OAS3 and RNase L integrate into higher-order antiviral condensates

ABSTRACT Oligoadenylate synthetase 3 (OAS3) and Ribonuclease L (RNase L) are components of a mammalian RNA decay pathway that is critical for combating viral infection. Nevertheless, the subcellular localization of OAS3 and RNase L during the antiviral response has remained elusive. Herein, we show that upon activation in response to double-stranded RNA or viral infection, OAS3 and RNase L integrate into higher-order cytoplasmic assemblies distinct from stress granules (SGs), RNase L-induced bodies (RLBs), or processing bodies (P-bodies). We identified these assemblies as double-stranded RNA-induced foci (dRIF), which also contain activated protein kinase R (PKR). We observed incorporation of RNase L, OAS3, and PKR in dRIFs during Zika virus and dengue virus infection. Our data support that condensation of dsRNA, OAS3 and RNase L at dRIFs promotes the homodimerization and oligomerization of RNase L required for its rapid activation, demonstrating a fundamental role for biological condensates in activating antiviral signaling.