2' 5'-Oligoadenylate synthetases (OAS) are interferon-stimulated proteins that act in the innate immune response to viral infection. Upon binding viral double-stranded RNA, OAS enzymes produce 2 '-5 '-linked oligoadenylates that stimulate RNase L and ultimately slow viral propagation. Truncations/mutations in the smallest human OAS isoform, OAS1, results in susceptibility to West Nile virus (WNV). We have previously demonstrated in vitro the interaction between OAS1 and the 5'-terminal region of the WNV RNA genome. Here we report that the 3'-terminal region is also able to mediate specific interaction with and activation of OAS1. Binding and kinetic experiments identified a specific stem loop within the 3'-terminal region that is sufficient for activation of the enzyme. The solution conformation of the 3'-terminal region was determined by small angle X-ray scattering, and computational models suggest a conformationally restrained structure comprised of a helix and short stem loop. Structural investigation of the 3'-terminal region in complex with OASI is also presented. Finally, we show that genome cyclization by base pairing between the 5'- and 3'-terminal regions, a required step for replication, is not sufficient to protect WNV from OAS1 recognition in vitro. These data provide a physical framework for understanding recognition of the highly structured terminal regions of a flaviviral genome by an innate immune enzyme. (C) 2015 Elsevier Inc. All rights reserved.