Abstract
The mechanism and role of RNA structure elements in the replication and translation of Caliciviridae remains poorly understood. Several algorithmically independent methods were used to predict secondary structures within the Norovirus, Sapovirus, Vesivirus and Lagovirus genera. All showed profound suppression of synonymous site variability (SSSV) at genomic 5' ends and the start of the sub-genomic (sg) transcript, consistent with evolutionary constraints from underlying RNA structure. A newly developed thermodynamic scanning method predicted RNA folding mapping precisely to regions of SSSV and at the genomic 3' end. These regions contained several evolutionarily conserved RNA secondary structures, of variable size and positions. However, all caliciviruses contained 3' terminal hairpins, and stem-loops in the anti-genomic strand invariably six bases upstream of the sg transcript, indicating putative roles as sg promoters. Using the murine norovirus (MNV) reverse-genetics system, disruption of 5' end stem-loops produced approximately 15- to 20-fold infectivity reductions, while disruption of the RNA structure in the sg promoter region and at the 3' end entirely destroyed replication ability. Restoration of infectivity by repair mutations in the sg promoter region confirmed a functional role for the RNA secondary structure, not the sequence. This study provides comprehensive bioinformatic resources for future functional studies of MNV and other caliciviruses.