VISA Is an Adapter Protein Required for Virus-Triggered IFN-β Signaling

Molecular Cell, Vol. 19, 727–740, September 16, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.molcel.2005.08.014

VISA Is an Adapter Protein Required for Virus-Triggered IFN-␤ Signaling Liang-Guo Xu,1,4 Yan-Yi Wang,2,4 Ke-Jun Han,1 Lian-Yun Li,2 Zhonghe Zhai,2 and Hong-Bing Shu1,2,3,* 1 Department of Immunology National Jewish Medical and Research Center 1400 Jackson Street Denver, Colorado 80206 2 Department of Cell Biology and Genetics College of Life Sciences Peking University Beijing 100871 China 3 College of Life Sciences Wuhan University Wuhan 430072 China

Summary Viral infection or stimulation of TLR3 triggers signaling cascades, leading to activation of the transcription factors IRF-3 and NF-␬B, which collaborate to induce transcription of type I interferon (IFN) genes. In this study, we identified a protein termed VISA (for virus-induced signaling adaptor) as a critical component in the IFN-␤ signaling pathways. VISA recruits IRF-3 to the cytoplasmic viral dsRNA sensor RIG-I. Depletion of VISA inhibits virus-triggered and RIG-Imediated activation of IRF-3, NF-␬B, and the IFN-␤ promoter, suggesting that VISA plays a central role in virus-triggered TLR3-independent IFN-␤ signaling. Our data also indicate that VISA interacts with TRIF and TRAF6 and mediates bifurcation of the TLR3-triggered NF-␬B and IRF-3 activation pathways. These findings suggest that VISA is critically involved in both virus-triggered TLR3-independent and TLR3mediated antiviral IFN signaling. Introduction Viral infection results in induction of type I IFNs, including IFN-β and IFN-α family cytokines (Durbin et al., 2000; Levy and Garcia-Sastre, 2001; Levy and Marie, 2004). Type I IFNs activate the JAK-STAT signal transduction pathways, leading to transcriptional induction of a wide range of genes. The induced downstream gene products, such as IRFs, PKR, IP10, ISG15, and OAS, orchestrate inhibition of viral replication and clearance of virus-infected cells and thus lead to antiviral responses (Durbin et al., 2000; Levy and GarciaSastre, 2001; Levy and Marie, 2004). Transcriptional activation of the promoters of type I IFN genes requires the coordinate activation of multiple transcription factors and their cooperative assembly into transcriptional enhancer complexes in vivo. For ex*Correspondence: [email protected] 4 These authors contributed equally to this work.

ample, the enhancer of the IFN-β gene contains a κB site recognized by NF-κB, a site for ATF-2/c-Jun, and two IFN-stimulated response elements (ISREs) recognized by phosphorylated IRF-3 and/or IRF-7. It has been shown that transcriptional activation of the IFN-β gene requires coordinate and cooperative assembly of an enhanceosome that contains all of these transcription factors (Maniatis et al., 1998; Wathelet et al., 1998). At least two distinct pathways for the activation of the innate immune responses by viral infection have been proposed (Levy and Garcia-Sastre, 2001; Levy and Marie, 2004; tenOever et al., 2002). First, viruses enter cells by membrane fusion at the plasma membrane or through an endocytic process, leading to release of viral nucleocapsids (or ribonucleoproteins) into the cytoplasm. The viral nucleocapsids trigger signaling cascades that lead to activation of IRF-3 and NFκB and subsequent transcription of type I IFNs (Levy and Garcia-Sastre, 2001; tenOever et al., 2002). Second, viral dsRNA, released upon lysis of infected cells, binds to TLR3 and triggers TRIF-mediated pathways, leading to IRF-3 and NF-κB activation (Alexopoulou et al., 2001; Han et al., 2004; Hoebe et al., 2003; Oshiumi et al., 2003a; Yamamoto et al., 2002, 2003). It has been proposed that TLR3 is important for systemic responses to viral infection but is not required for the initial, cell-autonomous recognition of viral infection that induces the first wave of type I IFN production (Honda et al., 2003; Levy and Marie, 2004; Yoneyama et al., 2004). Until recently, the molecular components involved in virus-triggered TLR3-independent signaling are largely undefined. However, during the past couple of years, major breakthroughs have been achieved. It has been shown that two serine/threonine kinases, TBK1 and IKK⑀, phosphorylate IRF-3 and IRF-7 and are involved in virus-triggered induction of type I IFNs in various cell types (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2003; Perry et al., 2004; Sharma et al., 2003). More recently, a CARD module-containing RNA helicase protein, RIG-I, has been identified as a sensor of cytoplasmic viral dsRNA (Levy and Marie, 2004; Yoneyama et al., 2004). Depletion of RIG-I inhibits IFN-β production in response to Newcastle disease virus, suggesting that RIG-I is required for virus-triggered signaling. It has been further shown that RIG-Imediated signaling is independent of TLR3 and TRIF (Yoneyama et al., 2004). The mechanisms of RIG-Imediated downstream signaling are unknown. The mechanisms of TLR3-mediated signaling have also begun to emerge. It has been shown that the TIR domain-containing adaptor protein TRIF is associated with TLR3 and critically involved in TLR3-mediated activation of NF-κB and IRF-3 and transcription of the IFN-β gene (Han et al., 2004; Jiang et al., 2004; Oshiumi et al., 2003a; Yamamoto et al., 2002, 2003). TRIF interacts with TRAF6, which activates NF-κB through TAK1 and IKK (Han et al., 2004; Jiang et al., 2004; Sato et al., 2003). TRIF is also associated with IRF-3 as well as TBK1 and IKK⑀ (Fitzgerald et al., 2003a; Han et al.,

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2004; Oshiumi et al., 2003a; Yamamoto et al., 2002). Several reports have indicated that TBK1 and IKK⑀ phosphorylate IRF-3 and IRF-7 and are critically involved in TLR3-mediated production of type I IFNs and cellular antiviral response (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2003; Perry et al., 2004; Sharma et al., 2003). In this study, we identified a protein termed VISA. VISA contains a CARD module and consensus TRAF binding motifs. VISA recruits IRF-3 to RIG-I and is required for virus-triggered TLR3-independent signaling. VISA also interacts with TRIF and TRAF6 and mediates bifurcation of the TLR3-mediated NF-κB and IRF-3 activation pathways. Our findings suggest that VISA is an adaptor protein critically involved in virus-triggered IFN-β signaling. Results Sequence and Expression Analysis of VISA In a large-scale screen for human proteins that activate NF-κB and MAPK signaling pathways, 25 uncharacterized proteins were identified to activate NF-κB (Matsuda et al., 2003). The mechanisms of action and biological significance of these proteins were unknown. We made expression plasmids for 12 of these 25 proteins and found that only one (designated as clone number 031N in the original report) could significantly activate NF-κB in reporter assays (data not shown). We have designated this protein as VISA. Alignment of available cDNA and EST sequences in the GenBank databases indicated that human VISA mRNA has two splice variants. The shorter one is w3.4 kb in length, and the longer one contains extra fragments at the 3# UTR (data not shown). However, both transcripts encode the same 540 amino acid (aa) protein (data not shown). Structural analysis with the EMBL-EBI InterProScan program indicated that VISA contains a CARD module at its N terminus (Figure 1A). The CARD module of VISA is mostly homologous to the CARDs of RIG-I and MDA5 (Figure 1B), two related RNA helicase proteins that have been shown to be involved in virus-triggered IFN signaling (Yoneyama et al., 2004). Other than the CARD module, VISA is not homologous to known proteins. Human VISA shares w45% sequence identity at the amino acid level with two uncharacterized sequences from mouse (Figure 1A) and rat (data not shown), respectively. Northern blot analysis indicated that human VISA mRNA is expressed in all examined tissues, including brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and peripheral blood leukocytes, as two transcripts of w3.4 and w9.5 kb in sizes (Figure 1C). We prepared two rabbit polyclonal antibodies against aa 1–180 and aa 181–360 of human VISA, respectively. Both of these antibodies could detect an endogenous w62 kDa protein in human embryonic kidney 293 and human sarcoma SAOS-2, but not in human lung cancer H1299 cells (Figure 1D and data not shown). These antibodies could also detect overexpressed HA-tagged VISA, which migrated slightly slower than the endogenous VISA because of the additional HA tag (Figure 1D).

