The human papillomaviruses (HPVs) are causally linked to a number of human cancers. Over 100 different HPVs have been identified and many are associated with lesions that are at risk to progress to cancer. The papillomaviruses are divided into different genera based on the sequence relatedness of their genomes. Some of the alpha genus HPVs are associated with cervical cancer, other anogenital cancers, and approximately 20% of head and neck cancers. There is also suggestive but not yet compelling evidence implicating some of the beta genus HPVs in some non-melanoma skin cancers. The papillomavirus E2 gene product is conserved among papillomaviruses and functions as a major regulator of viral transcription, viral replication and genome maintenance. The E2 protein was first described in the 1980s to be a transcription factor that can activate viral transcription through E2 responsive elements located within the viral genome. E2 has additional functions however, and can serve either as a transactivator or a repressor of viral transcription depending upon the location and context of its binding sites within the viral genome. E2 is required for vira DNA replication as an auxiliary DNA replication factor that helps recruit the viral E1 helicase to the viral DNA replication origin. In addition, E2 is essential for genome maintenance in infected cells and, for some papillomaviruses, E2 functions by binding and linking the viral DNA to host cell mitotic chromosomes. In 2004 my laboratory identified the bromodomain protein Brd4 as a major E2 interacting protein and established its function as a cellular tether for the BPV E2/DNA complex on host mitotic chromosomes. In the past funding cycle, this grant focused on E2 and Brd4 and established additional roles for Brd4 in mediating various E2 functions. This grant renewal application focuses on E2. The first aim recognizes that there is a broader need for understanding Brd4 itself and will further extend our knowledge of E2 and Brd4 functions. The second is a new area for this grant, proposing a broad non-biased analysis of HPV E2 protein interactions across 20 different HPV types. As an important multifunctional regulatory protein required for several different aspects of the viral life cycle, E2 is an attractive target for the development of HPV antivirals. An ultimate goal of these experiments is to gain further insights into the cellular proteins and pathways that both regulate and mediate E2 functions, with the expectation that one or more of them might have the potential to be exploited for the identification or development of small molecule inhibitors. Such compounds would be useful tools for further studies of HPV-host cell interactions and could serve as potential leads for modeling novel antivirals to treat papillomavirus infections.