The innate immune response is a critical component of host defense against infection. Defensins, one family of antimicrobial peptides, are an evolutionarily conserved class of innate immune effectors with well-described anti-bacterial activity; however, their role in anti-viral immunity is less well understood. The potent neutralization of diverse viruses by alpha-defensins has been described in vitro and in cell culture. To uncover general rules that dictate alpha-defensin activity against non-enveloped viruses, we will compare the molecular mechanisms of inhibition of human adenovirus and papillomavirus. These studies will combine biochemical, genetic, and structural studies to identify defensin binding determinants on the viral capsids. We will also identify the stage in human papillomavirus entry that is blocked by defensins. Using a novel 3D organoid culture system, we will then determine whether naturally secreted human and mouse alpha-defensins are anti- viral and block infection of primary intestinal epithelium. This investigation will provie a foundation for and aid in the interpretation of in vivo experiments to test the impact of alpha-defensin expression on viral pathogenesis. Thus, we will gain insight into the function of a critical component of the immune system that may be a common first line of defense against many viral pathogens. Development of 3D intestinal organoids, as part of these studies, will be valuable for dissecting a broad range of host interactions with enteric pathogens. And, these studies may aid in the development of alpha-defensins as therapeutics.