Mechanistic Studies of RNA Detection by the Cellular Immune Sensors LGP2 and MDA5 in Mammalian Antiviral Signaling.

摘要

Mammalian cells have the ability to recognize virus infection and mount a powerful antiviral response. Pattern recognition receptor proteins detect molecular signatures of virus infection and activate antiviral signaling. The RIG-I-like receptor (RLR) proteins are expressed in the cytoplasm of nearly all cells that specifically recognize virus-derived RNA species as a molecular feature discriminating the pathogen from the host. The RLR family is composed of three homologous proteins, RIG-I, MDA5, and LGP2, all with the ability to detect virus-derived dsRNA and hydrolyze ATP. While RIG-I and MDA5 contain the domains essential for activation of antiviral signaling, LGP2 lacks these domains, leaving the role of LGP2 in RLR-mediated antiviral signaling largely unresolved. LGP2 can enhance MDA5 signaling, but the mechanism of synergy had not been previously characterized.;The ATP hydrolysis activity of LGP2 is essential for proper antiviral signaling, but it has been unclear how the enzymatic properties of LGP2 regulate its biological response. The first section of this thesis research utilized biochemistry and single molecule RNA-binding experiments to investigate the role of ATP hydrolysis in the biological activity of LGP2. This analysis of LGP2 revealed high dsRNA-independent (basal) ATP hydrolysis activity. Mutant variants that dissociate basal from dsRNA-stimulated ATP hydrolysis were constructed and demonstrate LGP2 utilizes basal ATP hydrolysis to enhance and diversify its RNA recognition capacity. This property is required for LGP2 to synergize with MDA5 to potentiate IFNbeta transcription in vivo. These results demonstrated novel properties of LGP2 ATP hydrolysis and RNA interaction and provided the motivation for investigating the mechanism by which enzymatically active LGP2 enhances MDA5-mediated antiviral signaling.;MDA5 can form filaments along dsRNA that are critical for antiviral signaling. Experiments presented in chapter three reveal that LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly to produce more numerous, shorter MDA5-RNA filaments. These shorter filaments generate equivalent or greater signaling activity in vivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 co-activation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses.