Structure-first identification of conserved RNA elements that regulate dengue virus genome architecture and replication

The genomes of RNA viruses encode the information required for replication in host cells in both their linear sequence and in complex higher-order structures. A subset of these complex functional RNA genome structures show clear sequence conservation. However, the extent to which viral RNA genomes contain conserved structural elements – that cannot be detected by sequence alone – that nonetheless are critical to viral fitness is largely unknown. Here, we take a structure-first approach to identify motifs conserved across the coding sequences of the RNA genomes for the four dengue virus (DENV) serotypes. We used SHAPE-MaP to identify 22 candidate motifs with conserved RNA structures, but no prior association with viral replication. At least ten of these motifs are important for viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These conserved viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are constrained at the levels of both RNA structure and protein sequence and are potential resistance-refractory targets for antivirals and live-attenuated vaccines. Structure-first identification of conserved RNA structure is poised to guide efficient discovery of RNA-mediated regulation in viral genomes and other cellular RNAs. ### Competing Interest Statement K.M.W. is an advisor to and holds equity in Ribometrix. All other authors declare that they have no competing interests.