferase activity was measured 48 h posttransfection and normalized to an internal Renilla luciferase control. Error bars, ±SD. E, Wild-type MEFs were transfected with an expression vector encoding FADD. Forty-eight hours posttransfection, RNA was extracted from these cells and examined for endogenous Irf7 gene induction by real-time PCR.

 
Because FADD can robustly activate NF-{kappa}B, we next directly tested whether dsRNA-induced NF-{kappa}B activation was defective in Fadd–/– MEFs. Accordingly, Fadd+/– and Fadd–/– MEFs were transfected with dsRNA, and nuclear extracts prepared from these cells were examined by EMSA for binding activity to a radiolabeled NF-{kappa}B consensus sequence. As shown in Fig. 6B, both basal and dsRNA-induced NF-{kappa}B DNA-binding activity was found to be partially defective in early passage Fadd–/– MEFs compared with Fadd+/– MEFs. Supershift Ab analyses indicated that the NF-{kappa}B p65 subunit was predominantly involved in DNA complex formation in response to transfected dsRNA in MEFs (Fig. 6B). We therefore next examined direct p65 binding to its cognate consensus DNA sequence by ELISA. Nuclear extracts prepared as described above were analyzed for p65 DNA-binding activity using a p65 transcription factor ELISA kit. As shown in Fig. 6C, we again observed that Fadd–/– cells possessed significantly less basal as well as dsRNA-inducible p65-dependent NF-{kappa}B activity. These results demonstrate that FADD participates in the activation of NF-{kappa}B following dsRNA stimulation in fibroblasts.

We next tested whether FADD could directly activate the Irf7 promoter. To this end, we cotransfected 293T cells with expression vectors encoding FADD along with a luciferase reporter plasmid under control of a 1.6-kb region of the human Irf7 promoter (RL-1-Luc) (36). Luciferase activity measured 24 h posttransfection revealed that FADD can also modestly stimulate the Irf7 promoter (~5-fold; Fig. 6D). To extend these findings, we next overexpressed FADD in wild-type MEFs and examined endogenous murine Irf7 gene induction by real-time PCR 24 h posttransfection. In agreement with the luciferase reporter assay results, we found that overexpression of FADD could also activate Irf7 directly (~4-fold) (Fig. 6E).

Role of TRAF2 in RIG-I/MDA-5-dependent antiviral responses

Our findings indicate that FADD can potently activate NF-{kappa}B and appears to be at least partially required for NF-{kappa}B activation in response to cytoplasmic dsRNA. Reports based on recent data speculated a role for TRAF2 in signaling events upstream of FADD, specifically those involving NF-{kappa}B activation or triggered by IPS-1 (20, 38). To examine whether TRAF2 was indeed required for TLR3-dependent and -independent signaling, early passage Traf2+/+ and Traf2–/– MEFs, in tandem with MEFs lacking Traf6, or Trif, were treated with either IFN-{alpha}beta or IFN-{gamma} and subsequently infected with VSV at a m.o.i. of 10. In this assay, early passage primary MEFs deficient in Fadd, Ripk1, Tbk1, or Irf3 succumb to VSV-induced replication and cytolysis even in the presence of IFN pretreatment (10). Unlike the case with these cells, however, neither Traf2–/–, Traf6–/–, or Trif–/– MEFs appeared to be overtly sensitive to either VSV-induced cytopathic effect (Fig. 7A) or replication (Fig. 7B) compared with control cells, in the presence or absence of IFN pretreatment. Indeed, intracellular dsRNA-mediated activation of the IFN-beta promoter was found to be normal in cells deficient in either Traf6, Traf2, or Trif (Fig. 7C).


Figure 7
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  		FIGURE 7. Role of TRAF2, TRAF6, and TRIF in RIG-I/MDA-5-dependent innate immune antiviral responses. A, Traf2+/+ (wild-type; WT), Traf2–/–, Traf6–/–, or Trif–/– MEFs were infected with VSV (m.o.i. = 10), with and without pretreatment with IFN-{alpha}beta (100 U/ml) or IFN-{gamma} (5 ng/ml). Photomicrographs were taken 36 h postinfection. B, VSV progeny yield was determined from supernatants of cells treated as in A by standard plaque assay. C, IFN-beta-Luc was transfected into TRAF2, TRAF6, and TRIF+/+ and –/– MEFs. After 24 h, they were left unstimulated, or stimulated with poly(I:C) with and without transfection reagent (Lipofectamine; LF). Luciferase activity was measured after 6 h. D, Empty vector, TLR3, IRAK1, TRAF6, RIG-I (1–200), MDA-5 (1–220), TRIF, and IPS-1 were expressed along with the IFN-beta-Luc construct in Traf2+/+ and Traf2–/– MEFs. Luciferase values were measured after 24 h.

