The Structure of the Picornaviral 2C:RNA holoenzyme: Molecular Basis of RNA binding and specificity by a AAA+ protein

Picornaviruses are one of the leading agents of animal and human infectious disease with at least 8 billion infections a year and cause a range of symptoms including respiratory failure and acute flaccid myelitis ¹ . The most conserved nonstructural protein in picornaviruses is 2C ² , a member of the AAA+ family of ATPases that binds RNA, and a broad spectrum antiviral target ^3–5 . Despite its crucial role in the viral life cycle and as a clinical target, no structure of 2C bound to RNA has been structurally determined. Here we present the first structure of 2C as a hexamer bound to single stranded RNA in its central pore; a novel AAA+ protein:substrate interaction. Using the 2C:RNA holoenzyme complex structure, we characterize the mode that this AAA+ protein employs to specifically bind single stranded RNA, and demonstrate that mutations to key residues inhibit both RNA binding and viral replication in Apthovirus and Enterovirus systems, and show that the core residues responsible for binding are broadly conserved in viruses beyond Picornaviridae . Finally, we reveal that the 2C:RNA holoenzyme complex is conformationally more similar to a protein translocase adapted to bind RNA rather than other viral DNA binding SF3 helicases, underscoring how the AAA+ core module can be adapted for a variety of biochemical substrates.