PROJECT SUMMARY/ABSTRACT High-risk human papillomaviruses (HPVs) infect mucosal epithelia and cause nearly 5% of cancers worldwide. Other HPVs infect cutaneous epithelia and cause benign or pathogenic lesions. One hallmark of papillomavirus biology is the virus’ ability to establish persistent infections in the proliferative basal layer of stratified squamous epithelia. The mechanisms used by papillomaviruses to direct cells to be retained in the basal layer of stratified epithelia, which are dynamic tissues that are subject to constant cell turnover, are poorly understood. The goal of this proposal is to test whether specific activities of the HPV E6 and E7 oncoproteins from mucosal and cutaneous HPV genotypes drive cell retention in the basal epithelial layer. The work will use a powerful model developed in my lab to track the migration of epithelial cells in engineered three-dimensional tissue. We hypothesize that different papillomaviruses activate YAP1 and/or inhibit Notch signaling to promote cell persistence in the basal epithelial layer. We have identified several mechanisms by which HPV E6 and E7 proteins repress epithelial differentiation in response to a stimulus in two-dimensional cell culture models. Our data support that the ability of HPV E6 and E7 proteins to repress the commitment to epithelial differentiation is correlated with their ability to promote basal identity. We found that high-risk mucosal HPV E7 proteins bind and degrade the tumor suppressor PTPN14. PTPN14 degradation activates YAP1, a powerful driver of epithelial cell proliferation and basal identity. When high-risk HPV E7 proteins activate YAP1, cells resist differentiation signals and are more likely to be retained in the basal epithelial layer. Cutaneous HPV E6 proteins bind the transcriptional co-regulator MAML1. E6 binding to MAML1 blocks Notch signaling, repressing differentiation. We predict that E6 binding to MAML1 directs basal identity. The work will systematically determine which activities of E6 and E7 promote basal identity and whether interfering with such activities will prevent HPV-infected cells from being retained in the basal epithelial layer. The aims are 1) to test whether inhibition of HPV E7-mediated PTPN14 degradation limits basal cell retention and 2) to determine whether HPV E6 proteins promote basal cell retention by repressing Notch signaling. We have developed a robust and physiologically relevant assay for epithelial basal cell identity. We are ideally poised to apply the assay to identify the basis of the basal retention activities of HPV oncoproteins. Understanding the mechanisms by which HPV establish and maintain basal epithelial identity, which is required for persistent infections, will elucidate principles of epithelial biology and tumor virology. It will, in the long term, enable new therapeutic approaches to treat HPV-positive cancers and premalignant lesions.