A structural bridge between dengue virus tandem xrRNAs facilitates coordination of exonuclease resistance

Orthoflavivirus RNA genomes resist host 5′–3′ exoribonucleases to produce subgenomic flaviviral RNAs (sfRNAs). This resistance is conferred by exoribonuclease-resistant RNA (xrRNA) structures within the viral 3′ untranslated region that often occur in tandem, and whose function can be coupled. In dengue virus serotype 2 (DENV2), this coupling results in changing patterns of sfRNA identity and abundance associated with the ability of the virus to adapt to host vs. vector infections. The physical basis of this coupling was unknown. Using a combination of virology, biochemistry, bioinformatics, structural biology, and biophysics, we explored the structural and sequence determinants of tandem xrRNA coupling in DENV2. We discovered that the spatial proximity, order, and structural integrity of the tandem xrRNAs are all important for coupling. Furthermore, an unpaired A-rich linker that lies between the two xrRNAs is essential in stabilizing a specific structure that correlates to coupling. This A-rich sequence likely forms tertiary contacts with an adjacent stem-loop structure to form a physical bridge between the two xrRNAs, a finding that is supported by a mid-resolution cryo-electron microscopy (cryo-EM) map of the DENV2 tandem xrRNAs. Disruption of the structure of this bridge by mutation changes the relative orientation or spacing between the tandem xrRNAs, which is correlated to their functional coupling. These findings help provide an explanation for the coupling between tandem xrRNAs, suggesting a new mechanistic hypothesis in which the two tandem xrRNAs can simultaneously encounter Xrn1.