Papillomaviruses modify the growth properties of cells they infect as a normal consequence of the virus growth cycle, and thus provide useful models to study cell transformation. Our long term goal is to understand the biochemical mechanisms involved in papillomavirus induced cell transformation. This proposal is focused on the E5 oncogene of bovine papillomavirus type 1 (BPV1) which encodes the smallest known oncoprotein-an extremely hydrophobic protein of 44 amino acids. We have chemically synthesized the E5 oncoprotein and demonstrate that it functions to induce cellular DNA synthesis in growth arrested cells. Two peptide domains were identified in the E5 protein: the first located in the hydrophylic C-terminus is sufficient to induce cellular DNA synthesis; the second located in the hydrophobic central region confers about a 1000-fold higher DNA-synthesizing inducing activity to the molecule. We propose genetic and biochemical experiments to define structure-function relationships and to elucidate mechanism of action and cellular targets of E5 protein activity. First, we will chemically synthesize E5 deletion peptides and E5 single amino acid substitution peptides in order to delineate peptide domains and amino acid residues essential for biological activity. Second, we will determine the intracellular site of E5 function and the period during cell cycle progression when E5 acts. Third, we will use normal and mutant peptides and peptide-affinity protocols to identify and study cellular molecules that may serve as targets for E5 function. As a second investigation with a lower priority, we will attempt to map and identify the putative oncogene(s) encoded by human papillomavirus type 1 that may be involved in the transformation of rat embryo fibroblasts by transfection.
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