Date of Award
Doctor of Philosophy (PhD)
Philip E. Branton
Cellular transformation by human adenoviruses requires the expression of two early transcription units, E1A (0 to 4.5% of the genome) and E1B (4.5 to 11.2%). The products of both genes are required to disrupt normal cellular growth control and morphology. Early region 1A products alone can induce cellular immortalization, but establishment of fully transformed cells requires cooperation with E1B. Early region 1B produces two unrelated proteins, 496R and 176R, each of which can act independently with E1A to induce cellular transformation.
176R is a membrane associated protein whose molecular role in transformation is not known. Anti-peptide sera specific for the amino- and carboxy-termini of 176R were produced. These two sera were used for further purification and biochemical characterization of the protein. The 176R migrated as two species termed 18.5 and 19K on SDS-PAGE. The generation of the two species was not due to proteolysis, nor any post-translational modification investigated.
Two post-translational modifications of 176R were identified: phosphorylation and acylation. The phosphorylation site was mapped to serine residue 164, and a mutant virus in which this amino acid was altered, pm2204, was produced by oligonucleotide-directed mutagenesis. Characterization of pm2204 as well as an E1 plasmid carrying this mutation suggested that phosphorylation is not important for the biological activity of 176R. 176R was also found to contain covalently bound palmitate and myristate. Both tryptic and chymotryptic peptide mapping as well as CNBr cleavage were used to localize the acylation site. These experiments suggested that multiple acylation sites exist, and at least one site was localized to the amino terminal region by tryptic peptide mapping. The linkage of fatty acid to 176R was o found to be via an amide, or an unusually stable ester bond to an internal amino acid. Acylation of 176R likely facilitates its membrane association, but it was not possible to test this hypothesis without precise mapping of the acylation site(s) and construction of specific mutants.
McGlade, Catherine Jane, "Characterization of Human Adenovirus Type 5 Early Region 1B 176R Protein" (1990). Open Access Dissertations and Theses. Paper 3546.