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

The genomes of RNA viruses encode the information required for replication in host cells both in their linear sequence and in complex higher-order structures. A subset of these RNA genome structures show clear sequence conservation, and have been extensively described for well-characterized viruses. However, the extent to which viral RNA genomes contain functional structural elements – unable to be detected by sequence alone – that nonetheless are critical to viral fitness is largely unknown. Here, we devise a structure-first experimental strategy and use it to identify 22 structure-similar motifs across the coding sequences of the RNA genomes for the four dengue virus (DENV) serotypes. At least ten of these motifs modulate viral fitness, revealing a significant unnoticed extent of RNA structure-mediated regulation within viral coding sequences. These viral RNA structures promote a compact global genome architecture, interact with proteins, and regulate the viral replication cycle. These motifs are also thus 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 enables efficient discovery of pervasive RNA-mediated regulation in viral genomes and, likely, other cellular RNAs. Overall design: RNA gently extracted from purified virus particles (ex virion RNA) was treated with 1M7 or DMSO (control) for SHAPE-MaP RNA structure probing experiments. RNA within cells was treated with the bivalent chemical crosslinker SDA or DMSO (control) to assess engagement by proteins for RNP-MaP probing experiments. Chemically modified RNA samples from SHAPE-MaP and RNP-MaP experiments were subjected to MaP reverse transcription, which encodes the position of chemical adducts as non-templated nucleotides or deletions in the product cDNA. Sequencing libraries were generated using the Nextera XT workflow and sequenced on an Illumin MiSeq instrument. The ShapeMapper 2 software was used to align reads and call modification-induced mutations. Software analysis pipelines were employed to create SHAPE and RNP-MaP profiles and model RNA structures.