NMR structure of stem–loop D from human rhinovirus-14

The 5′-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem–loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3C pro . This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3D pol ) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3C pro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine–pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3C pro and SLD.