PROJECT SUMMARY/ABSTRACT High-risk human papillomavirus (HPV) is one of the most common sexually transmitted viruses and leads to a variety of human cancers causing significant mortality worldwide. HPV type 16 (HPV16) is the most common oncogenic genotype, accounting for over 50% of cervical cancer cases alone. It is well understood that viruses exploit many normal cellular processes to infect their host organisms, however, the mechanism of HPV16 endocytosis into epithelial cells has been shown to be completely independent of common canonical entry pathways including clathrin-, caveolin-, cholesterol-, flotillin-, and dynamin-dependent endocytosis. The discrete mechanisms of HPV16 endocytosis are poorly understood, but the process appears to be receptor- mediated and related to macropinocytosis with respect to actin dynamics. Although many membrane- associated molecules have been shown to be required for HPV16 endocytosis, to date, a mediator of this pathway is yet to be described. Our lab showed that the annexin A2 heterotetramer (A2t), a protein complex composed of two annexin A2 (A2) monomers and a S100A10 dimer, is required for the productive infection of HPV16 in epithelial cells. Both A2 and A2t have been implicated in many membrane-remodeling events including exocytosis, endosome biogenesis, and endosomal scaffolding through the association of A2 and A2t with F-actin, and in vitro studies have suggested an independent role for A2t in the endosomal escape of HPV16, specifically. Structurally, the annexin family of proteins are known to employ their disc-shape in order to to generate negative membrane curvature in a similar fashion to the curvature required to generate clathrin- coated pits, one of the most common mechanisms of endocytosis. Taken together, it is likely that A2t is involved in a distinct receptor-mediated endocytic pathway, and that this pathway is hijacked by HPV16. This study aims to determine the specific contributions of A2t in the endocytosis and intracellular trafficking of HPV16 with respect to morphology, composition, and subcellular distribution of HPV16-containing vesicles. In these studies A2t will be disrupted using CRISPR/Cas9 gene editing technology, and with a combination of cellular, molecular, and imaging based experiments, the role that A2t plays in the formation and processing of HPV-containing endosomes will be defined. Using HPV16 as a model ligand to study this previously undescribed pathway, findings from this study will shed light on the intracellular processing of the many other viruses with known A2t associations including HIV, cytomegalovirus, respiratory syncytial virus, and enterovirus 71. Ultimately, understanding the mechanics of this novel endocytic pathway may provide molecular targets for prevention strategies aimed at a variety of viral infections and therefore, have a broad impact on the field of virus-induced diseases.