 
Next, Traf2+/+ and Traf2–/– MEFs were transiently transfected with expression plasmids encoding TLR3, IRAK1, TRAF6, RIG-I (1–200), MDA-5 (1–220), TRIF or MAVS/IPS-1/VISA/Cardif, together with the IFN-beta luciferase reporter construct. Measurement of luciferase activity 24 h posttransfection revealed no significant differences in activation of the luciferase promoter by any of these polypeptides in the absence of TRAF2, implying a nonessential or redundant role for this molecule in dsRNA-triggered signaling events (Fig. 7D). Thus, FADD, but not TRAF2, TRAF6, or TRIF are required for IFN induction by intracellular dsRNA.

Regulation of dsRNA signaling by the FADD-interacting poxviral FLIP MC159

A number of viral proteins have been shown to inhibit TLR-independent dsRNA-induced antiviral signaling (for example, see Refs. 18, 39, 40, 41, 42). Viral FLIPs are a family of death effector domain-containing proteins encoded by gammaherpesviruses and poxviruses that have previously been shown to inhibit FADD- and caspase-8-dependent apoptotic signaling (43, 44). It is noteworthy that, of the various viral FLIPs, MC159, encoded by the mollscum contagiosum virus of the poxviridae family interacts specifically and strongly with FADD (45, 46). We hypothesized that, in addition to its role as an inhibitor of FADD-dependent apoptosis, MC159 could inhibit innate immune signaling by blocking FADD.

First, we examined whether MC159 inhibits dsRNA-mediated activation or IFN-beta. Hela cells were cotransfected with IFN-beta-Luc and either an empty vector control or with a vector encoding MC159. Such cells were stimulated with dsRNA 24 h posttransfection, and luciferase activity was measured 6 h later. As shown in Fig. 8A, we noticed that MC159 expression potently inhibited dsRNA-induced activation of the IFN-beta promoter. In empty vector-transfected cells, dsRNA stimulation resulted in a >250-fold activation of IFN-beta-Luc (Fig. 8A). In contrast, cells expressing MC159 manifested only ~20-fold activation of this reporter (Fig. 8A). To better elucidate the mechanism by which MC159 inhibits IFN-beta activation by dsRNA, 293T cells were cotransfected with the IFN-beta-Luc reporter plasmid, together with expression vectors encoding RIG-I (1–284), IPS-1, FADD, and IRF-7 (SA), along with increasing amounts of an expression vector encoding MC159. Expression of MC159 was confirmed by immunoblot analysis (Fig. 8B). As shown in Fig. 8C, MC159 was able to markedly inhibit signaling by RIG-I, IPS-1, FADD, and TBK-1 (by ~60, ~95, ~70, and ~80%, respectively). Importantly, MC159 had no inhibitory effect on IRF-7 (SA) (Fig. 8C). Furthermore, MC159 also inhibited FADD-mediated NF-{kappa}B activation by ~50% (Fig. 8D). These results describe a novel role for the FADD-interacting protein MC159 in specific inhibition of RIG-I-dependent host antiviral responses upstream of IRF-7 and reinforce the importance of FADD in innate antiviral signaling.


Figure 8
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  		FIGURE 8. Inhibition of cytosolic dsRNA signaling by the poxviral protein MC159. A, HeLa cells were transfected with an expression vector encoding MC159 together with the IFN-beta-Luc firefly reporter plasmid. Twenty-four hours posttransfection, such cells were further transfected with poly(I:C), or were left untreated. Firefly luciferase activity was measured 6 h posttreatment and normalized to an internal Renilla control. Error bars, ±SD. B, Empty vector (lane 1) or HA-tagged MC159 (250 ng-lane 2; 500 ng-lane 3) was transfected into 293T cells and expression was confirmed by Western blot analysis using an anti-HA Ab. beta-actin (lower panel) was used as a control to ensure equal loading. C, 293T cells were transfected with the indicated expression vectors (500 ng/sample), along with increasing amounts of the expression vector encoding MC159 (250 ng/sample, 500 ng/sample) and the IFN-beta-Luc firefly luciferase reporter plasmid. Total DNA concentrations were kept constant by adjustment with a control vector. Firefly luciferase activity was measured 48 h posttransfection and normalized to an internal Renilla control. Error bars, ±SD. D, 293T cells were transfected with an expression vector encoding FADD (500 ng/sample), together with a firefly luciferase reporter plasmid driven by a NF-{kappa}B-responsive promoter, and increasing amounts of an expression vector encoding MC159 (250 ng/sample, 500 ng/sample). Firefly luciferase activity was measured 48 h posttransfection, and normalized to an internal Renilla luciferase control.