These data suggest that the VISA cDNA we cloned encodes full-length VISA protein. VISA Potently Activates NF-␬B, ISRE, and the IFN-␤ Promoter To confirm whether VISA activates NF-κB, we transfected 293 cells with a VISA expression plasmid and performed NF-κB luciferase reporter assays. As shown in Figure 2A, VISA activated NF-κB more potently than the positive control IKKβ in these assays. These data suggest that overexpression of VISA activates NF-κB. We then determined whether VISA activates other transcription factors. We found that VISA could potently activate ISRE in reporter assays (Figure 2B). Consistently, overexpression of VISA caused a shift of IRF-3 to a slightly higher molecular weight band (Figure 2B), which is a hallmark of IRF-3 phosphorylation and activation (Lin et al., 1999; Servant et al., 2002; Weaver et al., 1998; Yoneyama et al., 1998; Zhang and Pagano, 2002). In vitro kinase assays using recombinant GSTIRF-3 as substrate confirmed that overexpression of VISA caused IRF-3 phosphorylation (Figure 2B). Both IRF-3 phosphorylation and ISRE activation caused by VISA were as potent as that mediated by IKK⑀, a kinase shown to be involved in virus-induced type I IFN signaling (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2003; Perry et al., 2004; Sharma et al., 2003). To further confirm that VISA activates NF-κB and ISRE, we performed gel-shift experiments with 32Plabeled consensus NF-κB and ISRE probes. These experiments indicated that overexpression of VISA caused NF-κB and ISRE activation (Figure 2C). Consistent with the observations that VISA activated both NFκB and IRF-3, overexpression of VISA potently activated the promoter of the human IFN-β gene (Figure 2D). In reporter assays, VISA did not activate CHOP, a transcription factor activated by p38 kinase (Figure 2E). These data suggest that VISA specially activated NFκB, ISRE, and the IFN-β promoter. VISA Is Required for Virus-Induced TLR3-Independent Signaling Because VISA potently activates NF-κB, ISRE, and the IFN-β promoter, we reasoned that VISA is involved in virus-triggered signaling. To test this, we made seven RNAi expression vectors containing different target sequences of the human VISA cDNA. Transient transfection and Western blot analysis indicated that these vectors could inhibit expression of endogenous VISA to varied levels in 293 cells (Figure 3A). To determine whether VISA plays a role in virus-triggered ISRE activation, we transfected the VISA RNAi vectors into 293 cells and performed reporter assays. As shown in Figure 3A, knockdown of VISA expression inhibited Sendai virus-induced ISRE activation. The degree of inhibition was correlated with the efficiency of knockdown of VISA expression by each RNAi vector (Figure 3A). Consistent with these data, knockdown of VISA expression by RNAi also significantly inhibited Sendai virus-induced IRF-3 phosphorylation (Figure 3B). Taken together, these data suggest that VISA is

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Figure 1. Sequence and Expression Analysis of VISA (A) Alignment of amino acid sequences of human and mouse VISA. The CARD module, TRAF2 binding motif (T2BM), N-terminal (T6BM1), and C-terminal (T6BM2) TRAF6 binding motifs are boxed. Identical amino acids are shaded and boxed. Similar amino acids are boxed only. (B) Alignment of amino acid sequences of the CARD modules of human VISA, RIG-I, and MDA5. (C) Tissue expression of human VISA mRNA. (D) Expression of human VISA protein in cells. Total lysates from HA-VISA-transfected 293 cells or untransfected 293, H1299, and SAOS-2 cells were analyzed by Western blot with a rabbit polyclonal antibody against human VISA-(181–360). Ten times less lysate from HA-VISAtransfected 293 cells was loaded.

required for virus-triggered IRF-3 phosphorylation and ISRE activation. We used the #3 VISA RNAi vector for all the experiments described below. Similar results were obtained with an independent VISA RNAi vector (#7) (data not shown). In reporter assays, VISA RNAi also dramatically inhib-

ited Sendai virus-induced activation of NF-κB (Figure 3C) and the IFN-β promoter (Figure 3D) in 293 cells. Previously, it has been shown that 293 cells do not express TLR3, and addition of poly(I:C) to the culture medium does not activate ISRE in reporter assays (Yoneyama et al., 2004). This observation was also confirmed by us (data not shown). Taken together, these data sug-

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Figure 2. VISA Activates NF-κB, ISRE, and the IFN-β Promoter, but Not CHOP (A) VISA activates NF-κB in reporter assays. 293 cells (w2 × 105) were transfected with an NF-κB luciferase plasmid (0.1 ␮g) and the indicated mammalian expression plasmids (0.5 ␮g each). Luciferase assays were performed 18 hr after transfection. (B) VISA activates IRF-3 and ISRE. Reporter assays (top) were similarly performed as in (A) except that ISRE-luciferase reporter plasmid was used. Cell lysates were analyzed by Western blots with anti-IRF-3 and anti-HA (for VISA and IKK⑀ expression) antibodies, respectively (middle panels). The unphosphorylated (unphos.), basally phosphorylated (basal-phos.), and super-phosphorylated (super-phos.) IRF-3 bands were indicated. In vitro kinase assays with recombinant GST-IRF-3(380–427) as substrate were also performed (bottom). (C) VISA activates NF-κB and ISRE in EMSAs. 293 cells were transfected with the indicated plasmids for 14 hr, and EMSAs were performed with NF-κB (lanes 1–3) or ISRE (lane 4–6) probes. (D) VISA activates the IFN-β promoter. The experiments were similarly performed as in (A) except that the IFN-β promoter reporter plasmid was used. (E) VISA does not activate CHOP. Detection of CHOP activity was carried out according to the PathDetect In Vivo Signal Transduction Pathway Trans Reporting Systems (Stratagene). 293 cells (w2 × 105) were transfected with pFR plasmid (0.5 ␮g), pFA-CHOP plasmid (0.05 ␮g), RSV-β-gal plasmid (0.1 ␮g), and the indicated mammalian expression plasmid (0.5 ␮g). Reporter assays were performed 18 hr after transfection. The results of reporter assays in (A), (B), (D), and (E) are averages of three replicates ± SD.

gest that VISA is required for virus-triggered TLR3indpendent signaling. VISA Is Required for RIG-I-Mediated Signaling VISA contains a CARD module that is homologous to those of RIG-I and MDA5 (Figure 1). In addition, both VISA (Figure 3) and RIG-I (Yoneyama et al., 2004) are required for virus-triggered IFN-β signaling. These results point to the possibility that VISA is an adaptor protein that links RIG-I to the downstream signaling components in the virus-triggered TLR3-independent pathways. To test this, we examined whether RIG-I interacts with VISA. In transient transfection and coimmunoprecipitation experiments, we found that VISA could interact with both RIG-I and IRF-3 (Figure 4A). In the same experiments, RIG-I could not interact with IRF-3. However, with the addition of VISA, RIG-I could efficiently interact with IRF-3 (Figure 4A). These results suggest that VISA recruits IRF-3 to RIG-I in mammalian overexpression system. To determine whether VISA interacts with RIG-I through their respective CARD domains, we made expression plasmids for VISA and RIG-I deletion mutants and performed coimmunoprecipitation experiments. The result indicated that the CARD of VISA could interact with the CARDs, but not the helicase domain, of RIG-I. Interestingly, both the N-terminal and the C-terminal CARDs of RIG-I were required for its interaction with the CARD of VISA (Figure 4B).

