Structure of coxsackievirus cloverleaf RNA and 3Cpro dimer establishes the RNA-binding mechanism of enterovirus protease 3Cpro

In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5′ end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3C^pro protease, and 3D^pol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C^pro domain, yet how 3C^pro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C^pro complex, wherein two 3C^pro molecules interact with cloverleaf stem-loop D. 3C^pro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C^pro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3C^pro. The structure provides insight into the RNA-binding mechanism of 3C^pro or 3CD with other cis-acting replication elements.