Essential Role of the N-terminal Domain in the Regulation of RIG-I ATPase Activity

Abstract

Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-␤ expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.Pathogen recognition receptors constitute one of the first lines of human host cell defense against viral infections. Among them, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) helicases are specialized sensors of viral nucleic acid. RIG-I was shown to be involved in the recognition of Sendai virus, Hepatitis C virus (HCV), 2 and vesicular stomatitis virus, whereas MDA-5 mediates the antiviral response to picornaviruses (1-3). Both RIG-I and MDA-5 are modular proteins of ϳ920 residues that contain an ATPase/helicase domain for RNA binding and two caspase activation and recruitment domains (CARDs) located in tandem at the N-terminal end of the protein (4). The CARDs, by way of homotypic interactions, are required for the signaling function between RIG-I and the newly discovered interferon-␤ promoter stimulator-1 (IPS-1), also called Cardif, MAVS, and VISA (for review see Ref. 5). The latter serves as an intermediate to the cascade leading to the activation of NF-B and IRF-3/7, which results in the production of antiviral cytokines such as type-1 interferons (6 -9). The receptor function of RIG-I is non-redundant and therefore essential to coordinate the signaling cascade leading to antiviral response (4), as confirmed by knock-out studies (10). Notably, the Huh7.5 cell line of hepatocytes is particularly permissive to HCV and vesicular stomatitis virus infection as the result of a defective RIG-I protein bearing a single mutation in its first CARD (11); the antiviral sig...

Evolutional Conservation of Molecular Structure and Antiviral Function of a Viral RNA Receptor, LGP2, in Japanese Flounder, Paralichthys olivaceus

“…8). Moreover, mutation studies revealed that a lysine (Lys) residue in motif I of the RNA helicase in human LGP2 and RIG-I (Lys30 in LGP2 or Lys270 in RIG-I) are involved in signal transduction (6,17). In the motif I of Japanese flounder LGP2, Lys30 of human LGP2 appears to be conserved in Japanese flounder LGP2, where it is Lys33 (Fig.…”

“…The DExD/H box RNA helicase domain includes two ATP-binding motifs (i.e., DExD/H box), which are also known as "Walker motif". The RNA helicase domain of RIG-I can bind to both dsRNA and ssRNA in an ATP-dependent manner (6)…”

“…The CARD of RIG-I and MDA5 interacts with CARD of IPS-1 (INF-b promoter stimulator 1, also known as MAVS, VISA, and Cardif) (3,4,7), and the interaction initiates downstream signaling to produce type I IFNs through promoting phosphorylation of IFN regulatory factor (IRF)-3 and IRF-7 (IRF-3/7) (2,8) and induction of NF-kB target genes (3). The CARDs usually bind to the regulatory domain (RD; also known as "repressor domain") to prevent the signal transduction in the absence of viral RNA (6,9). Recent structural analyses of C-terminal RLRs have shown that the C-terminal RD-which is conserved in RIG-I, MDA5, and LGP2-plays a role in RNA recognition (10)(11)(12).…”

Potential for all-trans retinoic acid [tretinoin] to enhance interferon-alpha treatment response in chronic myelogenous leukemia, melanoma, myeloma, and renal cell carcinoma

“…31,34-36 (G) P-IRF-3 or P-IRF-7 homodimerize. 22,23,37,38 Either homodimer is a promoter for Type 1 interferons' genes' transcription, 37,38 resulting in increased translation and increased Type 1 interferon protein. Note however that the spectrum of Type I interferons (and other genes) induced by each homodimer is somewhat different.…”

Cellular models for the screening and development of anti-hepatitis C virus agents

“…A recent report identified the PolyU/UC region of the 3'-NTR of HCV as the major pathogen-associated molecular patterns of this virus (Saito et al, 2008). Recent work suggested that in the absence of RNA, RIG-I is inhibited by intra-molecular interactions between the CARD and C-terminus domains, while RNA binding strongly stimulates ATPase activity and signaling (Gee et al, 2008). RIG-I activates the mitochondria-bound protein, IPS-1/MAVS/VISA/Cardif protein (Hiscott et al, 2006) (referred to here as MAVS) through the N-terminal tandem CARD of RIG-I and the Nterminal CARD of MAVS (T. Saito et al, 2007;Vitour & Meurs, 2007).…”