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 the effective interventions are lacking. Here, we identified Cys864-Cys869-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 Cys864-Cys869 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 IFN-I signaling. The corresponding C865S ‘knock-in’ caused defect of oligomerization and LLPS in mouse RIG-I, which inhibited innate immunity, resulting in increased viral load and mortality in mice. Through generating covalent Cys864-Cys869 linkage by unnatural amino-acid and developing interfering peptide to block Cys864-Cys869 residues, we bidirectionally regulate RIG-I activities in human diseases. These findings provide novel and in-depth insights on mechanism of RIG-I activation, develop methodologies that hold promising implications in clinics