ABSTRACT MULTIPLE ROLES OF BRD4 IN THE HUMAN PAPILLOMAVIRUS LIFE CYCLE Christine M. Helfer Jianxin You While human papillomavirus (HPV) vaccines protect against acquiring new infections, there is currently no antiviral treatment for eradicating persistent HPV infections. In this study, I demonstrated that the cellular chromatin binding protein, Brd4, in association with HPV E2 protein, is important for multiple HPV functions including replication, maintenance of viral genomes, and regulation of viral gene transcription. These studies suggest that the E2–Brd4 complex could be an effective target to disrupt the HPV life cycle. Using bimolecular fluorescence complementation, we demonstrate that E2 from high–risk HPV16 interacts with Brd4 on cellular chromosomes throughout mitosis while the BET bromodomain inhibitor, JQ1(+), dissociates Brd4–E2 complexes from mitotic chromosomes. These results suggest that Brd4 is important for tethering HPV16 E2 to mitotic chromosomes for stable viral genome maintenance and that abrogating Brd4's chromatin association might disrupt stable HPV genome maintenance. I also found that JQ1(+) treatment of cells stably maintaining papillomavirus genomes reduces viral mRNA levels, demonstrating that HPV association with cellular chromatin through Brd4 is essential for HPV transcription and further supporting the importance of Brd4 for the HPV life cycle. My work also identified a novel role of Brd4 in HPV16 DNA replication. Immunofluorescence analyses show Brd4 is recruited to nuclear foci actively replicating HPV16 genomes. Replication assays further confirm that Brd4 is essential for HPV16 genome replication. Interestingly, JQ1(+) treatment stimulates viral genome replication. Since HPV genome amplification is normally limited to upper epithelial layers, we predict premature stimulation of viral DNA amplification induced by JQ1(+) in basal epithelial cells might activate host immune responses to clear HPV infection. Finally, this work identified a specific function of Brd4 in papillomavirus transcription activation. Using ChIP analysis and an E2–responsive luciferase assay, we show Brd4 actively recruits P–TEFb to papillomavirus genomes to support E2 transactivation function. Together, this study uncovers two novel functions of Brd4 in the HPV life cycle, improving our understanding of this complex virus–host relationship. Furthermore, we identify the E2–Brd4 complex as a promising antiviral target for eliminating HPV persistent infection.