Characterization of the MDA5 2CARD Signaling Complex

Abstract

The mammalian innate immune system is the first line of defense against invading pathogens. The RIG-I like receptors (RLRs), RIG-I and MDA5 are critical for host recognition of viral RNAs. These receptors contain a helicase-like core domain, two caspase activation and recruitment domains at the N-terminus (2CARD) and a C-terminal regulatory domain. In RIG-I, RNA binding leads to exposure of the 2CARD domain and 2CARD oligomerization. The 2CARD signaling domain associates with the CARD domain of MAVS, a mitochondrial outer membrane protein, leading to oligomerization of MAVS, downstream signaling and interferon induction. K63-linked polyubiquitin chains (polyUb) play a role in RLR signaling by interacting with the 2CARD domain. PolyUb binding to RIG-I 2CARD induces formation of 2CARD tetramer. However, the nature of the signaling complex of MDA5 and the role of polyUb binding in MDA5 activation are not well understood. We have used sedimentation velocity analytical ultracentrifugation to characterize MDA5 2CARD oligomerization in absence and presence of polyUb. MDA5 2CARD forms a broad distribution of oligomers. The size distribution is concentration dependent, indicating reversibility. MDA5 2CARD forms complexes with polyUb, and complex formation is highly dependent on NaCl concentration. Multisignal sedimentation velocity experiments will probe the nature of the polyUb-2CARD complexes.