title: Kinetic discrimination of self/non-self RNA by the ATPase activity of RIG-I and MDA5.
abstract: BACKGROUND
  The cytoplasmic RIG-like receptors are responsible for the early detection of viruses and other intracellular microbes by activating the innate immune response mediated by type I interferons (IFNs). RIG-I and MDA5 detect virus-specific RNA motifs with short 5'-tri/diphosphorylated, blunt-end double-stranded RNA (dsRNA) and >0.5-2 kb long dsRNA as canonical agonists, respectively. However, in vitro, they can bind to many RNA species, while in cells there is an activation threshold. As SF2 helicase/ATPase family members, ATP hydrolysis is dependent on co-operative RNA and ATP binding. Whereas simultaneous ATP and cognate RNA binding is sufficient to activate RIG-I by releasing autoinhibition of the signaling domains, the physiological role of the ATPase activity of RIG-I and MDA5 remains controversial.
  
  
  RESULTS
  A cross-analysis of a rationally designed panel of RNA binding and ATPase mutants and truncated receptors, using type I IFN promoter activation as readout, allows us to refine our understanding of the structure-function relationships of RIG-I and MDA5. RNA activation of RIG-I depends on multiple critical RNA binding sites in its helicase domain as confirmed by functional evidence using novel mutations. We found that RIG-I or MDA5 mutants with low ATP hydrolysis activity exhibit constitutive activity but this was fully reverted when associated with mutations preventing RNA binding to the helicase domain. We propose that the turnover kinetics of the ATPase domain enables the discrimination of self/non-self RNA by both RIG-I and MDA5. Non-cognate, possibly self, RNA binding would lead to fast ATP turnover and RNA disassociation and thus insufficient time for the caspase activation and recruitment domains (CARDs) to promote downstream signaling, whereas tighter cognate RNA binding provides a longer time window for downstream events to be engaged.
  
  
  CONCLUSIONS
  The exquisite fine-tuning of RIG-I and MDA5 RNA-dependent ATPase activity coupled to CARD release allows a robust IFN response from a minor subset of non-self RNAs within a sea of cellular self RNAs. This avoids the eventuality of deleterious autoimmunity effects as have been recently described to arise from natural gain-of-function alleles of RIG-I and MDA5.
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France. jade.louber@gmail.com.|INSERM, U1111, Lyon, France. jade.louber@gmail.com.|Ecole Normale Supérieure de Lyon, Lyon, France. jade.louber@gmail.com.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France. jade.louber@gmail.com.|CNRS, UMR5308, Lyon, France. jade.louber@gmail.com.
authors.forename: Jade
authors.initials: J
authors.lastname: Louber
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France. Joanna.brunel@inserm.fr.|INSERM, U1111, Lyon, France. Joanna.brunel@inserm.fr.|Ecole Normale Supérieure de Lyon, Lyon, France. Joanna.brunel@inserm.fr.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France. Joanna.brunel@inserm.fr.|CNRS, UMR5308, Lyon, France. Joanna.brunel@inserm.fr.
authors.forename: Joanna
authors.initials: J
authors.lastname: Brunel
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, BP 181, 38042, Grenoble, Cedex 9, France. uchikawa@embl.fr.|Unit of Virus Host Cell Interactions, UJF-EMBL-CNRS, UMI 3265, 71 Avenue des Martyrs, BP 181, 38042, Grenoble, Cedex 9, France. uchikawa@embl.fr.
authors.forename: Emiko
authors.initials: E
authors.lastname: Uchikawa
authors.affiliation: European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, BP 181, 38042, Grenoble, Cedex 9, France. cusack@embl.fr.|Unit of Virus Host Cell Interactions, UJF-EMBL-CNRS, UMI 3265, 71 Avenue des Martyrs, BP 181, 38042, Grenoble, Cedex 9, France. cusack@embl.fr.
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
authors.affiliation: CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France. denis.gerlier@inserm.fr.|INSERM, U1111, Lyon, France. denis.gerlier@inserm.fr.|Ecole Normale Supérieure de Lyon, Lyon, France. denis.gerlier@inserm.fr.|Université Claude Bernard Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France. denis.gerlier@inserm.fr.|CNRS, UMR5308, Lyon, France. denis.gerlier@inserm.fr.
authors.forename: Denis
authors.initials: D
authors.lastname: Gerlier
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D000072640:Interferon-Induced Helicase, IFIH1; D053533:Keratin-8 / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic
chemical_list: C426203:KRT8 protein, human; D053533:Keratin-8; D012330:RNA, Double-Stranded; D011971:Receptors, Immunologic; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C452820:RIGI protein, human; C452815:IFIH1 protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1