 
Collectively, our data demonstrate that FADD is required for optimal primary innate immune responses triggered by activation of RIG-I, MDA-5, and IPS-1. In addition, secondary type I IFN-dependent responses are significantly compromised, explaining the severe loss of antiviral activity observed in Fadd–/– MEFs. Our observations of defective type I IFN production in Fadd–/– MEFs likely involve an impairment in dsRNA-activated Irf7 transcription.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
dsRNA produced during viral replication is considered a primary trigger of antiviral cytokine production and other innate and adaptive immune responses (3). In an emerging picture, extracellular dsRNA appears to signal via TLR3 and the adaptor molecule TRIF to activate type I IFN production, whereas intracellular dsRNA initiates a distinct signal transduction program mediated by RNA helicases RIG-I and MDA-5 (5). Although these two helicases share ~75% homology and can activate similar pathways, they seem to manifest distinct affinities for dsRNA and respond to different viruses. RIG-I appears important for IFN production after paramyxovirus, VSV, and influenza virus infection, whereas MDA-5 is necessary for protection against encephalomyocarditis virus and response to synthetic dsRNA in vivo (12, 13, 14). A number of studies now demonstrate that IPS-1 links these helicases to the induction of IFN-beta, although the underlying molecular mechanisms remain unclear (17, 18, 19, 20). One report suggest that IPS-1 can interact directly with RIG-I and MDA-5 and may signal via FADD to activate the NF-{kappa}B arm of dsRNA signaling (17), whereas another group reports that IPS-1 does not bind RIG-I or MDA-5 to any significant extent (19). A further study indicates that IPS-1 may bind to RIG-I, but not MDA-5, and can directly associate with members of the IKK family to trigger IFN-beta induction (18). Nevertheless, these reports and subsequent in vivo studies do indicate that IPS-1 is indeed required for optimal RIG-I and MDA-5 function (13, 14).

Our data presented in this study confirm a role for FADD downstream of RIG-I, MDA-5, and IPS-I in activation of IFN-beta by dsRNA. FADD may affect signaling downstream of IPS-1 by either direct interaction (17) or by participation in a signaling complex initiated by RIG-I/MDA-5 activation. For example, our preliminary data show that the FADD-interacting protein RIP1, homologous to the Imd gene product of Drosophila can interact with the CARDs of RIG-I and MDA-5, and associates with dsRNA-containing complexes in an IFN-dependent manner (data not shown). Given that FADD interacts strongly with RIP1, and that Ripk1–/– MEFs also display defective type I IFN (especially IFN-{alpha}) gene induction following virus infection or dsRNA stimulation (10), these data suggest possible physiologically relevant links between RIG-I, MDA-5, RIP1, and FADD.

Although a lack of FADD does not severely block virus-mediated activation of IFN-beta, loss of FADD does appear to have a detrimental effect on the kinetics of IFN-beta induction and on the activation of a subset of primary dsRNA-responsive genes. An analysis of the molecular determinants underlying this defect revealed that FADD appears to play a role in regulating the levels of the IRF-7 transcription factor following virus infection. Recent mouse knockout studies have revealed that IRF-7 is the master virus-responsive regulator of type I IFN production (32). In the absence of virus infection, IRF-7 in MEFs is found at very low levels. The levels of IRF-7, however, increase with passage number, underscoring the importance of performing experiments in early passage, nonimmortalized MEFs (47). Basal levels of IRF-7 seem to be maintained by low constitutive IFN production and signaling, and these basal levels of IRF-7 are important for the initial induction of primary genes in response to dsRNA (31). In a current model, dsRNA produced by the virus activates RIG-I or MDA-5, culminating in TBK1/IKK-{epsilon}-mediated formation of either IRF-7 homodimers or IRF-3/IRF-7 heterodimers and consequent induction of primary genes, including IFN-beta. Autocrine type I IFN activity then results in the production of greater amounts of IRF-7, its further activation by TBK1/IKK-{epsilon}, and full induction of the entire range of antiviral genes, including secondary IFN-{alpha}s, in a positive feedback loop (4). Our data indicate that FADD participates in the critical initial phase of primary type I IFN induction by dsRNA and virus. In the absence of FADD, we notice a significant delay in the production of IFN-beta by either dsRNA stimulation or virus infection. Because robust early induction of Irf7 is critical for the establishment of the positive feedback expression of IFN-{alpha} (and other secondary response genes), we propose that a delay in IFN-beta production prevents efficient establishment of this positive feedback loop and results in poor induction of Irf7 and consequent loss of secondary gene activation. Continual production of autocrine IFN during the course of a viral infection is critical for the protection of fibroblasts, despite pretreatment with exogenously supplied IFN, as we have shown previously (10).