The interactions of VISA with RIG-I and IRF-3 were confirmed in untransfected monocytic U937 cells in coimmunoprecipitation experiments. VISA interacted with RIG-I with or without virus infection (Figure 4C). The interaction between VISA and IRF-3 was detected 2 hr after virus infection but disappeared 12 hr after virus infection (Figure 4C). These data imply that VISA recruits IRF-3 to the RIG-I complex for activation in untransfected cells. Because VISA recruits IRF-3 to the RIG-I complex, we determined whether VISA is required for RIG-Imediated signaling. Previously, it has been shown that overexpression of the CARD domains of RIG-I potently activates activation of ISRE, NF-κB, and the IFN-β promoter, whereas overexpression of full-length RIG-I has minimal effects but can potentiate virus-triggered activation of ISRE, NF-κB, and the IFN-β promoter (Yoneyama et al., 2004). In reporter assays, VISA RNAi inhibited activation of ISRE, NF-κB, and the IFN-β promoter induced by overexpression of the CARD domains of RIG-I or by overexpression of full-length RIG-I in the presence of Sendai virus infection (Figure 4D-I). These data suggest that VISA is required for RIG-I-mediated signaling. Unambiguous Identification of VISA-Interacting Proteins To further explore the mechanism of action of VISA and the pathways in which VISA is involved, we attempted

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clones encoded the C-terminal polo box-containing region of Plk1, seven clones encoded full-length TRAF2 or its C-terminal TRAF domain-containing region, and one clone encoded the C-terminal TRAF domain of TRAF6. Plk1 is a serine/threonine kinase involved in regulation of the cell cycle (Barr et al., 2004). Our studies suggest that Plk1 is not involved in VISA-mediated NF-κB and ISRE activation pathways (data not shown). Whether VISA is involved in Plk1-mediated cell cycle regulation will be addressed in a separate study. VISA Interacts with TRAF2 and TRAF6 in Mammalian Cells Because VISA interacts with TRAF2 and TRAF6 in the yeast two-hybrid system, we determined whether they interact in mammalian cells. 293 cells were transfected with mammalian expression plasmids for HA-tagged VISA and FLAG-tagged TRAF2 or TRAF6. Coimmunoprecipitations indicated that VISA interacted with TRAF2 and TRAF6 (Figure 5B), but not with TRAF5 (data not shown).

Figure 3. VISA Is Required for Sendai Virus-Induced Signaling (A) Effects of VISA RNAi constructs on expression of endogenous VISA and on Sendai virus-induced ISRE activation. 293 cells (w2 × 105) were transfected with an ISRE luciferase plasmid (0.1 ␮g), pSuper control, or pSuper-VISA-RNAi plasmids (1 ␮g). 40 hr after transfection, cells were lysed and the lysates were analyzed by Western blot with anti-VISA and anti-β-tubulin antibodies (bottom) or cells were infected with Sendai virus or left uninfected for 8 hr before luciferase assays were performed (bottom). (B) Knockdown of VISA inhibits Sendai virus-induced IRF-3 phosphorylation. 293 cells (w2 × 105) were transfected with pSuper or pSuper-VISA-RNAi (#3) plasmid (2 ␮g). 12 hr after transfection, cells were selected with puromycin (1 ␮g/ml) for 24 hr, then infected with Sendai virus or left uninfected for 4 hr. Total cell lysates were analyzed by Western blot with anti-phospho-IRF-3 (Ser398) (top) or anti-IRF-3 (bottom) antibody. (C) VISA RNAi inhibits Sendai virus-induced NF-κB activation. 293 cells stably transfected with NF-κB luciferase plasmid were transfected with a control or VISA RNAi plasmid. The cells were selected with puromycin (1 ␮g/ml) for 30 hr, then infected with Sendai virus or left uninfected for 8 hr before luciferase assays were performed. (D) VISA RNAi inhibits Sendai virus-induced activation of the IFN-β promoter. The experiments were similarly performed as in (A) except that the IFN-β promoter reporter plasmid was used. The results of reporter assays in (A), (C), and (D) are averages of three replicates ± SD.

to unambiguously identify VISA-interacting proteins by yeast two-hybrid screens. By using full-length VISA as bait, we screened w2 × 107 clones from a combination of human B cell, 293 cell, and leukocyte cDNA libraries and obtained a total of 22 β-gal-positive clones. Blast searches of the GenBank databases indicated that 14

VISA interacts with TRAF2 and TRAF6 through Its Consensus TRAF2 and TRAF6 Binding Motifs, Respectively TRAF2 is involved in signaling by TNF receptor family members, whereas TRAF6 plays important roles in signaling by IL-1 receptors and TLRs (Bradley and Pober, 2001; Wajant et al., 2001). The functional specificity of the TRAFs is dictated by their ability to recognize and bind to distinct structural motifs. A consensus TRAF2 binding motif, PxQx(T/S), was identified in several TNF receptor family members, such as CD40 (Boucher et al., 1997) and LMP-1 (Brodeur et al., 1997), and was shown to be responsible for the interaction of these receptors with TRAF2. Analysis of the VISA aa sequence indicated that VISA contains such a motif (143PVQET-147) that matches perfectly with those found in CD40 and LMP1 (Figures 1A and 5A). Structural studies suggest that TRAF6 binds to a consensus motif defined as PxExx (aromatic/acidic residue) (Ye et al., 2002). Sequence analysis of VISA indicated that it contains two consensus TRAF6 binding motifs, 153-PGENSE-158 and 455-PEENEY-460 (Figures 1A and 5A). Because VISA contains these consensus TRAF2 and TRAF6 binding motifs, we determined whether these motifs are required for the interactions of VISA with TRAF2 and TRAF6, respectively. For the TRAF2 binding motif, we made an HA-tagged VISA mutant, designated as VISA⌬T2, in which the Q145 residue was changed to the similar residue asparagine (N) (Figure 5A). Coimmunoprecipitation indicated that this mutant protein had dramatically reduced ability to bind to TRAF2 (Figure 5B), suggesting the consensus TRAF2 binding motif is important for the interaction of VISA with TRAF2. Similarly, we made three HA-tagged VISA mutants in which either E155 or E457 or both residues were conservatively changed to aspartic acid (D). These mutants are designated as VISA⌬T6-1 (for E155D mutation), VISA⌬T6-2 (for E457D mutation), and VISA⌬T6-1+2 (for E144D and E457D double mutations), respectively (Fig-

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Figure 4. VISA Is Required for RIG-I-Mediated Signaling (A) VISA recruits IRF-3 to RIG-I. 293 cells (w2 × 105) were transfected with the indicated expression plasmids (5 ␮g each). 18 hr after transfection, cell lysates were immunoprecipitated with control mouse IgG (Ig) or anti-FLAG (αF) antibody as indicated. The immunoprecipitates were analyzed by Western blots with an HRP-conjugated anti-HA antibody (top panels). The expression levels of the transfected proteins in the lysates were analyzed by Western blots with anti-FLAG and anti-HA antibodies (middle and bottom panels). (B) VISA interacts with RIG-I through their respective CARDs. 293 cells were transfected with the indicated mutants. Immunoprecipitation and Western blots were done as in (A). (C) Endogenous interactions of VISA with RIG-I and IRF-3. U937 cells (w1 × 108) were infected with Sendai virus for 2 hr, 12 hr, or left uninfected. The lysates were immunoprecipitated with the indicated antibodies. Western blot analysis was performed with anti-VISA antibody. (D) VISA RNAi inhibits RIG-I-mediated ISRE activation. 293 cells (w2 × 105) were transfected with an ISRE luciferase plasmid (0.1 ␮g), an empty or RIG-I expression plasmid (0.5 ␮g), and a control or VISA RNAi plasmid (1.0 ␮g) as indicated. 40 hr after transfection, cells were infected with Sendai virus or left uninfected for 8 hr before luciferase assays were performed. (E) VISA RNAi inhibits RIG-I-mediated NF-κB activation. The experiments were done similarly as in (D) except that a 293-NF-κB-luciferase cell line was used. (F) VISA RNAi inhibits RIG-I-mediated activation of the IFN-β promoter. The experiments were similarly performed as in (A) except that the IFN-β promoter reporter plasmid was used. (G–I) VISA RNAi inhibits activation of ISRE (G), NF-κB (H), and the IFN-β promoter (I). The experiments were similarly done as in (D) and (E) but without Sendai virus infection. The results of reporter assays in (D)–(I) are averages of three replicates ± SD.

