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.
