A Fish MDA5 Integrates Canonical RIG‐I and MDA5 Ligand Recognition

RIG-I and MDA5 are cytosolic RNA sensors that recognize distinct viral ligands in mammals, yet RIG-I has been independently lost from multiple teleost lineages. How RIG-I-deficient vertebrates preserve recognition of RIG-I-associated viral RNA remains poorly understood. Here, we show that MDA5 from the large yellow croaker (Larimichthys crocea, Lc), a teleost species lacking RIG-I, recognizes both 5'-triphosphate dsRNA (5'ppp-dsRNA), the canonical ligand of RIG-I, and long dsRNA, the classical ligand of MDA5. Its C-terminal domain (CTD) is essential for the recognition of both ligands, with residues specifically required for sensing the 5'ppp moiety. Structural modeling, molecular dynamics simulations, and mutational validation support a model in which a flexible RNA-capping loop in the CTD confers LcMDA5 a conformational plasticity, allowing the CTD to cap the 5'ppp-dsRNA terminus for RIG-I-like recognition or orienting the CTD parallel to long-dsRNA axis for canonical MDA5 recognition. Disruption of 5'ppp recognition impaired LcMDA5-mediated antiviral activity against a RIG-I-sensitive virus. Downstream, LcMDA5 signals through an integrated MAVS-STING-TBK1/IRF3 axis to drive type I IFN responses to both RNA ligands. Our findings reveal an unexpectedly flexible architecture of vertebrate cytosolic RNA sensing in which a single RLR combines ligand-recognition and signaling properties classically partitioned between RIG-I and MDA5.