Supplementary Information

Supplementary Fig. 1. Absence of FADD sensitizes cells to infection by Encephalomyocarditis Virus (EMCV) and Influenza Virus (FLU). a. FADD-deficient MEFs are susceptible to EMCV-induced CPE despite IFN pre-treatment.FADD+/- and FADD-/- MEFs were infected with EMCV (m.o.i = 10) in the presence or absence of 12 hours IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) pre-treatment. Photomicrographs were taken 24 hours post-infection.b. FADD-deficient MEFs are not protected from EMCV-triggered cell death by IFN pre-treatment. FADD+/- and FADD -/- MEFs were infected with EMCV (m.o.i = 10) in the presence or absence of 12 hours IFN / (100 U/ml) or IFN- (0.5 ng/ml) pre-treatement. Cell viability was determined 24 hours post-infection by Trypan Blue exclusion analysis.c. FADD-deficient MEFs are susceptible to FLU-induced CPE despite IFN pre-treatment.FADD+/- and FADD-/- MEFs were infected with FLU (m.o.i = 10) in the presence or absence of 12 hours IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) pre-treatment. Photomicrographs were taken 24 hours post-infection.d. FADD-deficient MEFs are not protected from FLU-triggered cell death by IFN pre-treatment. FADD+/- and FADD-/-MEFs were infected with FLU (m.o.i = 10) in the presence or absence of 12 hours IFN / (100 U/ml) or IFN- (0.5 ng/ml) pre-treatement. Cell viability was determined 24 hours post-infection by Trypan Blue exclusion analysis. (Error bars: +/- standard deviation).

Supplementary Fig. 2. Caspase 8 is not required for protection against VSV. a.Caspase 8-deficient MEFs do not show increased susceptibility to VS- induced CPE. Caspase8+/+and caspase8-/- MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). Photomicrographs were taken 48 hours post-infection. b. Caspase8-/- MEFs are protected from VSV-induced cell death by IFN pre-treatment. Caspase8+/+and caspase8-/- MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). Cell viability was determined at the indicated times post-infection by Trypan Blue exclusion analysis. c. IFN pre-treatment efficiently inhibits VSV replication in both caspase8 +/+ and caspase8-/- MEFs. Caspase8+/+and caspase8-/- MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). At the indicated times post-infection, the medium was examined for progeny virion presence by standard plaque assay on BHK cells. d.Caspase 8 is not required for intracellular dsRNA signaling to the IFN-β promoter.Caspase8+/+and caspase8-/- MEFs were transfected with IFN--Luc. 24 hours later, these cells were transfected with poly (I:C) [6 μg/ml in Lipofectamine2000 (LF)] and luciferase activity measured after 6 hours. e.Caspase activity is required for FADD-dependent apoptosis. FADD+/- and FADD-/- MEFswere pre-treated with the broad-specificity caspase inhibitor z-VAD.fmk (100μM), or with DMSO alone, for 30 minutes after which they were treated with the anti-Fas agonistic antibody Jo2 (2μg/ml) in the presence of cycloheximide (2 μg/ml). 24 hours post-treatment, cell death was measured by Trypan Blue exclusion analysis.f.Caspase activity is not required for FADD-dependent induction of IFN-α by dsRNA. FADD+/- and FADD-/- MEFswere pre-treated with the broad-specificity caspase inhibitor z-VAD (100μM), or with DMSO alone, for 30 minutes after which they were transfected with poly (I:C) (6 μg/ml in Lipofectamine2000 [LF]). 24 hours later cell culture supernatants from these cells were examined for IFN-α by ELISA.

Supplementary Fig. 3.Normal IFN signaling in the absence of FADD. a.Normal STAT1 phosphorylation in the absence of FADD.FADD+/- and FADD-/- MEFs were treated with either IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) for the indicated times, and STAT1 phosphorylation status determined by immunoblotting using a STAT1 phospho-tyrosine 701-specific antibody. β-actin protein levels were also measured to ensure equivalent amounts of protein per lane. b. Nuclear translocation of STAT1 following IFN treatment occurs normally in the absence of FADD.FADD+/- and FADD-/- MEFs were transfected with a plasmid encoding a GFP-STAT1 fusion protein. 24 hours post-transfection, cells were treated with or without IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) for one hour and STAT1 localization was determined by GFP fluorescence microscopy. c. IFN-responsive promoters function normally in the absence of FADD.FADD+/- and FADD-/- MEFs were transfected with plasmids expressing luciferase under the control of either the interferon stimulated response element (ISRE-Luc) or the interferon gamma activated sequence (GAS-Luc). 24 hours later, cells were stimulated with or without IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) and luciferase activity measured 12 hours post treatment.

