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Abstract

RIG-I is a pattern recognition receptor that is responsible for the initiation of an antiviral response to a variety of virus infections. Cytoplasmic nucleic acid derived from virus replication is detected by RIG-I resulting in the initiation of a signalling cascade that ultimately leads to the activation of transcription factors, namely IRF3 and NFkB. Activation of these transcription factors leads to the production and secretion of type I and III interferons (IFN), which act in an auto- and paracrine manner to induce the expression of a large array of IFN stimulated genes (ISGs). In concert, these ISGs promote an antiviral state of the cell, limiting viral replication and spread. Much is known about the individual steps and proteins involved in the RIG-I signalling pathway, however, much less is understood about its regulation and dynamics. Upon virus entry into a cell, the initial response phase is critical to the outcome and determines if the infection is cleared or established. Understanding the dynamics of these processes will be key to comprehend and possibly predict the outcome of a viral infection. The main goal of this thesis, therefore, was to identify novel regulators and to kinetically characterize the rapid induction of the cellular antiviral signalling cascade. As for the identification of previously unrecognized regulators of the RIG-I pathway, we have performed an siRNA-based high-throughput screen of over 600 known and putative E3 ubiquitin ligase genes. Post-translational ubiquitination has been shown to constitute a major regulatory process in innate immune signalling. From our screening approach, we were able to identify several genes that significantly impacted IRF3 activation upon silencing during viral infection. The main part of this thesis deals with the temporal characterisation of RIG-I-initiated antiviral signalling. For that purpose, we developed an approach which permitted the completely synchronous stimulation of cells with virus-like double-stranded RNA. In contrast to authentic infections or classical liposome-based transfections, this method allows for a very high degree of time-resolution in measuring the flow of the signal along the cascade. Quantitative, time-resolved activation measurements of critical proteins in the RIG-I pathway showed that RIG-I signalling is rapid and, in contrary to previous reports, strictly deterministic with very little variability from cell to cell. Furthermore, we extensively characterized the transcriptional program triggered by RIG-I in a time-resolved manner by full-genomic transcriptional profiling. We found that the panel of genes upregulated directly by the primary IRF3 response is surprisingly large and congruent with the ISGs classically known to be induced only by IFN signalling, with only very few genes exhibiting a strict dependence on IFN/JAK/STAT signalling. This work represents the first detailed molecular characterization of the kinetics of host cellular processes triggered in the first few minutes after virus infection. The comprehensive quantitative and time-resolved data generated can serve as a solid basis for a mathematical model that combines viral replication dynamics and host antiviral responses. Such a model will be an unprecedented and powerful tool to study the principles governing the outcome of viral infection and to help understanding how certain viruses manage to overcome host immunity and cause fulminant disease or even establish life-long persistence.