Streit, L. V.; Onikubo, T.; Astore, M. A.; Cioppa, E. A.; Olinares, P. D. B.; Urnavicius, L.; Incarnato, D.; Bonilla, S. L.

Viral genomes encode dynamic RNA structures that are specifically recognized by proteins to regulate critical steps of the viral life cycle. Although these RNAs exist as multi-state conformational ensembles, structural studies have largely captured single conformations, limiting our mechanistic understanding of RNA-protein recognition and function. Here, using single-particle cryo-electron microscopy (cryo-EM), single-molecule Forster resonance energy transfer, and biochemical assays, we show how flaviviral genomes use structural dynamics encoded in their stem-loop A (SLA) to guide a conserved, multi-step mechanism of negative-strand synthesis initiation by non-structural protein 5 (NS5). Cryo-EM ensembles of SLAs from dengue, Zika, West Nile, and yellow fever viruses reveal a conserved conformational landscape dictated by alternative stacking configurations of a central three-way junction (3WJ). Cryo-EM structures of SLA-NS5 complexes from dengue and Zika show that NS5 engages a specific state within SLA 3D ensemble, one in which the 3WJ adopts a GRR/A tetraloop-like motif that we identify across diverse structural RNAs. Strikingly, mutations that substantially destabilize this state impair negative-strand synthesis without affecting NS5 binding, decoupling binding from function and ruling out a simple conformational-capture mechanism. Instead, the SLA's intrinsic structural dynamics direct a post-binding conformational search that converts an initial, conformationally heterogeneous encounter complex into a productive replication-initiation complex. These results demonstrate a mechanism of RNA-protein recognition in which the 3D conformational ensemble of an RNA guides formation of the functional state downstream of binding, and identify a ubiquitous, intrinsically dynamic 3D motif within the SLA's 3WJ as a critical determinant of flavivirus negative-strand synthesis.