Identification and characterization of signal-transducing and regulatory kinases involved in the RIG-I/IRF3-mediated induction phase of the innate antiviral immune-response

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Every cell (from bacterial to human) possesses innate mechanisms to perceive and promptly defend against intruding microorganisms. In case of virus infections in higher animals, the interferon system is the single major player. A panel of sensor molecules on and within cells detects invariant components of the pathogen and elicits a signaling cascade that culminates in the production and secretion of various cytokines, most prominently type I (and III) interferons. In both, the infected and surrounding cells, these messengers subsequently trigger an antiviral program, which by means of a multitude of molecular effectors impedes or even prevents viral replication and spread. Along with this antiviral program inflammatory processes are mounted, which, however, must be strictly regulated in order to avoid severe damage of the affected organ.The molecular mechanisms of regulation during induction of the interferon system are incompletely understood thus far. In the here proposed follow-up project, we are going to characterize factors, which significantly affect antiviral signal transduction and its regulation. For this, we will build upon our previous project, for which we had performed a high-throughput RNAi-based screen, in which we silenced the genes of 719 human kinases and assessed their impact on the activation of the essential antiviral transcription factor IRF-3. Those genes that exhibited the most significant effects have undergone robust validation, and basal functional characterization. One particularly impactful gene, the kinase DAPK1, has been investigated in detail and shown to function as a novel negative feedback regulator of the antiviral defense program of the cell.In the now proposed project, we are going to unravel the molecular mechanism of how the antiviral RIG-I/IRF3 signaling pathway activates its negative regulator DAPK1. We will further investigate how phosphorylation of RIG-I by DAPK1 affects the molecular recognition of virus infection. To this end, we have already observed that phosphorylation leads to a completely unexpected response-behavior of RIG-I towards viral RNA. We now plan to exploit this observation to study and better understand the mechanism of self-vs-non-self recognition of RNAs by RIG-I. In a second work package, we will investigate wether the closely related proteins DAPK2 and DAPK3 have a similar regulatory role for the RIG-I pathway as DAPK1. For this purpose, we will compare all three DAPK molecules functionally and also across different cell types.Lastly, within the proposed project, we are also going to deeper characterize further kinase genes that exhibited a strong phenotype in antiviral signaling in the first funding period of the project. For three of these kinases, we have already accumulated substantial amounts of data, which we will now deepen further and extend to full-fledged molecular characterizations.