Project Summary Antiviral systems are critical to the survival of their host. They protect from virally induced morbidity and mortality across all known domains of life and play an essential role in controlling the endogenized viral elements contained in genomes, the unchecked expression of which can lead to debilitating autoimmune diseases in humans and developmental problems in multicellular organisms. Many antiviral systems encode the exquisite ability to detect and modify or destroy specific nucleic acids, and this has made those components transformative tools for biotechnology and medicine. The directed discovery of antiviral systems in prokaryotes has exploded the number of antiviral systems that were known and reshaped how we think about the evolution of antiviral immune strategies. In eukaryotes, far less discovery research has been done even though there is significant lack of conservation between the well-studied antiviral systems of humans, plants, and insects with those of microbial eukaryotes such as amoeba. Not only are microbial eukaryotes the most abundant eukaryote, but they are also often hosts of the most enigmatic viruses discovered to date, the Megavirales family; giant viruses whose physical and genome size can rival that of bacteria. The machinery these microbial hosts use to control viral infection, and how these viruses subvert these responses remains almost entirely unknown. This proposal aims to address this unanswered question through first understanding how Acanthamoeba spp. defend themselves against members of the Megavirales family before expanding that understanding (and the tools used to arrive at it) to other microbial eukaryotes. We will combine principles of the virus-host arms race with cutting- edge AI-driven protein structure prediction and homology detection to uncover viral proteins that are involved in antagonizing currently known pathways of viral restriction in Acanthamoeba. We will further use environmental sampling to establish a collection of viruses and wild amoeba isolates that display a range of susceptibilities to our giant viruses to identify new systems of restriction within the Acanthamoeba genus. Finally, we will combine our environmental sampling with discovery-based functional metatranscriptomics to realize the hidden antiviral capacity of the abundant microbial eukaryotes. Ultimately, these discoveries will lead to the first comprehensive assessment of non-metazoan antiviral systems that are present in eukaryotes. This will not only dramatically expand our knowledge and challenge ideas of how eukaryotic antiviral systems evolved, but the results of this proposal will provide new molecules that can be used to better human life and life quality through their technological and direct biomedical applications, as have so many antiviral systems before.