Project Summary/Abstract Defensins are small, innate immune peptides with broad antimicrobial activity, yet some pathogens, including non-enveloped viruses, have evolved methods to evade or co-opt defensins to enhance their infectivity. Resistance or enhancement is most pronounced for enteric viruses, leading to the hypothesis that fecal/oral transmission facilitates viral evolution to escape defensin neutralization. The studies herein will determine the molecular, cellular, and evolutionary mechanisms of viral escape from defensin action. Using a library of chimeric defensin peptides, we will determine the structural and biochemical properties of defensins essential to their activity against human adenovirus, rotavirus, and human papillomavirus (Aim1). Testing defensins against representatives from multiple viral families will provide insight into which defensin structural elements are globally required for activity, versus which are pathogen-specific. To more clearly elucidate the drivers of viral resistance to defensins, we will leverage the biological diversity across enteroviruses (EVs) to study how defensin sensitivity is related to viral transmission (Aim 2). In parallel, we will determine the cellular mechanism of EV neutralization by defensins, as well as the mechanism of EV defensin escape (Aim 3). This work will provide insight into how viral pathogens bypass the host innate immune system to promote infectivity, expanding our understanding of the host/pathogen evolutionary arms race. Many of the viruses included in these studies cause serious human illnesses, and this work will also point to new routes for antiviral development. The proposed work is designed to provide training in several techniques of virology and cellular biology, including cell culture and viral assays; high-performance liquid chromatography and peptide purification; biophysical binding assays; cellular mechanism studies; as well as microscopy. In addition to scientific expertise, the proposed training plan also includes education in scientific communication; mentorship; responsible research conduct; diversity, equity, and inclusion; as well as a career development plan for becoming a successful independent researcher. This work will be conducted in the University of Washington Microbiology Department, which is a hub for infectious disease research. This richly collaborative environment includes several faculty members and core facilities that provide the necessary resources, equipment, and expertise to enable completion of the proposed training plan.