Regulation of Signal Transduction by Enzymatically Inactive Antiviral RNA Helicase Proteins MDA5, RIG-I, and LGP2

Abstract

Intracellular pattern recognition receptors MDA5, RIG-I, andLGP2 are essential components of the cellular response to virus infection and are homologous to the DEXH box subfamily of RNA helicases. However, the relevance of helicase activity in the regulation of interferon production remains elusive. To examine the importance of the helicase domain function for these signaling proteins, a series of mutations targeting conserved helicase sequence motifs were analyzed for enzymatic activity, RNA binding, interferon induction, and antiviral signaling. Results indicate that all targeted motifs are required for ATP hydrolysis, but a subset is involved in RNA binding. The enzymatically inactive mutants differed in their signaling ability. Notably, mutations to MDA5 motifs I, III, and VI and RIG-I motif III produced helicase proteins with constitutive antiviral activity, whereas mutations in RIG-I motif V retained ATP hydrolysis but failed to mediate signal transduction. These findings demonstrate that type I interferon production mediated by full-length MDA5 and RIG-I is independent of the helicase domain catalytic activity. In addition, neither enzymatic activity nor RNA binding was required for negative regulation of antiviral signaling by LGP2, supporting an RNA-independent interference mechanism.The first line of defense against virus infection is provided by the cellular antiviral response and innate immune system. The immediate response includes the induction of type I interferon (IFN␣ and IFN␤, referred to herein as IFN 2 ) and other cytokines. Virus infections are sensed by cellular receptors that can recognize pathogen-associated molecular patterns such as viral nucleic acids (1). In addition to the transmembrane Toll-like receptors (2), a family of cytoplasmic RNA helicases has been identified that can detect cytosolic non-self RNA. Two members of this group are MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I) (3).These proteins are characterized by the combination of two caspase activation and recruitment domain (CARD) motifs linked to an RNA helicase domain.MDA5 and RIG-I can detect foreign RNAs and transmit a signal through the CARD-containing mitochondrial adaptor molecule IPS-1 (also named Cardif, MAVS, and VISA) (4 -7). IPS-1 apparently serves as a scaffold for propagation of the signaling cascade that leads to the activation of transcription factors, including IRF-3 and NFB (4, 7-9) which are responsible for transcriptional activation of a variety of antiviral effectors, including the IFN␤ gene that is fundamental to the antiviral response.Despite their similarities in domain structure and amino acid sequence, MDA5 and RIG-I are nonredundant and are involved in recognition of different types of non-self RNAs and therefore different viruses (10 -12). RIG-I exhibits ligand specificity for short (Ͻ2 kbp) double-stranded RNA (dsRNA) and 5Ј-triphosphorylated single-stranded RNA (11,(13)(14)(15)(16)(17) and has been demonstrated to specifically recognize homopolyme...

MDA5 assembles into a polar helical filament on dsRNA

“…S6). 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.…”

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

“…The LGP2 mutant MI targets the Walker A motif and lacks ATP hydrolysis but retains RNA binding (16 -19). Mutant MIII is defective for both ATP hydrolysis and RNA binding (16). Three other LGP2 mutations were designed to target residues that available structural information indicated might be important for biological activity (20 -23).…”

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

“…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 RIG-I and MDA5 (11,12). In addition, induction of ATP hydrolysis by dsRNA has been shown to correlate with stimulation of interferon signaling (6,13).…”