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Figure 5. Structural Determinants of VISA-Mediated NF-κB and ISRE Activation (A) A schematic presentation of VISA mutants and their expression. Mammalian expression plasmids for HA-tagged VISA and its point mutants were constructed (left). Expression of these plasmids in 293 cells was examined by transient transfection and Western blots anti-HA antibody (right). (B) VISA interacts with TRAF2 and TRAF6 through its consensus TRAF2 and TRAF6 binding motifs. 293 cells (w2 × 106) were transfected with the indicated mammalian expression plasmids. 18 hr after transfection, cell lysates were immunoprecipitated with control mouse IgG (Ig) or anti-FLAG antibody (αF). The immunoprecipitates were analyzed by Western blot with anti-HA antibody (top panels). The soluble lysates (s) and insoluble fractions (ins) were equilibrated to the same volumes by SDS-PAGE loading buffer and analyzed by Western blots with anti-FLAG (middle panels) and anti-HA antibodies (bottom panels). (C) Effects of the TRAF binding mutants of VISA on activation of NF-κB and ISRE. 293 cells (w2 × 105) were transfected with the indicated luciferase reporter construct (0.1 ␮g) and mammalian expression plasmids (0.5 ␮g each). Luciferase assays were performed 18 hr after transfection. (D) Effects of VISA deletion mutants on activation of NF-κB and ISRE. The experiments were similarly performed as in (C). The results of reporter assays in (C) and (D) are averages of three replicates ± SD.

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ure 5A). Coimmunoprecipitation experiments indicated that both VISA⌬T6-1 and VISA⌬T6-2 could still interact with TRAF6 (data not shown), whereas VISA⌬T6-1+2 had dramatically reduced ability to bind to TRAF6 (Figure 5B). These data suggest that both consensus TRAF6 binding motifs can independently mediate the interaction of VISA with TRAF6. VISA Causes Accumulation of TRAF2 and TRAF6 in Insoluble Fractions The interaction between VISA and TRAF2 or TRAF6 was relatively weak in our coimmunoprecipitation experiments (Figure 5B). During our experiments, we found that a major fraction of overexpressed VISA existed in insoluble cellular fractions (data not shown). Overexpression of VISA also caused increased accumulation of TRAF2 and TRAF6 in the insoluble fractions (Figure 5B). Mutation of the consensus TRAF2 and TRAF6 binding motifs of VISA abolished its ability to cause accumulation of TRAF2 and TRAF6 in the insoluble fractions, respectively (Figure 5B). These data suggest that VISA-mediated accumulation of TRAF2 and TRAF6 in insoluble fractions requires its binding to TRAF2 and TRAF6. These observations suggest a functional connection between VISA and TRAF2 or TRAF6. In this context, several reports have demonstrated that activation of TNF receptor family members caused accumulation of cytoplasmic TRAF2 in insoluble aggregates (Arch et al., 2000; Brown et al., 2001; Fotin-Mleczek et al., 2002). Structural Determinants of VISA-Mediated NF-␬B and ISRE Activation To determine the functional significance of the interaction between VISA and TRAF2 or TRAF6, we examined whether the TRAF binding motif mutants of VISA could activate NF-κB and ISRE in reporter assays. As shown in Figure 5C, VISA⌬T2T6, a VISA mutant in which all three TRAF binding motifs are mutated, completely lost its ability to activate NF-κB, whereas VISA⌬T2, VISA⌬T6-1, VISA⌬T6-2, and VISA⌬T61+2 all had reduced ability to activate NF-κB. These data suggest that VISA activates NF-κB through its interactions with TRAF2 and TRAF6, and all three TRAF binding motifs contribute to this ability. Among the TRAF binding motifs of VISA, it seems that the C-terminal TRAF6 binding motif is most important for its ability to activate NF-κB, because mutation of this motif had a more dramatic effect (Figure 5C). Similarly, we performed ISRE reporter assays with the VISA mutants. VISA⌬T2 and VISA⌬T6-1 did not affect the ability of VISA to activate ISRE. VISA⌬T6-2, VISA⌬T6-1+2, and VISA⌬T2/T6 had reduced, but not abolished, ability to activate ISRE activation (Figure 5C). These data suggest that VISA-induced ISRE activation is not dependent on TRAF2 and TRAF6. Mutation of the C-terminal TRAF6 binding motif reduced the ability of VISA to activate ISRE, which can be explained by the observation that the C terminus of VISA is important for its ability to activate ISRE (see below). We also made mammalian expression plasmids for a series of deletion mutants of VISA, including aa 1–180, aa 1–360, aa 181–360, aa 181–540, and aa 361–540

(Figure 5A). Reporter assays indicated that the mutants containing any one of the TRAF binding motifs could activate NF-κB but to a lower degree in comparison to the full-length VISA (Figure 5D). One exception is the aa 181–540 mutant, which contains the C-terminal TRAF6 binding motif but did not activate NF-κB (Figure 5D). The simplest explanation is that aa 181–360 has an inhibitory role on NF-κB activation when linked with the C terminus of VISA. In reporter assays, the C terminus of VISA (aa 361– 540) was sufficient to activate ISRE, whereas the mutants that lack this domain did not significantly activate it (Figure 5D). These data suggest that the CARD domain is not required and the C-terminal domain (aa 361–540) is sufficient for activating ISRE. Again, it seems that aa 181–360 had an inhibitory role on ISRE activation when linked with the C terminus of VISA (Figure 5D).

VISA Is Involved in TLR3-, but Not TLR4- and TNF-, Triggered Signaling Pathways Because VISA interacts with TRAF2 and TRAF6, two components that are involved in signaling mediated by TNF receptor and TLR family members, we determined whether knockdown of VISA has any effects on these pathways. In reporter assays, VISA RNAi significantly inhibited poly(I:C)-induced activation of NF-κB, ISRE, and the IFN-β promoter in a TLR3-expressing 293 cell line (293-TLR3) (Figure 6A). In contrast, VISA RNAi did not inhibit LPS-induced activation of NF-κB and ISRE in an LPS-responsive 293 cell line (293-TLR4/MD2/CD14) stably expressing TLR4, MD2, and CD14 (Figure 6B). VISA RNAi also did not inhibit TNF-induced NF-κB activation in 293 cells (Figure 6C). These data suggest that VISA is specifically involved in TLR3-mediated signaling.

