PROJECT SUMMARY Ribonuclease L (RNase L) is a key component of the mammalian innate antiviral response. For decades, RNase L was presumed to reduce viral protein synthesis by cleaving ribosomes to arrest translation. However, we and others recently demonstrated that RNase L-cleaved ribosomes are translation-competent, and that pathogenic viruses can synthesize proteins despite activating RNase L. These observations have revealed a significant gap in knowledge regarding how RNase L functions and how viruses evade it. We have demonstrated that RNase L rapidly degrades nearly all cellular mRNAs upon activation. This activity regulates three cellular processes that have expanded our understanding of RNase L and that have elucidated how pathogenic viruses evade and potentially hijack RNase L functions. First, RNase L reprograms translation to an antiviral state by degrading constitutively expressed cellular mRNAs while sparing host mRNAs encoding antiviral proteins (e.g., type I interferons), which permits antiviral protein synthesis. Importantly, the mRNAs encoded by several pathogenic viruses (e.g., dengue virus) similarly evade RNase L-mediated mRNA decay, thus permitting viral protein synthesis. This observation has elucidated how pathogenic viruses synthesize proteins despite activating RNase L. This application proposes to characterize the RNase L-mediated mRNA decay pathway and determine how host and viral mRNAs evade it. Second, RNase L activation triggers the inhibition of nuclear mRNA export. This is a critical antiviral mechanism that antagonizes influenza A virus protein synthesis, but it also downregulates the expression of host antiviral proteins (e.g., type I interferons). Importantly, pathogenic viruses (e.g., dengue virus) activate this RNase L-dependent pathway, resulting in sequestration of host antiviral mRNAs in the nucleus. This observation suggests that viruses potentially hijack this function of RNase L to limit host antiviral protein production. This application aims to determine how RNase L inhibits mRNA export, the breadth of viruses it antagonizes, how it impacts host antiviral gene expression during pathogenic viral infections. Third, RNase L regulates the assembly of cytoplasmic antiviral ribonucleoprotein complexes. Specifically, RNase L inhibits the assembly of stress granules and promotes the assembly of an alternative stress granule-like ribonucleoprotein complex termed RNase L-dependent body. RNase L-dependent bodies are the predominant antiviral granule assembled in response to SARS-CoV-2 or dengue virus infection, yet their function is completely unknown. This application aims to determine the function of antiviral stress granules and RNase L-dependent bodies and to determine how their regulation by RNase L alters the antiviral response. Understanding the mechanisms and functions of these cellular processes will advance our understanding of the OAS/RNase L pathway, innate immune antiviral gene induction, and virol...