Regulation of HPV Replication.

项目总结

PROJECT SUMMARY Human papillomaviruses (HPV) infect stratified squamous epithelia and link their productive life cycles to the differentiation of the host cell. HPVs infect cells in the basal layer of stratified epithelia and establish genomes as low copy nuclear episomes. When HPV infected cells migrate from the basal layer, they re-enter S/G2 phases in the most differentiated layers to allow for vegetative viral DNA replication in a process referred to as amplification. HPV replication is regulated by viral proteins as well as cellular factors that control cell cycle progression, differentiation and DNA damage repair pathways. Our recent studies have shown that activation of both the ataxia telangiectasia (ATM) pathway as well as the ataxia telangiectasia and Rad3-related (ATR) pathway is necessary for differentiation-dependent amplification of viral genomes and we have identified many members of these pathways that provide important functions. Our studies further indicate that enhanced levels of DNA breaks in HPV positive cells are responsible for activation of these pathways but the mechanism behind this increase has not been identified. DNA breaks can be induced by exposure to exogenous DNA damaging agents or through endogenous pathways such as through the action of topoisomerases. We have recently demonstrated that the type II topoisomerase TOP2is responsible for generating over 50% of DNA breaks in HPV positive cells. Topoisomerases regulate higher order chromatin structures through the transient breaking and re-ligation of one or both strands of the phosphodiester backbone of duplex DNA. Our studies have shown that the levels of TOP2 are increased by a 3 to 5-fold in cells with high-risk HPV genomes. Importantly, knockdown of TOP2 blocks HPV genome replication and moderately reduces viral transcription but has no effect on cell proliferation. Furthermore, knockdown of TOP2reduced the amount of DNA breaks in HPV positive cells which results in decreased DDR nuclear repair foci. While our assays demonstrated that over half of total DNA breaks in HPV positive cells were induced by TOP2 other factors must be responsible for the remaining breaks. Strong candidates for this activity are two other topoisomerases, TOP2and TOP1, and investigation of this possibility as well as an examination of the role of these enzymes in viral replication is the focus of this R21 application. These studies will provide important insights into how the differentiation-dependent HPV life cycle is regulated.