Structure of Essential RNA Regulatory Elements in the West Nile Virus 3’-Terminal Stem Loop

Flaviviruses, such as West Nile and Dengue Virus, pose a significant and growing threat to global health. Central to the flavivirus life cycle are highly structured 5'- and 3'-untranslated regions (UTRs), which harbor conserved cis-acting RNA elements critical for viral replication and host adaptation. Despite their essential roles, detailed molecular insights into these RNA elements have been limited. By employing nuclear magnetic resonance (NMR) spectroscopy in conjunction with SAXS experiments, we determined the three-dimensional structure of the West Nile Virus (WNV) 3'-terminal stem-loop core, a highly conserved element critical for viral genome cyclization and replication. Single nucleotide mutations at several sites within this RNA abolish the ability of the virus to replicate. These critical sites are located within a short 18-nucleotide hairpin stem, a substructure notable for its conformational flexibility, while the adjoining main stem-loop adopts a well-defined extended helix interrupted by three non-Watson-Crick pairs. This study enhances our understanding of several metastable RNA structures that play key roles in regulating the flavivirus lifecycle, and thereby also opens up potential new avenues for the development of antivirals targeting these conserved RNA structures. In particular, the structure we observe suggests that the plastic junction between the small hairpin and the tail of the longer stem-loop could provide a binding pocket for small molecules, for example potentially stabilizing the RNA in a conformation which hinders the conformational rearrangements critical for viral replication.