MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure of MDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.

Ubiquitin (Ub) has important roles in a wide range of intracellular signalling pathways. In the conventional view, ubiquitin alters the signalling activity of the target protein through covalent modification, but accumulating evidence points to the emerging role of non-covalent interaction between ubiquitin and the target. In the innate immune signalling pathway of a viral RNA sensor, RIG-I, both covalent and non-covalent interactions with K63-linked ubiquitin chains (K63-Ubn) were shown to occur in its signalling domain, a tandem caspase activation and recruitment domain (hereafter referred to as 2CARD)1,2. Non-covalent binding of K63-Ubn to 2CARD induces its tetramer formation, a requirement for downstream signal activation3. Here we report the crystal structure of the tetramer of human RIG-I 2CARD bound by three chains of K63-Ub2. 2CARD assembles into a helical tetramer resembling a ‘lock-washer’, in which the tetrameric surface serves as a signalling platform for recruitment and activation of the downstream signalling molecule, MAVS. Ubiquitin chains are bound along the outer rim of the helical trajectory, bridging adjacent subunits of 2CARD and stabilizing the 2CARD tetramer. The combination of structural and functional analyses reveals that binding avidity dictates the K63-linkage and chain-length specificity of 2CARD, and that covalent ubiquitin conjugation of 2CARD further stabilizes the Ub–2CARD interaction and thus the 2CARD tetramer. Our work provides unique insights into the novel types of ubiquitin-mediated signal-activation mechanism, and previously unexpected synergism between the covalent and non-covalent ubiquitin interaction modes.

MDA5, an RIG-I-like helicase, is a conserved cytoplasmic viral RNA sensor, which recognizes dsRNA from a wide-range of viruses in a length-dependent manner. It has been proposed that MDA5 forms higher-order structures upon viral dsRNA recognition or during antiviral signaling, however the organization and nature of this proposed oligomeric state is unknown. We report here that MDA5 cooperatively assembles into a filamentous oligomer composed of a repeating segmental arrangement of MDA5 dimers along the length of dsRNA. Binding of MDA5 to dsRNA stimulates its ATP hydrolysis activity with little coordination between neighboring molecules within a filament. Individual ATP hydrolysis in turn renders an intrinsic kinetic instability to the MDA5 filament, triggering dissociation of MDA5 from dsRNA at a rate inversely proportional to the filament length. These results suggest a previously unrecognized role of ATP hydrolysis in control of filament assembly and disassembly processes, thereby autoregulating the interaction of MDA5 with dsRNA, and provides a potential basis for dsRNA length-dependent antiviral signaling. (1). Upon viral RNA recognition, RIG-I and MDA5 interact with a common signaling adaptor, MAVS and activate NF-kB and IRF3/7 signaling pathways, resulting in the expression of type I interferon and proinflammatory cytokines (2).Previous studies suggested that RIG-I and MDA5 recognize largely distinct groups of viruses through their divergent RNA specificity (3, 4). RIG-I detects a variety of positive and negative strand viruses through recognition of a 5′ triphosphate group and blunt ends of genomic RNAs (3, 5, 6). By contrast, MDA5 detects several positive strand and dsRNA viruses such as picornaviruses and reoviruses through recognition of dsRNA replication intermediates and genomic dsRNAs (3,4,7). This viral RNA recognition by MDA5 is independent of 5′ triphosphate or blunt end, but instead depends on dsRNA length in a range of approximately 1-7 kb (8).The precise mechanism for length-dependent dsRNA recognition by MDA5 is poorly understood. Previous studies suggest that ATP hydrolysis could play an important role in both interferon signaling and RNA specificity (1, 2). The current model of MDA5 (or RIG-I) mediated signal activation is that binding of viral RNA to MDA5 triggers ATP hydrolysis in the conserved helicase domain, which then alters the receptor conformations or accessibility of the signaling domain (a tandem caspase activation recruitment domain) to allow its interaction with MAVS (1, 9). The proposed role of ATP hydrolysis as a conformational switch for signaling is supported by the requirement of ATP in the reconstituted signaling system of RIG-I (9, 10) and the observation that mutations in the active site for ATP hydrolysis either constitutively activate or inactivate interferon signaling by 12). In addition, induction of ATP hydrolysis by dsRNA has been shown to correlate with stimulation of interferon signaling (6, 13).It has been proposed that MDA5 (and RIG-I) forms a higher-order st...