My career goal is to become a leading independent scientist in the DNA tumor virus field. During the mentored phase of the Pathway to Independence award, I will work to further establish my publication record in the HPV field which will lay the groundwork for the research I will do as a faculty member. This project will provide training with new techniques (confocal microscopy, flow cytometry, gel shifts) that will be applicable to the research I perform as an independent investigator. The expertise of Dr. Laimins, as well as other faculty members in the Microbiology-Immunology Department, will be invaluable in learning the necessary skills to become a successful independent researcher. Human papillomaviruses are small, DNA viruses that are considered the etiological agents of cervical cancer. The infectious life cycle of HPV is dependent on cellular factors and epithelial differentiation. Differentiation triggers the productive phase of the life cycle, which includes viral genome amplification and virion production. The long-term goal of this project is to understand the mechanisms that regulate the activation of differentiation-dependent viral events. I recently demonstrated that HPV-31 stimulates a low level of caspase activation upon differentiation that is characteristic of the mitochondrial apoptotic pathway and necessary for amplification of viral genomes through cleavage of the viral replication protein E1. Based on the finding that caspase activation could be stimulated by both E6 and E7, and the identification of caspase cleavage sites in several HPV proteins, I hypothesize that: HPV, through E6 and E7, induces caspase activation in differentiating cells to modify viral protein function as a mechanism to activate late viral events. In specific Aim 1, to be initiated during the mentored phase, I will identify the mechanism(s) by which HPV proteins activates caspases in differentiating cells by examining the effect of E6 and E7 on mitochondrial membrane integrity and identifying domains in E6 and E7 that are necessary for caspase activation. In Specific Aim 2, to be initiated during the independent phase, I will determine how caspase cleavage of HPV proteins contributes to differentiation-dependent events in the life cycle by using in vitro and in vivo studies. Delineating the pathway by which E6 and E7 mediate caspase activation in differentiating epithelia to result in cleavage of viral proteins will offer insight as to how HPV modulates apoptotic machinery to facilitate not only the viral life cycle, but possibly transformation as well. RELEVANCE: Human papillomaviruses are the causative agents of cervical cancer. Understanding how the viral oncoproteins contribute to regulation of the viral life cycle will ultimately offer insight into how HPV causes disease and provide potential therapeutic targets.