Abstract
Enteroviral replication-linked cloverleaf RNAs recruit the viral 3CD protein, a fusion of 3C protease and 3D RNA-dependent RNA-polymerase, for negative-strand synthesis during genome replication. However, the structures and mechanisms of this virological process remain unclear. Using the coxsackievirus B3 model, we determine the crystal structures of both intact cloverleaf-3C and isolated sD-3C complexes at 2.69 Å and 2.41 Å resolutions, respectively. Our structures reveal that the sD stem-loop is the sole determinant for binding two 3C monomers, with each monomer recognizing the lateral surface of the sD stem either upstream (toward the apical tetraloop) or downstream (near the dinucleotide bulge) of the Py•Py helix. Binding studies with structure-guided cloverleaf and 3C mutants further clarify the roles of specific nucleotides and residues involved in the interactions between cloverleaf and 3C, explaining earlier virological observations. Through comparative structural and binding studies of 3C, 3D, and 3CD with cloverleafs from seven different enteroviral species, we demonstrate that while the 3D domain does not contribute to cloverleaf binding, the sD sequence and its structural pattern govern 3CD-cloverleaf interactions through the 3C domain. Our work establishes a high-resolution structural framework for understanding enteroviral replication mechanisms, which will aid in developing antivirals targeting this platform.