The innate immune response plays a crucial role in recognising and combating viral infections. Double-stranded RNA (dsRNA) is a potent proinflammatory signal, and its recognition by MDA5 and its coregulatory factor, LGP2, is essential for initiating antiviral responses. This study aimed to investigate the structure and function of MDA5 and LGP2.

I showed the cooperative nature of ATP hydrolysis in MDA5-dsRNA filaments. I discovered that adjacent MDA5 subunits in a filament hydrolyse ATP cooperatively, leading to coupled filament disassembly which allows MDA5 to displace tightly bound proteins from dsRNA. Unlike MDA5, LGP2 exhibits noncooperative ATP hydrolysis and ATP dependent internal binding to dsRNA which promotes the assembly of shorter MDA5 filaments, potentially enhancing filament stability or expanding the range of dsRNA ligands that MDA5 can interact with.

The M854K MDA5 variant is associated with interferonopathy. I revealed that the M854K mutation abolishes ATPase activity, stabilises MDA5-dsRNA complexes, increases IFN-β transcription, and disrupts signal transduction. Cryo-electron microscopy structures of wild-type (WT) and M854K MDA5-dsRNA filaments provided detailed insights into how the M854K mutation hinders conformational changes necessary for ATP hydrolysis and dissociation from endogenous RNAs, resulting in constitutive activation of signalling pathways.

In contrast, the partial loss-of-function I923V variant of MDA5 is type 1 diabetes (T1D) protective but increases vulnerability to viral infections. I showed how the I923V mutation causes hyperactive ATP hydrolysis, resulting in shorter and less stable filaments that have a relaxed grip on dsRNA. The I923V mutation disrupts the molecular brake function of residue 923, leading to accelerated ATP hydrolysis and expansion of the RNA binding footprint.

This work provides structural and functional data of MDA5 variants and its regulatory factors during the innate immune response. Understanding the mechanisms of MDA5 variants has the potential to inform the development of novel therapeutics and enhance personalised medicine approaches.