VISA Is Associated with Multiple Proteins in the TLR3-Mediated Signaling Pathways In our earlier experiments, we found that VISA could interact with TRAF6 in a mammalian expression system. However, TRAF6 is only involved in TLR3-mediated activation of NF-κB, but not ISRE (Han et al., 2004; Jiang et al., 2004). To determine the molecular mechanisms of the involvement of VISA in TLR3-mediated NFκB and ISRE activation pathways, we examined whether VISA could interact with other components in the TLR3mediated signaling pathways. In transient transfection and coimmunoprecipitation experiments, we found that VISA was associated with TRIF, TAK1, TBK1, IRF-3, and IRF-7, but not with IRF-1 (Figure 7A). We also attempted to detect endogenous interactions between VISA and signaling components of the TLR3- and virus-triggered pathways. In 293-TLR3 cells, endogenous VISA interacted with endogenous TRIF and TRAF6 and these interactions were not significantly affected by poly(I:C) treatment (Figure 7B). These data suggest that VISA is a scaffolding protein that is associated with multiple signaling components in the TLR3-mediated pathways.

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Figure 6. VISA Is Required for TLR3-, but Not TLR4- and TNF-, Induced Signaling (A) VISA-RNAi inhibits poly(I:C)-induced activation of NF-κB, ISRE, and the IFN-β promoter. 293-TLR3 cells (w2 × 105) were transfected with the indicated luciferase construct (0.1 ␮g) and a control or VISA RNAi plasmid (1 ␮g each). 40 hr after transfection, cells were treated with poly(I:C) (25 ␮g/ml) for 8 hr before luciferase assays were performed. (B) VISA RNAi does not inhibit LPS-induced activation of ISRE and NF-κB. 293-TLR4/ MD2/CD14 cells (w2 × 105) were transfected with the indicated luciferase reporter construct (0.1 ␮g) and control or VISA RNAi plasmid (1 ␮g each). 40 hr after transfection, cells were treated with LPS (10 ␮g/ml) or left untreated for 8 hr before luciferase assays were performed. (C) VISA RNAi did not inhibit TNF-induced NF-κB activation. 293 cells (w2 × 105) were transfected with an NF-κB luciferase construct (0.1 ␮g) and a control or VISA RNAi plasmid (1 ␮g each). 40 hr after transfection, cells were treated with TNF (10 ␮g/ml) or left untreated for 8 hr before luciferase assays were performed. The results of reporter assays in (A)–(C) are averages of three replicates ± SD.

A Molecular Order for VISA-Mediated NF-␬B and ISRE Activation Pathways Because VISA activates both NF-κB and ISRE and is associated with multiple components in the TLR3mediated pathways, we attempted to determine a molecular order for the involvement of VISA in these signaling pathways. In reporter assays, overexpression of VISA could activate both NF-κB and ISRE to a similar degree in TRIF−/− and wild-type (wt) mouse embryonic fibroblasts (MEFs) (Figures 8A and 8B). In addition, VISA⌬T2T6 inhibited TRIF-, but not TRAF6-, induced NFκB activation in a dose-dependent manner (Figure 8E), whereas VISA RNAi inhibited TRIF-induced ISRE activation (Figure 8G). These data suggest that VISA functions downstream of TRIF. Mutation of the TRAF6 binding motifs reduced the ability of VISA to activate NF-κB (Figure 5), suggesting

that TRAF6 functions downstream of VISA in NF-κB activation. To confirm this, we performed NF-κB reporter assays with TRAF6−/− MEFs. As shown in Figure 8C, VISA-mediated NF-κB activation was dramatically decreased in TRAF6−/− MEFs in comparison to wt control MEFs, suggesting that TRAF6 is important for VISAmediated NF-κB activation. Consistently, dominantnegative mutants of TRAF6, TAK1, and IKKβ, but not TRIF, inhibited VISA-mediated NF-κB activation (Figure 8F). These data suggest that VISA signals NF-κB activation through a TRAF6-TAK1-IKK-dependent pathway. In reporter assays, overexpression of VISA activated ISRE in both wt and TRAF6−/− MEFs (Figure 8D), suggesting that TRAF6 is not required for VISA-mediated ISRE activation. Dominant-negative mutants of IRF-3, but not those of TRIF and TBK1, inhibited VISA-induced ISRE activation (Figure 8H). On the other hand, VISA Figure 7. VISA Interacts with Signaling Components of the TLR3-Mediated Pathways (A) VISA is associated with TRIF, TAK1, TBK1, IRF-3, and IRF-7, but not IRF-1. 293 cells (w2 × 106) were transfected with the indicated plasmids (5 ␮g each). 18 hr after transfection, coimmunoprecipitation and Western blots were performed with the indicated antibodies. The expression levels of the transfected proteins in the lysates were comparable as indicated by Western blot analysis (data not shown). (B) Endogenous interactions between VISA and TRIF or TRAF6. 293-TLR3 cells (w2 × 107) were treated with poly (I:C) (25 ␮g/ml) or left untreated for 10 min. Coimmunoprecipitation and Western blots were performed with the indicated antibodies. Protein A-HRP was used as the secondary antibody in these experiments.

Molecular Cell 736

RNAi significantly inhibited ISRE activation induced by overexpression of TRIF and TBK1, but not IRF-3 (Figure 8G). These data suggest that VISA functions downstream of TRIF and upstream of IRF-3 in TLR3-induced ISRE activation pathways. Discussion Both viral infection and TLR3 engagement cause induction of type I IFNs. In this study, we identified an adaptor protein that is critically involved in both virus-triggered TLR3-independent and TLR3-mediated IFN-β signaling.

Figure 8. Molecular Position of VISA in TLR3-Mediated Signaling Pathways (A) VISA activates NF-κB in both TRIF-deficient and wt MEFs. TRIF+/+ or TRIF−/− MEFs (w1 × 105) were transfected with an NFκB luciferase plasmid (0.5 ␮g) and an empty control (empty bars) or VISA expression plasmid (filled bars) (1 ␮g each). Reporter assays were performed 18 hr after transfection. (B) VISA activates ISRE in both TRIF-deficient and wt MEFs. The experiments were performed similarly as in (A) except that an ISRE luciferase plasmid was used. (C) Deficiency of VISA-mediated NF-κB activation in TRAF6−/− MEFs. The experiments were performed similarly as in (A) except that TRAF6−/− MEFs were used. (D) VISA activates ISRE in both TRAF6−/− and wt MEFs. The experiments were performed similarly as in (A) except that TRAF6−/− MEFs were used. (E) Effects of VISA⌬T2T6 on TRIF- and TRAF6-mediated NF-κB activation. 293 cells were transfected with an NF-κB luciferase plasmid (0.1 ␮g), an empty or TRIF (0.2 ␮g, filled bars) or TRAF6 (0.2 ␮g, empty bars) expression plasmid, and increased amounts of VISA⌬T2T6 expression plasmid (0, 0.1, 0.2, and 0.4 ␮g). Reporter assays were performed 18 hr after transfection. (F) Effects of various dominant-negative mutants on VISA-mediated NF-κB activation. 293 cells (w2 × 105) were transfected with an NFκB luciferase plasmid (0.1 ␮g), an empty or VISA expression plasmid (0.2 ␮g), and expression plasmids for the indicated dominantnegative mutants (0.2 ␮g). Reporter assays were performed 8 hr after transfection. (G) Effects of VISA RNAi on TRIF-, TBK1-, and IRF-3-mediated ISRE activation. 293 cells stably transfected with pSuper or pSuperVISA-RNAi were transfected with ISRE luciferase plasmid (0.1 ␮g) and the indicated expression plasmids (0.5 ␮g each). Reporter assays were performed 18 hr after transfection.

