Project Summary Oligo adenylate synthetase (OAS) proteins are a family of interferon stimulated RNA binding proteins important for restricting viral replication. Upon sensing viral RNA, the catalytically active OAS proteins generate the second messenger 2-5A, which activates the latent ribonuclease RNaseL. Activated RNaseL potently restricts viral replication by degrading cellular RNAs, effectively blocking host translation, and inducing apoptosis. Of the human OAS proteins, only OAS1, OAS2, and OAS3 are catalytically active and capable of synthesizing 2- 5A. Human OAS1 is spliced into several different isoforms, but if these OAS1 isoforms are functionally different is not understood. A SNP in the splice acceptor site of exon 7 in the OAS1 gene (A>G, rs10774671) is associated with West Nile Virus resistance. The susceptibility allele (A) controls p42 expression and the resistance allele (G) controls p46 expression, which suggests OAS1 isoforms have different functions in antiviral immunity. The goal of this proposal is to uncover those differential functions and describe their mechanisms. Interestingly, p46 contains a C-terminal membrane-targeting CaaX motif. Proteins containing CaaX motifs at their C-termini are prenylated and targeted to membranes. We have found the two major isoforms of human oligo adenylate synthetase 1 (OAS1) localize to unique subcellular compartments. The p46 isoform localizes to the Golgi, while p42 is cytosolic. Subcellular targeting of antiviral proteins is important for their specificity. Since positive-strand RNA viruses replicate on modified cellular membranes, termed viral replication organelles (VROs), we hypothesize targeting antiviral proteins to these membranes enhances their antiviral activity. Our preliminary data suggests p46 is recruited to VROs during West Nile Virus infection. We hypothesize the membrane-targeting CaaX motif on p46 allows the protein to infiltrate VROs, giving it access to viral RNA otherwise shielded from detection by the OAS/RNaseL pathway. Consistent with our hypothesis, preliminary data with encephalomyocardidits virus (EMCV) shows p46 might be broadly antiviral against RNA viruses that replicate on cellular membranes and suggests p46 is antiviral independent of synthetase activity. Based on this preliminary data, we propose p42 has specificity for different RNA viruses which do not replicate on cellular membranes.