The RIG-I-like Receptor LGP2 Recognizes the Termini of Double-stranded RNA

Abstract

The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded RNA with 5 triphosphates and doublestranded RNA (dsRNA) to initiate innate antiviral immune responses.LGP2, a homolog of RIG-I and MDA5 that lacks signaling capability, regulates the signaling of the RLRs. To establish the structural basis of dsRNA recognition by the RLRs, we have determined the 2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of dsRNA with minimal contacts between the protein molecules. Gel filtration chromatography and analytical ultracentrifugation demonstrated that LGP2 binds blunt-ended dsRNA of different lengths, forming complexes with 2:1 stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do not stimulate the activation of RIG-I efficiently in cells. Surprisingly, full-length LGP2 containing mutations that abolish dsRNA binding retained the ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading pathogens; it is the ubiquitous system of defense against microbial infections (1). Toll-like receptors (TLRs) 3 and RIG-I (retinoic acid-inducible gene 1)-like receptors (RLRs) play key roles in innate immune response toward viral infection (2-5). Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the endosome following phagocytosis of the pathogens (6). RIG-I-like receptors RIG-I and MDA5 detect viral RNA from replicating viruses in infected cells (3,7,8). Stimulation of these receptors leads to the induction of type I interferons (IFNs) and other proinflammatory cytokines, conferring antiviral activity to the host cells and activating the acquired immune responses (4, 9).RIG-I discriminates between viral and host RNA through specific recognition of the uncapped 5Ј-triphosphate of singlestranded RNA (5Ј ppp ssRNA) generated by viral RNA polymerases (10, 11). In addition, RIG-I also recognizes doublestranded RNA generated during RNA virus replication (7,12). Transfection of cells with synthetic double-stranded RNA stimulates the activation of RIG-I (13, 14). Synthetic dsRNA mimics, such as polyinosinic-polycytidylic acid (poly(I⅐C)), can activate MDA5 when introduced into the cytoplasm of cells. Digestion of poly(I⅐C) with RNase III transforms poly(I⅐C) from a ligand for MDA5 into a ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I recognizes short dsRNA (15). Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of these receptors in antiviral immune responses and demonstrated that they sense different sets of RNA viruses (12, 16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N termini, a DEX(D/H) box RNA helicase domain, and a C-terminal regulatory or repressor domain (CTD). The helicase domain and the CTD are responsible for viral RNA binding, whereas the CARDs are required for signaling (3, 8). The current model of RIG-I activation suggests that under resting ...

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

“…The FLAG‐tagged CTD of LGP2 was able to co‐IP Dicer, unlike two different NTD fragments (Fig 2B). Mutation of a conserved lysine residue near the C‐terminus of LGP2 (K634), which has previously been shown to prevent RNA binding (Li et al , 2009; Pippig et al , 2009), reduced the association of the CTD of LGP2 with Dicer (Fig 2C). However, mutation of the same residue within the context of the full‐length protein weakened but did not abolish binding to Dicer, suggesting that the N‐terminal helicase domain, although by itself unable to bind Dicer, stabilises the interaction between the CTD and Dicer.…”

“…Importantly, the inhibitory effect was unique to LGP2, as inclusion of FLAG‐tagged MDA5 or FLAG‐tagged RIG‐I had little effect on dicing efficiency (Figs 4C and EV3C). In contrast to full‐length LGP2, purified LGP2 CTD only modestly decreased in vitro dicing of dsRNA despite its ability to associate with Dicer and to bind dsRNA with high affinity (Li et al , 2009; Pippig et al , 2009; Figs 4D and EV3D). Furthermore, there was little difference between the LGP2 CTD and a CTD K634E mutant, which completely lacks RNA binding.…”

The TLR‐independent DNA recognition pathway in murine macrophages: Ligand features and molecular signature

“…Given the facts that RIG-I detects 5 0 -triphosphate-containg RNA [6,7] and Lgp2 binds to the termini of dsRNA [19], we raised the hypothesis that the 5 0 -end of DNA may be a critical determinant for cytosolic recognition. To test this hypothesis, we generated four different ISD variants: (i) ISD containing a free 5 0 -hydroxyl group (ISD-OH), (ii) ISD in vitro phosphorylated at the 5 0 -end (ISD-P; [20]), (iii) ISD blocked at the 5 0 -end by addition of a biotin group (ISD-Bio) and (iv) ISD blocked at the 5 0 -end by addition of an amino group (ISD-NH2).…”

ATP Hydrolysis Enhances RNA Recognition and Antiviral Signal Transduction by the Innate Immune Sensor, Laboratory of Genetics and Physiology 2 (LGP2)

“…LGP2 mutant K651E was based on the observation that mutation of LGP2 lysine 651 to glutamic acid completely disrupts RNA recognition by the isolated C-terminal RD (20).…”