VISA Is Required for Virus-Triggered TLR3-Independent Signaling Recently, it has been demonstrated that cytoplasmic viral dsRNA is recognized by the RNA helicase protein RIG-I. RIG-I contains two N-terminal CARD modules and a C-terminal RNA helicase domain. It has been proposed that binding of viral dsRNA to the RNA helicase domain of RIG-I causes its interaction with a putative CARD domain-containing adaptor protein, which recruits downstream signaling components to RIG-I (Levy and Marie, 2004; Yoneyama et al., 2004). In this study, we have provided several lines of evidences suggesting that VISA is such an adaptor protein. VISA contains a CARD module that is mostly homologous to those of RIG-I and MDA5. In transient transfection and coimmunoprecipitation experiments, VISA interacted with RIG-I through their respective CARD domains. In these experiments, RIG-I did not interact with IRF-3. However, with the addition of VISA, RIG-I could strongly interact with IRF-3 (Figure 4). These results suggest that VISA recruits IRF-3 to RIG-I in a mammalian overexpression system. In reporter assays, overexpression of VISA activated ISRE, NF-κB, and the IFN-β promoter (Figure 2). Depletion of VISA inhibited Sendai virus-induced activation of ISRE, NF-κB, and the IFN-β promoter in cells lacking TLR3 (Figure 3). In addition, depletion of VISA inhibited RIG-I-mediated activation of ISRE, NF-κB, and the IFN-β promoter (Figure 5). These findings suggest that VISA is an adaptor protein functioning downstream of RIG-I in virus-triggered TLR3-independent signaling. VISA Is Involved in TLR3-Mediated Signaling Viral dsRNA released after lysis of infected cells binds to TLR3 and triggers IFN signaling. Our studies suggest that VISA is also involved in TLR3-mediated signaling. In addition to its N-terminal CARD module, VISA also contains consensus TRAF6 binding motifs. Yeast twohybrid screens unambiguously identified TRAF6 as a

(H) Effects of various dominant-negative mutants on VISA-mediated ISRE activation. 293 cells were transfected with an ISRE luciferase reporter plasmid (0.1 ␮g), an empty or VISA expression plasmid (0.2 ␮g), and the indicated dominant-negative mutant plasmids (0.2 ␮g each). Reporter assays were performed 18 hr after transfection. The results of reporter assays in (A)–(H) are averages of three replicates ± SD.

VISA Is a Component of Virus-Induced Signaling 737

VISA-interacting protein. This interaction was confirmed in both a mammalian overexpression system and untransfected cells (Figures 5 and 7). In addition, VISA also interacts with TRIF, TBK1, and IRF-3, components in the TLR3-mediated pathways (Figure 7). These data suggest that VISA is a scaffolding protein that helps to assemble a complex for activation of NF-κB and ISRE in the TLR3-mediated pathways. Knockdown of VISA expression by RNAi inhibited TLR3-mediated activation of NF-κB, ISRE, and the IFN-β promoter in poly(I:C)-responsive 293-TLR3 cells (Figure 6). These results suggest that VISA is involved in TLR3-mediated signaling leading to NF-κB and ISRE activation. Molecular Mechanisms of VISA-Mediated Activation of IRF-3 Previously, it has been demonstrated that TBK1 directly phosphorylates IRF-3 and is critically involved in virustriggered TLR3-independent IFN signaling pathways (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2003; Perry et al., 2004; Sharma et al., 2003). In coimmunoprecipitation experiments, we found that VISA could interact with both IRF-3 and TBK1 (Figures 4 and 7), suggesting that VISA may function as a scaffolding protein for TBK1 and IRF-3. Upon viral infection, VISA may recruit these proteins to the RIG-I complex in which IRF-3 is phosphorylated by TBK1. In the absence of VISA, IRF-3 may not be efficiently recruited to TBK1 for phosphorylation. Consistent with this hypothesis, knockdown of VISA significantly inhibited TBK1-mediated activation of ISRE (Figure 8G). VISA may play a similar scaffolding role in TLR3mediated signaling pathways. In TRIF-deficient MEFs, VISA activated both NF-κB and ISRE as potently as in wt control cells (Figures 8A and 8B). On the other hand, VISA mutant or RNAi inhibited TRIF-mediated activation of NF-κB and ISRE, respectively (Figures 8E and 8G). These results suggest that VISA functions downstream of TRIF in TLR3-mediated signaling pathways. Previously, it has been shown that TBK1 and IKK⑀ are also involved in TLR3-mediated IFN signaling by their association with TRIF (Fitzgerald et al., 2003a; Hemmi et al., 2004; McWhirter et al., 2003; Perry et al., 2004; Sharma et al., 2003). Taken together, these observations suggest that VISA recruits IRF-3 to the TRIF complex in which IRF-3 is phosphorylated by TBK1 and/ or IKK⑀. Because the kinase-inactive mutant of TBK1 or IKK⑀ (or their combination) could not significantly inhibit VISA-mediated ISRE activation in reporter assays (Figure 8H and data not shown), this points to the possibility that VISA may be capable of activating an additional unidentified IRF-3 and/or IRF-7 kinase(s). Reporter assays with deletion mutants of VISA indicated that the N-terminal CARD domain was not required and the C-terminal domain (aa 360–540) was sufficient for activating ISRE (Figure 5D). One explanation for these observations is that the C-terminal domain of VISA can simultaneously recruit IRF-3 and TBK1/IKK⑀ and is therefore capable of activating ISRE when overexpressed in cells. Although the CARD domain of VISA can interact with RIG-I, this mutant may not be able to recruit IRF-3 and TBK1/IKK⑀ and there-

fore barely activates ISRE when overexpressed (Figure 5D). Previous studies have demonstrated that both IRF-3 and IRF-7 are required for efficient production of type I IFNs (Servant et al., 2002; Zhang and Pagano, 2002). However, their roles are different in these processes. In the early phase of viral infection, preexisting IRF-3 is activated and induces expression of IFN-β and IFN-α4. These early produced IFNs transcriptionally induce IRF-7, and upon viral infection, the induced high-level IRF-7 is activated and transactivates multiple IFN genes, leading to robust production of IFNs in response to viral infection. Recently, gene knockout studies have confirmed that IRF-7 plays a critical role in robust production of type I IFNs induced by viral infection in various cell types and by engagement of TLR9 in differentiated plasmacytoid dendritic cells (Honda et al., 2005a, 2005b). Previously, it has been demonstrated that IRF-7 is also phosphorylated by TBK1 and IKK⑀ (Han et al., 2004; tenOever et al., 2004). In the present study, we found that VISA could also interact with IRF-7 (Figure 7). These observations point to the possibility that VISA is also involved in signaling that leads to IRF-7 activation. Molecular Mechanisms of VISA-Mediated Activation of NF-␬B Our results suggest that VISA is also involved in virustriggered and TLR3-mediated activation of NF-κB. Several observations suggest that TRAF6 is required for VISA-mediated NF-κB, but not ISRE activation. Mutation of the TRAF binding motifs abolished the ability of VISA to activate NF-κB (Figure 5), suggesting that TRAF6 functions downstream of VISA in NF-κB activation. In TRAF6−/− MEFs, VISA-mediated NF-κB activation was dramatically inhibited in comparison to wt control MEFs (Figure 8C). In addition, dominant-negative mutants of TRAF6, TAK1, and IKKβ inhibited VISAmediated NF-κB activation (Figure 8F). These results suggest that VISA signals NF-κB activation through the TRAF6-TAK1-IKK cascade. In contrast, VISA still activated ISRE in TRAF6−/− MEFs, suggesting that TRAF6 is not required for VISAmediated ISRE activation (Figure 8D). In fact, VISA activates ISRE more potently in TRAF6−/− MEFs than in wt control MEFs (Figure 8D). It is possible that VISA-mediated NF-κB and ISRE activation pathways can compete with each other. Deficiency of TRAF6 causes loss of VISA-mediated NF-κB activation and enhancement of VISA-mediated ISRE activation. Taken together, our data suggest that VISA mediates bifurcation of virus-triggered and TLR3-mediated NFκB and ISRE activation pathways. VISA Is Not Involved in TLR4and TNF-Induced Signaling Knockdown of VISA did not inhibit TLR4-mediated IFN-β signaling (Figure 6). This is surprising because it was reported that TLR4 signals IFN-β activation through a TRIF and TRAM-containing complex (Fitzgerald et al., 2003b; Oshiumi et al., 2003b; Yamamoto et al., 2003). It is possible that the TRIF-TRAM complex can signal without VISA.

