Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing

Maintaining innate immune homeostasis is critical for preventing infections and autoimmune diseases but effective interventions are lacking. Here we identified C864-C869-mediated intermolecular disulfide-linkage formation as a critical step for human RIG-I activation that can be bidirectionally regulated to control innate immune homeostasis. The viral-stimulated C864-C869 disulfide linkage mediates conjugation of an SDS-resistant RIG-I oligomer, which prevents RIG-I degradation by E3 ubiquitin-ligase MIB2 and is necessary for RIG-I to perform liquid-liquid phase separation to compartmentalize downstream signalsome, thereby stimulating type I interferon signalling. The corresponding C865S 'knock-in' caused an oligomerization defect and liquid-liquid phase separation in mouse RIG-I, which inhibited innate immunity, resulting in increased viral load and mortality in mice. Using unnatural amino acids to generate covalent C864-C869 linkage and the development of an interfering peptide to block C864-C869 residues, we bidirectionally regulated RIG-I activities in human diseases. These findings provide in-depth insights on mechanism of RIG-I activation, allowing for the development of methodologies that hold promising implications in clinics.