Project Summary/Abstract Viruses have evolved sophisticated mechanisms to hijack host cellular machinery. For their part, hosts have developed their own intricate defense systems. Defining how these opposing strategies have co-evolved increases our understanding of infectious diseases and provides new opportunities to discover unexplored areas of biology. Our understanding of host cellular defense systems has been limited due to genetic redundancy in host genomes. A further complication is that successful pathogens are able to inactivate host antiviral networks during infection. We have recently established a new genetic screening platform that overcomes these roadblocks. First, we bypass genetic redundancy in host genomes through a gain of function screen that identifies antiviral genes. Second, we use both virulent and attenuated strains of viruses in our screening pipeline. Importantly, these attenuated viruses have deletions or mutations in critical, yet poorly understood, immune evasion proteins that antagonize host antiviral restriction factors. We predict that the avirulent strains of these viruses will become sensitive to restriction factors that have otherwise defied molecular identification. Using this strategy, one project will be to identify ancient antiviral genes that reveal unappreciated areas of host- pathogen conflict. For example, we are unmasking how the influenza immune evasion protein, NS1 orchestrates a complex, multifaceted rewiring of host antiviral networks using comparative screens with wild-type and NS1-deficient viruses. We are extending these studies to other virulent-attenuated virus pairings as well. Another project takes newly identified antiviral molecules that inhibit disparate virus families and define the molecular mechanism underlying viral inhibition. Here, we focus on the JADE family of proteins that regulate histone acetylation. We aim to uncover functional redundancy in the JADE family and to determine if it assembles a concerted antiviral epigenetic program. Another project is to leverage newly identified host-pathogen conflicts to discover the physiological functions of host- proteins that are poorly understood. In this regard, we are focusing on the CD300 family of receptor proteins. CD300 genes are undergoing rapid positive selection, yet their physiological function remains largely unexplored. We previously demonstrated that CD300lf is a protein receptor for murine norovirus. We will employ the biochemical, genetic, and cellular tools we developed for studying norovirus-CD300 interactions to define the physiological ligands of CD300 receptors. Determining the ligands for these orphan receptors will provide functional insight into these rapidly evolving proteins. Taken together, we plot a road map for discovering new host-pathogen interfaces, defining their molecular interactions during host defense, and how this relates to the physiological functions of these proteins and pathways.