Cooperative assembly and dynamic disassembly of MDA5 filaments for viral dsRNA recognition

Abstract

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...

MDA5 assembles into a polar helical filament on dsRNA

“…Third, given that filament formation is inhibited by the ATPase activity of MDA5 (8), and that ATPase-deficient mutants of MDA5 exhibit constitutive signaling in cells (18), filaments-or filamentlike structures-may be involved in signaling. Similarly, a natural partial loss-of-function mutant in the MDA5 CTD (I923V) reduces binding cooperativity and filament formation in vitro (9) while reducing signaling in vivo (19,20), supporting a physiological role for filaments in signaling. The discovery that MDA5 polymerizes into helical filaments along dsRNA supports a signal transduction mechanism in which the helical array of MDA5 CARDs on the outside of MDA5-RNA filaments recruits MAVS and nucleates its assembly into an active fibril form.…”

“…RNA binding through the helicase domain and CTD (5, 6) releases the CARDs, which then recruit unanchored lysine 63-linked polyubiquitin chains and activate the signaling adaptor mitochondrial antiviral-signaling protein (MAVS, also called interferon-β promoter stimulator 1, or IPS-1) (7). In contrast, MDA5 does not sequester its CARDs (8) and cooperatively assembles into ATP-sensitive filaments on dsRNA (8,9). Moreover, the MDA5 CTD is required for cooperative filament assembly, but not for RNA binding (8,10,11).…”

Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy

“…In contrast to the previous model in which IFI16·dsDNA complexes resemble noninteracting beads on a string (9), our binding experiments suggested that IFI16 assembles into distinct oligomeric clusters on dsDNA. These two models can be distinguished by monitoring formation of FAM-dsDNA·IFI16 complexes in the presence of increasing amounts of unlabeled DNA using native gel electrophoretic mobility-shift assays (EMSAs): the clustering mechanism would show a concerted transition without resulting in significant intermediates, whereas the noninteracting mechanism would clearly display intermediate species (25,26). We first performed EMSAs with various FAM-dsDNA fragments.…”

“…Conventionally, assembling filaments on foreign nucleic acids has been considered as a defense strategy reserved for sensing intracellular RNA (28,42). For example, a principal mechanism by which melanoma differentiation associated protein (MDA)5 distinguishes self-from nonself-RNA is via cooperatively assembling into filaments along the length of long dsRNA with its two CARDs forming clusters tracing the center of filaments in a helical trajectory (25,26,(42)(43)(44)(45). IFI16 and MDA5 are unrelated proteins, and, not surprisingly, mechanisms allowing these proteins to assemble into filaments are significantly different.…”

Viral counterattack against the host innate immune system

“…We found that MDA5 assembles into linear filaments along the length of dsRNA and that this filament formation is required for antiviral signal activation as it promotes oligomerization of the signaling domain, caspase activation recruitment domain (CARD) [4,5]. Furthermore, filament formation allows MDA5 to regulate its interaction with dsRNA according to the length of dsRNA as it disassembles from filament termini during ATP hydrolysis [6].…”