title: Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors.
abstract: During viral infection, the innate immune RIG-I like receptors (RLRs) recognize viral double stranded RNA (dsRNA) and trigger filament assembly of the adaptor protein Mitochondrial Anti-viral Signaling protein (MAVS). The MAVS filament then activates anti-viral signaling events including the up-regulation of type I interferon expression. In recent years, much insight has been gained into how RLRs recognize dsRNA, but the precise mechanism of how activated RLRs stimulate MAVS filament formation remains less understood. In this chapter, we describe an in vitro reconstitution assay that we have previously developed to study the RLR-catalyzed filament assembly of MAVS. We provide technical guidance for purifying the caspase activation recruitment domain (CARD) of MAVS (MAVS(CARD)) as a functional monomer and also preformed filament seed. We also describe the methods to monitor the monomer-to-filament transition of MAVS(CARD) upon stimulation. This protocol provides a minimalist approach to studying RLR signaling events and can potentially be applied to elucidate signaling mechanisms of other innate immune receptors, such as Toll-like receptors and inflammasomes, that involve higher order assemblies of CARDs or related domains for their downstream signal activation.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, 3 Blackfan Circle, 3rd Floor, Boston, MA, 02115, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, 3 Blackfan Circle, 3rd Floor, Boston, MA, 02115, USA.
authors.forename: Yu-San
authors.initials: YS
authors.lastname: Huoh
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA. Sun.Hur@childrens.harvard.edu.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, 3 Blackfan Circle, 3rd Floor, Boston, MA, 02115, USA. Sun.Hur@childrens.harvard.edu.
authors.forename: Sun
authors.initials: S
authors.lastname: Hur
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D011485:Protein Binding; D011971:Receptors, Immunologic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C505371:MAVS protein, human; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases
keywords: Caspase activation recruitment domain (CARD); Filament assembly; Filament formation; IFNα/β signaling pathway; Innate immune signaling; MAVS; MDA5; Protein refolding; RIG-I