High-risk human papillomavirus (HPV) infection is responsible for nearly 5% of the global cancer burden. HPV infect stratified squamous epithelia and virus replication depends upon epithelial differentiation. To enable virus replication in differentiating cells, HPV-encoded proteins manipulate the cellular environment to promote proliferation and inhibit apoptosis. Much of this cellular reprogramming is enabled by the high-risk HPV E6 and E7 oncoproteins. High-risk HPV E7 bind and degrade the retinoblastoma tumor suppressor (RB1) to promote S- phase specific gene expression, thus driving proliferation and enabling viral genome replication. RB1 degradation is necessary, but not sufficient, for the oncogenic activity of high-risk HPV E7. Consequently, researchers have long sought to identify additional transforming activities of high-risk HPV E7. One candidate to account for this cooperative transforming activity is the protein tyrosine phosphatase PTPN14. PTPN14 is a putative tumor suppressor that is targeted for proteasome-mediated degradation by high-risk HPV E7 but not low-risk HPV E7. It inhibits the transcriptional co-regulator YAP1. We recently discovered that PTPN14 potently promotes epithelial differentiation. HPV E7 proteins inhibit differentiation and preliminary data suggests that this correlates with oncogenic activity and with PTPN14 degradation. This proposal tests the hypothesis that high-risk HPV E7 degrade PTPN14 to activate YAP1 and impair epithelial differentiation. The goal of the work is to integrate PTPN14 degradation into an updated model of oncogenic transformation by high-risk HPV. The aims are 1) to define the contribution of E7-mediated PTPN14 degradation to transformation and differentiation and 2) to determine the mechanism by which PTPN14 degradation represses differentiation. The current understanding of oncogenic transformation by HPV E6/E7 has not identified transforming activities that cooperate with RB1 inactivation nor does it explain how high-risk HPV oncoproteins impair differentiation. Our work will address both questions and we anticipate that it will define an additional activity required for HPV- mediated transformation. Because there are no antivirals available to treat existing HPV infections, identifying any new essential step in HPV-mediated transformation could have therapeutic potential. The ability to inhibit YAP1 and/or restore differentiation in HPV-positive cancers is especially promising. Understanding the mechanisms by which high-risk HPV inhibit differentiation will, in the long term, enable new therapeutic approaches in HPV-positive cancers and premalignant lesions.