Viral RNA recognition by RIG-I-like receptors and CARD mediated signalling – CARDINNATE

Cell intrinsic innate immunity is the first and crucial barrier that controls virus infection upon entry into a target cell. Recognition of viral infection very often involves detection of virus specific RNA moieties by a specific class of pattern recognition receptor (PRR), the RIG-like helicase (RLH) family. RLH comprises three members, RIG-I, MDA5 and LGP2. Upon recognition of their cognate RNA ligand, RIG-I and MDA5 activate the anti-viral interferon response by binding to MAVS, a central signalling node, while LGP2 modulates the activation of the other two RLHs. Double stranded RNA (dsRNA) particularly produced by RNA viruses such as paramyxoviruses is a common feature recognize by the RLHs and a 5’ triphosphate group is specifically recognised by RIG-I. However, the natural viral RNA agonists are very poorly defined. The RLH have a similar modular architecture associating tandem N-terminal CARD domains (absent in LGP2), a central DECH helicase (Hel) domain and a C-terminal domain (CTD). RNA binding site maps to both Hel and CTD domains. In a recent Cell paper, S. Cusack’s group (P2), in collaboration with D. Gerlier’s group (P1), has reported the first and only full length crystal structure of RIG-I. Together with structures of dsRNA containing complexes and functional analysis, they have defined the mechanism that triggers the switch from the auto-repressed resting state to the activated dsRNA/ATP bound state. The activated state transduces the signal via the resulting exposure of the CARD domains that become free to interact with the MAVS-CARD domain. The aim of this proposal is to get further insight into the recognition of viral RNA by RLHs and the CARD/CARD mediated recruitment of MAVS. To this end, three tasks will be undertaken. The first one will be devoted to the identification of measles virus RNAs that act as agonists of RIG-I and MDA5 and how their production is regulated by the viral C protein, a virulence factor. The second aim will be to analyse how MDA5 binds to dsRNA and obtain crystal structures of MDA5 alone and in complex with RNA so as to be able to derive and subsequently test a structure-based model for MDA5 activation. This analysis will be complemented by solving the structure of MDA5 in complex with paramyxovirus V protein, an MDA5 inhibitor and virulence factor. The third aim will be to delineate the various posttranscriptional modifications of RIG-I CARD domains according to its resting and activated state and uses this information to analyse biochemically and biophysically the interaction of RIG-I and MDA5 CARD domains with the MAVS CARD domain and to solve the crystal structure of the CARDs/CARD complex. The complementary expertise of the two partners will cover all the technical needs required for the successful completion of the project from structural and biophysical (S Cusack) to functional analysis in a model of a paramyxovirus (measles) infection (D. Gerlier).