Solution Structure of a Consensus Stem-Loop D RNA Domain that Plays Important Roles in Regulating Translation and Replication in Enteroviruses and Rhinoviruses^†,‡

Stem-loop D from the cloverleaf RNA is a highly conserved domain within the 5‘-UTR of enteroviruses and rhinoviruses. Interaction between the stem-loop D RNA and the viral 3C or 3CD proteins constitutes an essential feature of a ribonucleoprotein complex that plays a critical role in regulating viral translation and replication. Here we report the solution NMR structure of a 38-nucleotide RNA with a sequence that encompasses the entire stem-loop D domain and corresponds to the consensus sequence found in enteroviruses and rhinoviruses. Sequence variants corresponding to Poliovirus type 1 and Coxsackievirus B3 have virtually the same structure, based on small differences in chemical shifts. A substantial number (136) of ¹H−¹³C one-bond residual dipolar coupling (RDC) values were used in the structure determination in addition to conventional distance and torsion angle restraints. Inclusion of the RDC restraints was essential for achieving well-defined structures, both globally and locally. The structure of the consensus stem-loop D is an elongated A-type helical stem capped by a UACG tetraloop with a wobble UG closing base pair. Three consecutive pyrimidine base pairs (two UU and one CU pair) are present in the middle of the helical stem, creating distinctive local structural features such as a dramatically widened major groove. A dinucleotide bulge is located near the base of the stem. The bulge itself is flexible and not as well defined as the other parts of the molecule, but the flanking base pairs are intact. The peculiar spatial arrangement of the distinctive structural elements implies that they may work synergistically to achieve optimal binding affinity and specificity toward the viral 3C or 3CD proteins.