Summary In this proposal, we will explore the role of prenylation in antiviral immunity. Prenylation is a post-translational modification that renders proteins hydrophobic, thus targeting them to the endomembrane. While the role of prenylation in immunity is unclear, our preliminary data shows compelling evidence that prenylation is a modification widely used in innate antiviral immunity. As both the host and virus utilize prenylation and the cholesterol biosynthetic pathway, we propose a dynamic host-pathogen interaction at this interface. We hypothesize that prenylation could be a potent host innovation to counter viral pathogenesis. This is based on in silica analysis that revealed an enrichment of immunity-related proteins with a prenylation motif. We followed in-silica analyses with a biochemical screen and identified several prenylated antiviral proteins that are involved in interferon responses and virus restriction, with potential to affect virus entry, replication, and egress. In this grant, we propose to identify the functional consequences of prenylated proteins on subcellular localization, interferon signaling, and antiviral function. By perturbing specific enzymes in the prenylation pathway, we will study its effect on interferon response, antiviral effectors, and virus control. This work will provide novel insights into cell-intrinsic antiviral countermeasures and the development of novel antiviral drug targets.