Molecular Cell 738

In a mammalian overexpression system, VISA can interact with TRAF2 (Figure 5). However, RNAi knockdown experiments suggest that VISA is not involved in TNF-induced NF-κB activation (Figure 6). Whether VISA interacts with TRAF2 under physiological conditions and whether VISA plays a role in signaling of unidentified pathways will be investigated in a separate study. Concluding Remarks In virus-triggered TLR3-independent signaling pathways, VISA interacts with RIG-I through its respective CARD modules and functions as an adaptor to recruit downstream components, such as IRF-3, to the RIG-I complex. In TLR3-mediated signaling pathways, VISA interacts with TRIF and TRAF6 and mediates bifurcation of the NF-κB and ISRE activation pathways. Our findings suggest that VISA plays essential roles in antiviral responses by participating two virus-triggered IFN signaling pathways. Experimental Procedures Reagents Recombinant human TNF (R&D Systems), poly(I:C) (Invivogen), Sendai virus (Charles River Laboratories), LPS (Sigma), monoclonal antibodies against FLAG (Sigma) and HA (Covance) epitopes, rabbit polyclonal anti-TRAF6 antibody (Santa Cruz Biotechnology), goat polyclonal anti-TRIF antibody (Imgenex), SAOS-2 and H1299 (ATCC), and 293-TLR4/MD2/CD14 cells (Invivogen) were purchased from the indicated manufacturers. Rabbit anti-RIG-I (Tadaatsu Imaizumi), 293 cells (Dr. Zhaodan Cao), 293-TLR3 cells (Drs. Katherine Fitzgerald and Tom Maniatis), and TRAF6−/− and wt MEFs (Drs. Tak Mak and Wen-Chen Yeh) were provided by the indicated investigators. TRIF-deficient mice were provided by Dr. Bruce Beutler, and TRIF−/− MEFs were prepared from embryos at day 10 of gestation.

were performed as previous described (Han et al., 2004; Jiang et al., 2004). Coimmunoprecipitation and Western Blot Analysis Transient transfection, coimmunoprecipitation, and Western blot analysis were performed as previous described (Shu et al., 1996). In Vitro Kinase Assay To determine whether IRF-3 is phosphorylated after overexpression of VISA and IKK⑀, 293 cells (w2 × 106) were cotransfected with expression plasmids for HA-VISA or HA-IKK⑀ (5 ␮g/each) for 16 hr. Cells were lysed, and the lysate was incubated with 25 ␮l of GSTIRF-3(380–427) bound glutathione Sepharose beads for 1 hr at 4°C. The beads were washed with lysis buffer and then kinase buffer (20 mM HEPES, [pH 7.4], 10 mM MgCl2, 0.1 mM sodium orthovanadate, 20 mM β-glycerophosphate, 1 mM DTT, and 50 mM NaCl). The beads were then incubated with 30 ␮l of kinase buffer in the presence of 1 ␮l of [32P]-γ-ATP at 30°C for 30 min. Kinase reactions were resolved by 10% SDS-PAGE. The gel was fixed in 30% methanol/10% acetic acid, dried, and exposed to X-ray film. EMSAs EMSAs were performed as previously described (Wu et al., 2003). The consensus NF-κB and ISRE oligonucleotides were purchased from Santa Cruz Biotechnology. RNAi Constructs The human VISA RNAi constructs were made by using the pSuper. retro vector (OligoEngine) according to protocols recommended by the manufacturer. The target sequences for the human VISA constructs were: #1, 5#-GACAAGACCTATAAGTATA-3#; #2, 5#-GACCTA TAAGTATATCTGC-3#; #3, 5#-GTATATCTGCCGCAATTTC-3#; #4, 5#ATGCAGAGAAACAGGAGTC-3#; #5, 5#-TTTCAGCAATTTTTGCAAT3#; #6, 5#-TTTTTGCAATGTGGATGTT-3#; and #7, 5#-GACAAGACC TATAAGTATA-3#.

Acknowledgments

Constructs NF-κB luciferase reporter construct was provided by Dr. Gary Johnson. ISRE and CHOP luciferase reporter constructs were purchased from Stratagene. Mammalian expression plasmids for TRAF2, TRAF6, IKKβ, and IKKβ-KA (Drs. David Goeddel and Zhaodan Cao); TBK1, TBK1-KA, IKK⑀, and IKK⑀-KA (Dr. Uli Siebenlist); and IRF-3 (Dr. John Hiscott) were provided by the indicated investigators. Mammalian expression plasmids for IRF-7, TRIF, and TRIF(1–394) (Han et al., 2004) and the IFN-β promoter luciferase reporter plasmid (Han et al., 2004) have been described. Mammalian expression plasmids for human HA- or FLAG-tagged VISA and RIG-I and their mutants were constructed by standard molecular biology techniques.

We thank Laurel Lenz for reading the manuscript and Bruce Beutler, Tak Mak, Wen-Chen Yeh, Tadaatsu Imaizumi, David Goeddel, John Hiscott, Rongtuan Lin, Gary Johnson, Uli Siebenlist, Katherine Fitzgerald, and Tom Maniatis for reagents. This work was supported by grants from the National Institutes of Health (R01 AI062739) and the Chinese High-Technology Program (#2003AA221030).

Antibody Preparation Human VISA-(1–180) and VISA-(181–360) cDNAs were constructed in pET32a vector (Novagen). The recombinant proteins were expressed in E. coli and purified by nickel column chromatography. The recombinant proteins were injected into rabbits to produce antisera against VISA-(1–180) and VISA-(181–360), respectively.

Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A. (2001). Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732–738.

Northern Blot Hybridization Human multiple tissue mRNA blot was purchased from Clontech. The blot was hybridized with P32-dCTP-labeled cDNA probe corresponding to human VISA coding sequence. Hybridization was performed in the Rapid Hybridization Buffer (Clontech) under highstringency conditions. Reporter Gene Assays 293 cells and their derivatives (w2 × 105) were seeded in 12-well dishes and transfected the following day by standard calcium phosphate precipitation method. Wt, TRAF6−/−, and TRIF−/− MEFs (w1 × 105) were seeded in 24-well dishes and transfected the following day with Lipofectamine 2000 (Invitrogen). Reporter assays

Received: July 26, 2005 Revised: August 8, 2005 Accepted: August 15, 2005 Published online: September 8, 2005 References

Arch, R.H., Gedrich, R.W., and Thompson, C.B. (2000). Translocation of TRAF proteins regulates apoptotic threshold of cells. Biochem. Biophys. Res. Commun. 272, 936–945. Barr, F.A., Sillje, H.H., and Nigg, E.A. (2004). Polo-like kinases and the orchestration of cell division. Nat. Rev. Mol. Cell Biol. 5, 429– 440. Boucher, L.M., Marengere, L.E., Lu, Y., Thukral, S., and Mak, T.W. (1997). Binding sites of cytoplasmic effectors TRAF1, 2, and 3 on CD30 and other members of the TNF receptor superfamily. Biochem. Biophys. Res. Commun. 233, 592–600. Bradley, J.R., and Pober, J.S. (2001). Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20, 6482–6491. Brodeur, S.R., Cheng, G., Baltimore, D., and Thorley-Lawson, D.A. (1997). Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs. J. Biol. Chem. 272, 19777–19784.

