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

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 protease, and 3D polymerase. The interaction between cloverleaf and 3CD is mediated by the 3C domain, yet how 3C promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3C complex, wherein two 3C molecules interact with cloverleaf stem-loop D. 3C dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3C 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. The structure provides insight into the RNA-binding mechanism of 3C or 3CD with other cis-acting replication elements.