PROJECT SUMMARY The zoonotic transmission of viral pathogens from animals to humans contributes to the majority of emerging infectious diseases, which pose a substantial and increasing threat to human health. While host innate immunity—namely the production of Type I Interferon (IFN) and the subsequent production of Interferon Stimulated Genes (ISGS) with antiviral activities—plays an important role in determining viral tropism and limiting the cross-species transmission of viruses, much work remains on identifying and characterizing ISGs limiting viral tropism. Our lab has determined that while the human immunodeficiency virus (HIV-1) and non-human primate simian immunodeficiency viruses (SIV) are able to at least partly evade IFN-mediated defenses in cells from their cognate host, they remain exquisitely sensitive to such defenses in cells from unnatural hosts. Since HIV-1 and HIV-2 both arose from the cross-species transmission of diverse SIV species, the human and primate immunodeficiency viruses are a rich model system for studying the relationship between host innate immunity and viral adaptation. In order to identify human genes inhibiting SIV infection, we have modified a recently described high-throughput, CRISPR-based screening assay to identify human ISGs with activity against SIVmac239. Our preliminary data suggests that our model system is working as designed, as had identified a number of candidate genes that inhibit SIVmac infection in human cells. In Aim 1, we will determine the mechanism of action of novel ISGs with confirmed activity against SIVmac239. We will perform stepwise mechanistic studies to dissect the point of inhibition in the viral lifecycle. We will additionally perform co- immunoprecipitation and co-localization studies to identify viral components and cellular cofactors relevant to mechanism of inhibition. In Aim 2, we will use our established pipeline to screen for ISGs inhibiting diverse SIV strains. Preliminary data strongly suggests that we will be able to directly use the system we have developed to screen diverse strains of SIV. We will assess multiple SIV strains for sensitivity to IFN and screen those that are sensitive for inhibitory ISGs. We hypothesize that there will be both conserved and distinct inhibitory factors targeting the various SIV species. Combined, these approaches will reveal novel restriction factors that inhibit diverse SIVs in human cells, providing insight into the evolutionary adaptations that HIV-1 and HIV-2 made in order to successfully colonize humans.