Abstract
Subgenomic flavivirus RNAs (sfRNAs) are structured RNA elements encoded in the 3'-UTR of flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze the production of sfRNAs using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) and super-resolution microscopy during West Nile virus, Zika virus, or Dengue virus serotype 2 infection. We show that sfRNAs are initially localized diffusely in the cytosol or in processing bodies (P-bodies). However, upon activation of the host antiviral endoribonuclease, Ribonuclease L (RNase L), nearly all sfRNAs re-localize to antiviral biological condensates known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery in P-bodies, which coincides with increased viral RNA decay. These findings establish a role of RLBs in promoting viral RNA decay, demonstrating the complex host-pathogen interactions at the level of RNA decay and biological condensation.