Structural basis for RIG-I and MDA-5 mediated antiviral response

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We address the structural and biochemical mechanisms of activation, regulation and inhibition of RIG-I like helicases in the antiviral interferon response. RIG-I and the related MDA5 protein detect viral RNA and initiate a signal transduction cascade to stimulate innate immunity. The molecular basis for virus versus self RNA differentiation by RIG-I and MDA5 is not understood, but of central importance to understand intrinsic antiviral functions of our cells. We use a combination of X-ray crystallography, small angle X-ray scattering and biochemical techniques to understand key principles of how RIG-I recognizes viral RNA patterns, how ATP binding and hydrolysis by RIG-I is used in the process of pattern recognition and finally, how viral protein inhibitors interfere with pattern recognition and activation. Based on existing, X-ray diffracting crystals we aim at deriving in the first aim a structure of the helicase domain of RIG-I. This structure will guide the analysis of the mechanism of activation of RIG-I by viral patterns. We will aim at deriving molecular determinants for the recognition of 5' triphosphate RNA as well as double stranded RNA by the ATPase and regulatory domains of RIG-I. We will address how regulatory domains as well as ATPase domain of RIG-I are mechanistically linked and test the hypothesis that RIG-I integrates several patterns into an active "signal on" conformation. We will then aim at deriving a detailed molecular and mechanistic picture of this "signal on" conformation of RIG-I using a multidisciplinary and collaborative approach. This apprach will include the interlink between ATP dependent pattern recognition and posttranslational modification of RIG-I and we will address whether these posttranslational modifications manifest the "signal on" conformation. Finally, we will aim at revealing how viruses counteract MDA5 signalling by using a proteinaceous inhibitor against MDA5. All in all, the expected outcome will advance our understanding of the specific and proofread pattern recognition of viral RNA by RIG-I like helicases at the molecular mechanistic and atomic level.