Alternatively, our preliminary data also suggest that FADD may directly impinge upon the Irf7 promoter following dsRNA stimulation or virus activation. IFN-independent activation of Irf7 is not without precedent. For example, studies on the Irf7 promoter show that it can be activated by TNF-{alpha} and TPA in a manner dependent on NF-{kappa}B (36). In addition, EBV-encoded latent membrane protein-1 can also induce Irf7 by mechanisms that also may depend on NF-{kappa}B signaling (48). It is thus conceivable that FADD, which can potently stimulate NF-{kappa}B activity, may regulate the Irf7 promoter though similar mechanisms following dsRNA stimulation or virus infection. In agreement with such a role for FADD, we show that NF-{kappa}B p65 binding to a consensus NF-{kappa}B binding site following dsRNA stimulation is significantly impaired in the absence of FADD. FADD-mediated NF-{kappa}B-dependent activity would represent a more direct parallel to IMD signaling in Drosophila, where a FADD-dependent innate-immune cascade uses NF-{kappa}B-like signaling to directly transactivate innate immune genes. In the IMD pathway, the RIP1 homologue IMD binds dFADD to activate innate immune responses (11).

Although FADD is required for optimal induction of IFN-beta following dsRNA stimulation, its requirement for the production of IFN-beta after infection with either VSV, SeV, or Newcastle disease virus appears less critical (23). Virally infected cells thus appear to possess a FADD-independent mechanism(s) of IFN-beta gene induction that is at least partially distinct from synthetic dsRNA-activated signaling, and requires TBK1, because Tbk1–/– MEFs are almost completely defective in IFN-beta induction by either virus or dsRNA (49, 50). Also of interest is the fact that TRIF (as well as other members of the TLR signaling pathway) can activate IFN-beta normally in Fadd–/– MEFs, whereas RIG-I, MDA-5, and IPS-1 cannot. Recent studies have begun to highlight significant differences between RIG-I-mediated and TLR-3-, 7-, or 9-mediated induction of type I IFN (5). For example, TLR3 activates IFN-{alpha}beta gene expression via a TRIF-NAP1-TBK1-dependent pathway, whereas the TLR9 subfamily appears to directly recruit and phosphorylate IRF-7 via MyD88 and the IRAK family of kinases to induce type I IFN gene expression (4, 5). Although these examples provide possible explanations for FADD-independent TLR signaling in fibroblasts, the fact that these cells express undetectable endogenous levels of TLR3 and are essentially unresponsive to untransfected dsRNA make them poor cells of choice to examine FADD’s role in TLR3-dependent signaling. This is emphasized by the recent demonstration of a critical role for FADD in dsRNA-mediated proliferative responses in B cells, indicating that, whereas FADD seems dispensable for TLR3-mediated IFN-beta activation in fibroblasts, it is nevertheless required for TLR3-dependent signaling in other more physiologically relevant cell types (51). Whether FADD plays a broader role in TLR signaling events, for example by participating in TLR9-subfamily mediated IRF7-dependent induction of IFN-{alpha} in plasmacytoid DCs, remains to be seen.

