Doctor of Philosophy
Microbiology and Immunology
Dr. Joe Mymryk
Although remarkable biological diversity is exhibited by viruses, as obligate intracellular parasites, they rely on host cell functions. As such, viruses typically must overcome a set of host barriers that prevent infection. For human papillomaviruses (HPV) one of these barriers is the state of terminal differentiation of the host cell. For that purpose HPVs encode two major oncoproteins, E6 and E7, which combine their efforts to effectively uncouple cellular differentiation from the cell cycle arrest. The E7 proteins have no intrinsic enzymatic activity or DNA binding ability, but they bind and manipulate numerous host proteins. E7 is a modular oncoprotein and contains three conserved regions (CR), of which CR3 is a highly structured zinc-binding domain, and is the primary focus of this thesis. In order to study this highly structured portion of E7, we created a panel of surface-exposed mutants with the primary aim of preserving the structure of CR3, but disrupting potential cellular interactions. Although it was known that E7 CR3 exists as a dimer, the functional significance of dimerization had not been established. Using our panel of novel E7 mutants, we established that E7 does not need to dimerize in order to transform primary baby rat kidney cells. Furthermore, we utilized the same panel of surface-exposed mutants to characterize the contribution of CR3 in deregulation of one of the most vital interacting partners of E7, the retinoblastoma tumor suppressor (pRb). We establish that CR3 binds pRb independently of the well characterized high-affinity binding site in vivo, and show that this interaction is functionally important in overcoming the cell cycle arrest. We also find that E7 has additional mechanisms for targeted pRb degradation, aside from the known pathway. Additionally, we identify a novel binding partner of E7, the p190RhoGAP, and characterize this interaction using the surface-exposed mutants. We find that the interaction of E7 with p190RhoGAP contributes to deregulation of the RhoA GTPase and the actin cytoskeleton, and therefore likely represents an important aspect of HPV induced tumorigenesis. Considered together, these studies have expanded our knowledge of known processes and illuminated novel pathways utilized by HPV E7 in carcinogenesis.
Todorovic, Biljana, "Systematic Analysis of Residues in Conserved Region 3 of the Human Papillomavirus 16 E7 Oncoprotein" (2013). Electronic Thesis and Dissertation Repository. 1290.