Ubiquitin-Induced Oligomerization of the RNA Sensors RIG-I and MDA5 Activates Antiviral Innate Immune Response

Abstract

SUMMARY RIG-I and MDA5 detect viral RNA in the cytoplasm and activate signaling cascades leading to the production of type-I interferons. RIG-I is activated through sequential binding of viral RNA and unanchored lysine-63 (K63) polyubiquitin chains, but how polyubiquitin activates RIG-I and whether MDA5 is activated through a similar mechanism remain unresolved. Here we showed that the CARD domains of MDA5 bound to K63 polyubiquitin and that this binding was essential for MDA5 to activate the transcription factor IRF3. Mutations of conserved residues in MDA5 and RIG-I that disrupt their ubiquitin binding also abrogated their ability to activate IRF3. Polyubiquitin binding induced the formation of a large complex consisting of four RIG-I and four ubiquitin chains. This hetero-tetrameric complex was highly potent in activating the antiviral signaling cascades. These results suggest a unified mechanism of RIG-I and MDA5 activation and reveal a unique mechanism by which ubiquitin regulates cell signaling and immune response.

Toward a crystal-clear view of the viral RNA sensing and response by RIG-I-like receptors

“…7,42,43 Ubiquitin or phosphorylation-mediated post-translational modifications further potentiate RIG-I activation. 16,40,44 Lastly, multiple copies of activated RIG-I:RNA complex are probably needed to activate the adaptor protein MAVS.…”

“…47,50 This cooperativity is necessary for activation of MDA5, as MDA5 displays poor cellular activity on short RNA species. 40,45,46 The structure of MDA5 lacking the CARDs in complex with a 12mer RNA duplex exhibits several differences from those of similar RIG-I:RNA complexes: first, the MDA5 CTD in the structure binds only to the backbone of the RNA duplex but not to the end (Fig. 1); 50 second, unlike the RIG-I CTD, mutations that remove the RNA capping loop from the CTD of MDA5 do not weaken RNA binding of MDA5.…”

MDA5 assembles into a polar helical filament on dsRNA

“…Moreover, the MDA5 CTD is required for cooperative filament assembly, but not for RNA binding (8,10,11). The MDA5 CARDs have been proposed to nucleate the assembly of MAVS into its active fibril form (8,12) in a process involving free polyubiquitin chains (13). The prion-or amyloidlike properties of MAVS fibrils amplify signaling, culminating in activation of NF-κB (12).…”

RNF122 suppresses antiviral type I interferon production by targeting RIG-I CARDs to mediate RIG-I degradation

“…Riplet has been shown to mediate the K63-linked polyubiquitination of the C-terminal region of RIG-I. In addition, TRIM25 and MEX3C have both been shown to mediate the K63-linked ubiquitination of RIG-I CARDs at lysine 172, 99, or 169, respectively (10,(16)(17)(18)(19). Different E3 ubiquitin protein ligases mediate different ubiquitination sites of RIG-I, indicating that the coordinated regulation of these molecules is required for the RIG-I-mediated antiviral immune responses.…”

“…E3 ubiquitin ligases have been reported to be involved in the regulation of RIG-I signaling (14,15). For example, E3 ubiquitin ligases TRIM25, Riplet, and MEX3C can mediate K63-linked polyubiquitination of human RIG-I (10,(16)(17)(18)(19), thus positively regulating RIG-I-mediated signaling. On the contrary, RING finger protein (RNF) 125 (RNF125) or c-Casitas B lineage lymphoma (c-Cbl) catalyzes K48-linked ubiquitination and promotes proteasomal degradation of RIG-I, thus negatively regulating RIG-I-mediated signaling (20,21).…”