Structural and functional integrity of the coxsackievirus B3 oriR: spacing between coaxial RNA helices.

The enterovirus oriR is composed of two helices, X and Y, anchored by a kissing (K) interaction. For proper oriR function, certain areas of these helices should be specifically oriented towards each other. It was hypothesized that the single-stranded nucleotides bridging the coaxial helices (Y-X and K-Y linkers) are important to determine this orientation. Spatial changes were introduced by altering the linker length between the helices of the coxsackievirus B3 oriR. Changing the linker lengths resulted in defective RNA replication, probably because of an altered oriR geometry. The identity of the linker residues also played a role, possibly because of sequence-specific ligand recognition. Although each point mutation altering the primary sequence of the Y-X spacer resulted in defective growth at 36 degrees C, the mutations had a wild-type phenotype at 39 degrees C, indicating a cold-sensitive phenotype. The results show that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between the coaxial RNA helices.