Since the discovery that the v-sis oncogene of Simian Sarcoma Virus encodes a homolog of platelet-derived growth factor (PDGF), the sis/PDGF system has yielded important information concerning signal transduction and cellular transformation. This proposal examines structural aspects of the E5 oncoprotein of bovine papilloma virus (BPV), which mimics PDGF and activates PDGF receptors during autocrine transformation. These experiments will include the E5 proteins encoded by the clinically important human papillomaviruses (HPVs). The first specific aim will continue our extensive analysis of the structure and function of the BPV B5 oncoprotein based on many deletion and point mutants. The ability of BPV E5 to specifically activate the PDGF alpha- and beta-receptors, as well as other receptor tyrosine kinases, will be examined. Molecular modeling techniques will be utilized to formulate structural models of the E5 oncoprotein and, lastly, X-ray crystallography will be employed to achieve a complete structural determination. In the second specific aim, transforming B5 derivatives with heterologous transmembrane domains will be characterized. Our goal is to probe the structural features of the transmembrane domain of the E5 oncoprotein that contribute to its biological potency. In successful preliminary experiments, pools of degenerate oligonucleotides have generated large numbers of transmembrane substitutions between the BPV E5 oncoprotein and either the neu receptor or glycophorin A. These experiments will explore how small transmembrane peptides activate receptors resulting in biological transformation. In the third specific aim, we will build upon preliminary results demonstrating reversion of transformation by dominant negative E5 mutants. In the experiments proposed here, we will undertake a more detailed analysis of this dominant negative inhibition and, importantly, generalize this approach to novel E5-derived oncoproteins containing heterologous transmembrane domains. Our goal is to design effective dominant negative inhibitors that function by interacting with specific transmembrane domains required for the activity of transmembrane oncoproteins. Finally, we will examine the receptor specificity of HPV E5 proteins. HPV infection represents one of the most significant risk factors identified for ovarian cancer. These experiments will explore whether the HPV E5 proteins may function to stimulate the proliferation of cells through activation of a growth factor receptor. These experiments may thus be expected to increase our understanding of the molecular mechanisms employed by the HPVs to deregulate the control of cellular proliferation.
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