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Brown, K.D., Hostager, B.S., and Bishop, G.A. (2001). Differential signaling and tumor necrosis factor receptor-associated factor (TRAF) degradation mediated by CD40 and the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1). J. Exp. Med. 193, 943–954. Durbin, J.E., Fernandez-Sesma, A., Lee, C.K., Rao, T.D., Frey, A.B., Moran, T.M., Vukmanovic, S., Garcia-Sastre, A., and Levy, D.E. (2000). Type I IFN modulates innate and specific antiviral immunity. J. Immunol. 164, 4220–4228. Fitzgerald, K.A., McWhirter, S.M., Faia, K.L., Rowe, D.C., Latz, E., Golenbock, D.T., Coyle, A.J., Liao, S.M., and Maniatis, T. (2003a). IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496. Fitzgerald, K.A., Rowe, D.C., Barnes, B.J., Caffrey, D.R., Visintin, A., Latz, E., Monks, B., Pitha, P.M., and Golenbock, D.T. (2003b). LPSTLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J. Exp. Med. 198, 1043–1055. Fotin-Mleczek, M., Henkler, F., Samel, D., Reichwein, M., Hausser, A., Parmryd, I., Scheurich, P., Schmid, J.A., and Wajant, H. (2002). Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8. J. Cell Sci. 115, 2757–2770. Han, K.J., Su, X., Xu, L.G., Bin, L.H., Zhang, J., and Shu, H.B. (2004). Mechanisms of the TRIF-induced interferon-stimulated response element and NF-kappaB activation and apoptosis pathways. J. Biol. Chem. 279, 15652–15661. Hemmi, H., Takeuchi, O., Sato, S., Yamamoto, M., Kaisho, T., Sanjo, H., Kawai, T., Hoshino, K., Takeda, K., and Akira, S. (2004). The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. J. Exp. Med. 199, 1641–1650. Hoebe, K., Du, X., Georgel, P., Janssen, E., Tabeta, K., Kim, S.O., Goode, J., Lin, P., Mann, N., Mudd, S., et al. (2003). Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424, 743–748. Honda, K., Sakaguchi, S., Nakajima, C., Watanabe, A., Yanai, H., Matsumoto, M., Ohteki, T., Kaisho, T., Takaoka, A., Akira, S., et al. (2003). Selective contribution of IFN-alpha/beta signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100, 10872–10877. Honda, K., Ohba, Y., Yanai, H., Negishi, H., Mizutani, T., Takaoka, A., Taya, C., and Taniguchi, T. (2005a). Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434, 1035–1040. Honda, K., Yanai, H., Negishi, H., Asagiri, M., Sato, M., Mizutani, T., Shimada, N., Ohba, Y., Takaoka, A., Yoshida, N., and Taniguchi, T. (2005b). IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434, 772–777. Jiang, Z., Mak, T.W., Sen, G., and Li, X. (2004). Toll-like receptor 3-mediated activation of NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-beta. Proc. Natl. Acad. Sci. USA 101, 3533–3538. Levy, D.E., and Garcia-Sastre, A. (2001). The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. Cytokine Growth Factor Rev. 12, 143–156. Levy, D.E., and Marie, I.J. (2004). RIGging an antiviral defense—it’s in the CARDs. Nat. Immunol. 5, 699–701. Lin, R., Mamane, Y., and Hiscott, J. (1999). Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol. Cell. Biol. 19, 2465– 2474. Maniatis, T., Falvo, J.V., Kim, T.H., Kim, T.K., Lin, C.H., Parekh, B.S., and Wathelet, M.G. (1998). Structure and function of the interferonbeta enhanceosome. Cold Spring Harb. Symp. Quant. Biol. 63, 609–620. Matsuda, A., Suzuki, Y., Honda, G., Muramatsu, S., Matsuzaki, O., Nagano, Y., Doi, T., Shimotohno, K., Harada, T., Nishida, E., et al. (2003). Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways. Oncogene 22, 3307–3318.

McWhirter, S.M., Fitzgerald, K.A., Rosains, J., Rowe, D.C., Golenbock, D.T., and Maniatis, T. (2003). IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl. Acad. Sci. USA 101, 233–238. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T., and Seya, T. (2003a). TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat. Immunol. 4, 161–167. Oshiumi, H., Sasai, M., Shida, K., Fujita, T., Matsumoto, M., and Seya, T. (2003b). TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta. J. Biol. Chem. 278, 49751–49762. Perry, A.K., Chow, E.K., Goodnough, J.B., Yeh, W.C., and Cheng, G. (2004). Differential requirement for TANK-binding kinase-1 in type I interferon responses to toll-like receptor activation and viral infection. J. Exp. Med. 199, 1651–1658. Sato, S., Sugiyama, M., Yamamoto, M., Watanabe, Y., Kawai, T., Takeda, K., and Akira, S. (2003). Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) associates with TNF receptorassociated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-kappa B and IFN-regulatory factor-3, in the Toll-like receptor signaling. J. Immunol. 171, 4304– 4310. Servant, M.J., Tenoever, B., and Lin, R. (2002). Overlapping and distinct mechanisms regulating IRF-3 and IRF-7 function. J. Interferon Cytokine Res. 22, 49–58. Sharma, S., tenOever, B.R., Grandvaux, N., Zhou, G.P., Lin, R., and Hiscott, J. (2003). Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151. Shu, H.B., Takeuchi, M., and Goeddel, D.V. (1996). The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc. Natl. Acad. Sci. USA 93, 13973–13978. tenOever, B.R., Servant, M.J., Grandvaux, N., Lin, R., and Hiscott, J. (2002). Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J. Virol. 76, 3659–3669. tenOever, B.R., Sharma, S., Zou, W., Sun, Q., Grandvaux, N., Julkunen, I., Hemmi, H., Yamamoto, M., Akira, S., Yeh, W.C., et al. (2004). Activation of TBK1 and IKKvarepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity. J. Virol. 78, 10636– 10649. Wajant, H., Henkler, F., and Scheurich, P. (2001). The TNF-receptorassociated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. Cell. Signal. 13, 389–400. Wathelet, M.G., Lin, C.H., Parekh, B.S., Ronco, L.V., Howley, P.M., and Maniatis, T. (1998). Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol. Cell 1, 507–518. Weaver, B.K., Kumar, K.P., and Reich, N.C. (1998). Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol. Cell. Biol. 18, 1359–1368. Wu, M., Xu, L.G., Zhai, Z., and Shu, H.B. (2003). SINK is a p65interacting negative regulator of NF-kappaB-dependent transcription. J. Biol. Chem. 278, 27072–27079. Yamamoto, M., Sato, S., Mori, K., Hoshino, K., Takeuchi, O., Takeda, K., and Akira, S. (2002). Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J. Immunol. 169, 6668–6672. Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., Sugiyama, M., Okabe, M., Takeda, K., and Akira, S. (2003). Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301, 640–643. Ye, H., Arron, J.R., Lamothe, B., Cirilli, M., Kobayashi, T., Shevde, N.K., Segal, D., Dzivenu, O.K., Vologodskaia, M., Yim, M., et al. (2002). Distinct molecular mechanism for initiating TRAF6 signalling. Nature 418, 443–447.

Molecular Cell 740

Yoneyama, M., Suhara, W., Fukuhara, Y., Fukuda, M., Nishida, E., and Fujita, T. (1998). Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J. 17, 1087–1095. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., and Fujita, T. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNAinduced innate antiviral responses. Nat. Immunol. 5, 730–737. Zhang, L., and Pagano, J.S. (2002). Structure and function of IRF-7. J. Interferon Cytokine Res. 22, 95–101. Accession Numbers The GenBank accession numbers for the human and mouse VISA mRNAs are DQ167126 and DQ167127, respectively.