High Resolution Kinetic Characterization and Dynamic Mathematical Modeling of the RIG-I Signaling Pathway and the Antiviral Responses

ABSTRACT The pattern recognition receptor RIG-I is essential for the recognition of viral dsRNA and the activation of a cell-autonomous antiviral response. Upon stimulation, RIG-I triggers a signaling cascade leading to the expression of cytokines, most prominently type I and III interferons (IFNs). IFNs are secreted and signal in an auto- and paracrine manner to trigger the expression of a large variety of IFN-stimulated genes, which in concert establish an antiviral state of the cell. While the topology of this pathway has been studied quite intensively, the dynamics, particularly of the RIG-I-mediated IFN induction, is much less understood. Here, we employed electroporation-based transfection to synchronously activate the RIG-I signaling pathway, enabling us to characterize the kinetics and dynamics of cell-intrinsic innate immune signaling to virus infections. By employing an A549 IFNAR1/IFNLR deficient cell line, we could analyze the difference between the primary RIG-I signaling phase and the secondary signaling phase downstream of the IFN receptors. We further used our quantitative data to set up and calibrate a comprehensive dynamic mathematical model of the RIG-I and IFN signaling pathways. This model accurately predicts the kinetics of signaling events downstream of dsRNA recognition by RIG-I as well as the feedback and signal amplification by secreted IFN and JAK/STAT signaling. We have furthermore investigated the impact of various viral immune antagonists on the signaling dynamics experimentally, and we utilized the here described modelling approach to simulate and in silico study these critical virus-host interactions. Our work provides a comprehensive insight into the signaling events occurring early upon virus infection and opens up new avenues to study and disentangle the complexity of the host-virus interface.