Characterization of Papillomavirus E6 Proteins that Bind Cellular E6AP

Papillomaviruses infect vertebrate epithelia and induce squamous papillomas in order to replicate. The papillomavirus E6 oncoprotein is important not only in maintenance of the papillomavirus genome but also in disrupting cellular functions to enable viral genome replication. E6 proteins exert their function by interacting with cellular proteins, one of which is the E3 ubiquitin ligase E6AP via its LXXLL motif (LQELL). E6 proteins from high- risk papillomaviruses hijack E6AP ubiquitin ligase activity to target the degradation of cellular p53, along with other cellular proteins. E6 proteins from low-risk papillomaviruses can bind E6AP and stimulate its ubiquitin ligase activity, but cellular degradation targets of the low-risk E6-E6AP complex are unclear. A cellular protein targeted for degradation by both the high and low-risk E6-E6AP complex has yet to be described. Although stimulating the ubiquitin ligase activity of E6AP is an important function of these E6AP-interacting E6 proteins, little is known regarding the precise mechanism(s) responsible. Finally, although both high and low-risk E6 proteins can bind full-length E6AP, only high-risk E6 can bind the short LQELL E6AP peptide. This indicates that there may be other regions within the E6AP protein that are important in mediating the formation of the E6- E6AP complex outside of the well-characterized E6AP LQELL motif.

In this dissertation we describe an E6AP-dependent degradation target common to both high and low-risk E6 proteins: NHERF1. The targeted degradation of NHERF1 by E6AP-binding E6 proteins is not only conserved among high and low-risk E6 proteins, but also among E6 proteins from papillomaviruses discovered in other vertebrate mammals. We identify a novel region within E6 that is required for NHERF1 degradation as well as a domain in NHERF1 that is both necessary and sufficient for E6-mediated degradation. We further find that E6 degradation of NHERF1 augments canonical Wnt/beta-catenin, a complex signaling pathway involved in regulating cell proliferation.

In this thesis we also examine regions within E6AP, outside of the LQELL motif, that are important in mediating the E6-E6AP interaction (which we termed E6AP auxiliary regions). We further identify one of these auxiliary regions as being required for both high and low-risk E6 proteins to stimulate E6AP E3 ubiquitin ligase activity to initiate degradation of cellular proteins. Our findings indicate there are two types of E6-E6AP interactions: those that can bind the isolated E6AP LQELL motif (Type I) and those that cannot and instead depend more heavily on E6AP auxiliary regions (Type II). Overall, our work has numerous therapeutic implications as NHERF1 may serve as a viable target for both high and low-risk infections and our identification of E6AP auxiliary regions will inform the design of therapies directed at disrupting the interaction of E6 and E6AP.