Supplementary Fig. 4.Confirmation of a requirement for FADD for optimal induction of IFN-β following intracellular dsRNA treatment.a. Reconstitution of murine (m) FADD into FADD-/- MEFs can partially rescue dsRNA-mediated activation of the IFN-β promoter. FADD-/- MEFs were transfected with either the empty vector or with a plasmid encoding full-length mFADD, along with IFN--Luc. 24 hours later, cells were transfected with poly (I:C) [6 μg/ml in Lipofectamine2000 (LF)] and luciferase activity measured 6 hours thereafter. b.RNAi-mediated knockdown of FADD abolishes activation of the IFN-β promoter by intracellular dsRNA. HeLa cells were transfected with siRNA sequences targeting murine FADD (mFADD) or human FADD (hFADD) and knockdown of human FADD confirmed by immunoblotting (inset). These cells were then transfected with IFN--Luc, and 24 hours later, were further transfected with poly (I:C) (2 μg/ml in Lipofectamine2000 [LF]). Luciferase activity was measured 6 hours thereafter.c. RNAi-mediated knockdown of hFADD significantly reduces intracellular dsRNA induction of IFN-β mRNA. HeLa cells were treated with siRNA sequences from mFADD or hFADD for 72 hours. These cells were then transfected with poly(I:C) (6 μg/ml in Lipofectamine2000) for 6 hours and induction of IFN-β mRNA was analyzed by TaqMan real time RT-PCR. Expression levels are normalized to basal levels found in HeLa cells not treated with poly(I:C), which was arbitrarily set at 1. d.RNAi-mediated knockdown of FADD diminishes production of IFN-β by intracellular dsRNA. HeLa cells were transfected with siRNA sequences representing mFADD or hFADD, and subsequently transfected with the indicated amounts of poly (I:C) (using Lipofectamine2000 [LF]). 24 hours later cell culture supernatants were examined for IFN-β protein by ELISA.

Supplementary Fig. 5.TLR3 and PKR independent signaling by intracellular dsRNA.a..RNAi-mediated knockdown of PKR and TLR3 does not effect on intracellular dsRNA signaling. HeLa cells were treated with siRNA representing mFADD, PKR and TLR3. 48 hours post siRNA treatment, cells were transfected with IFN--Luc. 24 hours later, these cells were treated with poly (I:C) (2 μg/ml in Lipofectamine2000 [LF]) and luciferase activity measured 6 hours post treatment.b.Quantitation of siRNA mediated gene reduction. Knockdown of the respective gene products were confirmed by TaqMan real time RT-PCR.c. PKR deficiency does not significantly alter the ability of intracellular dsRNA to activate the IFN-β promoter. PKR+/+ and PKR-/- (derived from knockout mice produced independently by the J. Bell and C. Weissmannlabs) MEFs weretransfected with IFN-β-Luc. 24 hours later, these cells were treated with poly (I:C) alone [100ug/ml] or transfected with poly (I:C) (6μg/mL in Lipofectamine2000 [LF]) and luciferase activity was measured after 6 hours. d. Induction of several anti-viral genes by intracellular dsRNA is not defective in the absence of PKR. PKR+/+ and PKR-/- (J. Bell knockout mouse) MEFs were transfected with poly (I:C) (6μg/ml in Lipofectamine2000 [LF]). 3 hours post-transfection, mRNA from these cells was subjected to DNA microarray analysis. Induction profiles of a selection of key anti-viral genes are displayed. Induced levels of these genes in wild type (PKR+/+) cells was arbitrarily set to 1 (yellow).

Supplementary Fig. 6. Analysis of Ripk1-/- MEFs. a.IFN pre-treatment cannot efficiently inhibit virus replication in the absence of RIP1.Ripk1+/+ and Ripk1-/- MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). 48 hours post-infection, the medium was examined for progeny virion production. b.RIP1 is not required for IFN-induced gene induction. Ripk1+/+ and Ripk1-/- MEFs were treated with or without IFN-/ (100 U/ml) or IFN- (0.5 ng/ml) for 12 hours. Lysates prepared from these cells were subject to immunoblot analysis for the indicated proteins. β-actin protein levels were also measured to ensure equivalent amounts of protein per lane. c.RIP1 is not required for TLR3 activation of the IFN-β promoter. Ripk1+/+ and Ripk1-/-were transfected with plasmids encoding TLR3, IRAK-1 or TRAF6, along with IFN--Luc, and luciferase activity was measured 24 hours post-transfection.

Supplementary Fig. 7. Quantitation of cell death and virus yield from Tbk1/Ikkδ+/+ and Tbk1/Ikkδ-/-, as well as Irf3+/+and Irf3-/- MEFs. a. TBK-1/IKKδ-deficient MEFs are not protected from VSV-triggered cell death by IFN pre-treatment.Tbk1/Ikkδ+/+ and Tbk1/Ikkδ-/-MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). Cell viability was determined by Trypan Blue exclusion analysis. b. IFN pre-treatment cannot inhibit virus replication in the absence of TBK-1/IKKδ.Tbk1/Ikkδ+/+ and Tbk1/Ikkδ-/- MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). 48 hours post infection, the medium was examined for progeny virion production.c. IRF-3-deficient MEFs are not protected from VSV-triggered cell death by IFN pre-treatment.Irf3+/+and Irf3-/-MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). Cell viability was determined by Trypan Blue exclusion analysis. b. IFN pre-treatment cannot inhibit virus replication in the absence of IRF-3.Irf3+/+and Irf3-/-MEFs were infected with VSV (m.o.i = 10) in the presence or absence of 12 hours pre-treatment with IFN-/ (100 U/ml) or IFN- (0.5 ng/ml). 48 hours post infection, the medium was examined for progeny virion production.

Supplementary Fig. 8. FADD, RIP1 and IRF-3 are required for IFN-α production in 293 cells. FADD, RIP1 and IRF-3 expression was specifically knocked down in 293 cells by siRNA treatment for 72 hours, following which these cells were transfected with the indicated amounts of poly (I:C) in Lipofectamine2000 (LF). Tissue culture supernatants were collected 24 hours later, and IFN-α measured by ELISA.

Supplementary Fig. 9.The RIP1/FADD/TBK-1 (RIFT) cascade. This schematic highlights the major known dsRNA signaling components in mammalian cells that result in production of IFN, including the putative recruitment of FADD and RIP1 into an ‘innateosome’ that governs activation of TBK-1/IKKδ by such dsRNA receptors as RIG-I.

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