Volume 19, Issue 6, Pages 727-740 (September 2005) VISA Is an Adapter Protein Required for Virus-Triggered IFN-β Signaling  Liang-Guo Xu, Yan-Yi Wang, Ke-Jun Han, Lian-Yun Li, Zhonghe Zhai, Hong-Bing Shu  Molecular Cell  Volume 19, Issue 6, Pages 727-740 (September 2005) DOI: 10.1016/j.molcel.2005.08.014 Copyright © 2005 Elsevier Inc. Terms and Conditions

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-VISA-transfected 293 cells was loaded. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼2 × 105) were transfected with an NF-κB luciferase plasmid (0.1 μg) and the indicated mammalian expression plasmids (0.5 μg 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 IKKϵ expression) antibodies, respectively (middle panels). The unphosphorylated (un-phos.), 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 (∼2 × 105) were transfected with pFR plasmid (0.5 μg), pFA-CHOP plasmid (0.05 μg), RSV-β-gal plasmid (0.1 μg), and the indicated mammalian expression plasmid (0.5 μg). 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. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼2 × 105) were transfected with an ISRE luciferase plasmid (0.1 μg), pSuper control, or pSuper-VISA-RNAi plasmids (1 μg). 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 (∼2 × 105) were transfected with pSuper or pSuper-VISA-RNAi (#3) plasmid (2 μg). 12 hr after transfection, cells were selected with puromycin (1 μg/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 μg/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. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 VISA Is Required for RIG-I-Mediated Signaling (A) VISA recruits IRF-3 to RIG-I. 293 cells (∼2 × 105) were transfected with the indicated expression plasmids (5 μg 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 (∼1 × 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 (∼2 × 105) were transfected with an ISRE luciferase plasmid (0.1 μg), an empty or RIG-I expression plasmid (0.5 μg), and a control or VISA RNAi plasmid (1.0 μg) 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. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼2 × 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 (∼2 × 105) were transfected with the indicated luciferase reporter construct (0.1 μg) and mammalian expression plasmids (0.5 μg 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. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼2 × 105) were transfected with the indicated luciferase construct (0.1 μg) and a control or VISA RNAi plasmid (1 μg each). 40 hr after transfection, cells were treated with poly(I:C) (25 μg/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 (∼2 × 105) were transfected with the indicated luciferase reporter construct (0.1 μg) and control or VISA RNAi plasmid (1 μg each). 40 hr after transfection, cells were treated with LPS (10 μg/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 (∼2 × 105) were transfected with an NF-κB luciferase construct (0.1 μg) and a control or VISA RNAi plasmid (1 μg each). 40 hr after transfection, cells were treated with TNF (10 μg/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. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼2 × 106) were transfected with the indicated plasmids (5 μg 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 (∼2 × 107) were treated with poly (I:C) (25 μg/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 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

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 (∼1 × 105) were transfected with an NF-κB luciferase plasmid (0.5 μg) and an empty control (empty bars) or VISA expression plasmid (filled bars) (1 μg 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 VISAΔT2T6 on TRIF- and TRAF6-mediated NF-κB activation. 293 cells were transfected with an NF-κB luciferase plasmid (0.1 μg), an empty or TRIF (0.2 μg, filled bars) or TRAF6 (0.2 μg, empty bars) expression plasmid, and increased amounts of VISAΔT2T6 expression plasmid (0, 0.1, 0.2, and 0.4 μg). Reporter assays were performed 18 hr after transfection. (F) Effects of various dominant-negative mutants on VISA-mediated NF-κB activation. 293 cells (∼2 × 105) were transfected with an NF-κB luciferase plasmid (0.1 μg), an empty or VISA expression plasmid (0.2 μg), and expression plasmids for the indicated dominant-negative mutants (0.2 μg). 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 pSuper-VISA-RNAi were transfected with ISRE luciferase plasmid (0.1 μg) and the indicated expression plasmids (0.5 μg each). Reporter assays were performed 18 hr after transfection. (H) Effects of various dominant-negative mutants on VISA-mediated ISRE activation. 293 cells were transfected with an ISRE luciferase reporter plasmid (0.1 μg), an empty or VISA expression plasmid (0.2 μg), and the indicated dominant-negative mutant plasmids (0.2 μg each). Reporter assays were performed 18 hr after transfection. The results of reporter assays in (A)–(H) are averages of three replicates ± SD. Molecular Cell 2005 19, 727-740DOI: (10.1016/j.molcel.2005.08.014) Copyright © 2005 Elsevier Inc. Terms and Conditions