Numerous viruses encode products that inhibit dsRNA-initiated signaling events. For example, paramyxovirus V proteins have been shown to directly interact with MDA-5 and inhibit its activity (39). Several groups have also shown that the hepatitis C virus-encoded NS3/4a protease cleaves and inactivates IPS-1, thereby neutralizing RIG-I and MDA-5 signaling (18, 40, 52, 53, 54). Additionally, Borna disease virus P protein binds to and inhibits TBK1 (55). In this study, we examined the effects of molluscum contagiosum poxvirus-encoded v-FLIP MC159 on FADD-dependent dsRNA signaling. MC159 binds strongly to FADD via its death effector domains, and inhibits FADD-dependent apoptotic events. We show in this study that MC159 is also a potent inhibitor of dsRNA-activated RIG-I and IPS-1 signaling, adding it to a growing list of virally encoded inhibitors of innate immune signaling.

In summary, our studies here indicate that FADD is required for RIG-I, MDA-5, and IPS-1-mediated production of type I IFN. Although the exact mechanism(s) by which FADD participates in these signaling events remains unclear, it appears that FADD may lie downstream of IPS-1 and upstream of IRF-7 in the "classical" (i.e., TLR-independent) antiviral gene induction pathway required for the production of type I IFNs, especially the IFN-{alpha}s. In the initial stages of a viral infection, a lack of FADD impairs establishment of a positive feedback loop required for production of IFN-{alpha} and the robust induction of an antiviral state. Taken together with the fact that IFN-{alpha}s are crucial components of antiviral host defense, these findings help explain the remarkable susceptibility of Fadd–/– fibroblasts to virus infection.


   Acknowledgments
 
We are grateful to Drs. Shizuo Akira (Osaka University, Osaka, Japan), Joan Durbin (Columbus Children’s Research Institute, Columbus, OH), Wen-Chen Yeh (University of Toronto, Toronto, Canada), John Hiscott (McGill University, Montreal, Canada), Paula Pitha-Rowe (Johns Hopkins University, Baltimore, MD), Tom Maniatis (Harvard University, Cambridge, MA), and Joanna Shisler (University of Illinois, Urbana, IL) for various cells and plasmids.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


Figure 9
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  		FIGURE 9. Schematic of cytosolic dsRNA-triggered signaling events. Cytosolic viral dsRNA is detected by PKR and 2',5'-oligoadenylate synthase leading to translational shutdown to restrict viral growth, or by the RNA helicases RIG-I and MDA-5, which initiate a signaling cascade leading to the activation of an antiviral program. RIG-I and MDA-5 require IPS-1 to active IFN-beta, and may transduce their signal via complex formation with molecules such as FADD, RIP1, and TRAF3. Stimulation of IPS-1 ostensibly leads to activation of TBK-1 and IKK, consequent nuclear translocation of IRF-3, IRF-7 and NF-{kappa}B homo- and heterodimers, and activation of primary antiviral genes, including IFN-beta. IFN-beta binds to IFNAR and induces Irf7 through the JAK/STAT pathway leading to a secondary antiviral response, including the induction of the CARD-less RIG-I/MDA-5 homologue, LGP2, which may dampen dsRNA signaling in a negative feedback loop. Viruses have evolved various mechanisms to disrupt cytosolic dsRNA-activated innate immune signaling. For example, the paramyxovirus V proteins inhibit MDA-5, hepatitis C virus NS3/4A inhibits IPS-1, molluscum contagiosum poxviral FLIP MC159 inhibits FADD and TBK-1, and the Borna disease virus P protein blocks TBK-1.

 

   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Defense Advanced Research Projects Agency and National Institutes of Health Grant R01GM068448 (to P.B.F.). Back

2 S.B. and T.V. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Glen N. Barber, Room 511, Papanicolaou Building, 1550 Northwest Tenth Avenue, University of Miami School of Medicine, Miami, FL 33136. E-mail address: gbarber{at}med.miami.edu Back

4 Abbreviations used in this paper: PKR, protein kinase R; FADD, Fas-associated death domain-containing protein; IMD, immune deficiency; RIG-I, retinoic acid-inducible gene I; MDA, melanoma differentiation Ag; CARD, caspase recruitment domain; TBK, tank binding kinase; IKK, I{kappa}B kinase; IPS, IFN-beta promoter stimulator; IRF, IFN regulatory factor; TRAF, TNFR-associated factor; MEF, murine embryonic fibroblast; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-beta; SA, superactive; IRAK, IL-1R-associated kinase; VSV, vesicular stomatitis virus; SeV, Sendai virus; m.o.i., multiplicity of infection; HA, hemagglutinin. Back

Received for publication August 30, 2006. Accepted for publication November 28, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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