| Phosphorylation of a protein frequently serves to regulate the activity of the protein. Because the understanding of its regulation can be the key to understanding the function of the protein, efforts are often made to map the patterns of phosphorylation. VP16, the activator of the immediate early genes of herpes simplex, type 1, is phosphorylated during infection. This activator is viewed as a key factor not only in activation of the immediate early genes, but also in the control of the virion host shut-off function and in the assembly of virion. Because these functions occur at different stages in infection, it raises the question of how the virus program manages them during infection. Phosphorylation is a likely possibility and the identification of the phosphorylation sites of VP16 during infection could improve our understanding of how herpes simplex bypasses the host cells defense mechanisms and how it recruits the cellular systems to ensure its own survival.; The main question addressed in this thesis was whether specific phosphorylation events were involved in regulation of the function of VP16 during infection. In order to answer that question, it was first necessary to map sites of phosphorylation during infection. The mapping positively identified three sites of phosphorylation (Ser18, Ser353, Ser452) and indicated a fourth (Ser411) as a possible site. A site that had been reported to be phosphorylated outside of the context of infection (Ser375) was found not to be phosphorylated under the conditions tested.; The functions of the Ser375 and Ser411 in infection were tested. A virus strain with the Ser375Ala mutation displayed a significant reduction of immediate early activation when protein synthesis was inhibited. In contrast, when protein synthesis was allowed, the immediate early gene expression was only marginally affected, as compared to the wild type. The strain did not exhibit discernible deficiencies in virus proliferation, nor did the mutation of the site affect the phosphorylation of VP16 derived from infection by this virus. It was concluded that while the mutation affects one mode of activation by VP16, the IE genes are redundantly activated. The results favors a model in which Ser375 plays a functional role but does not serve as a phosphorylation site, and is therefore probably not as a site of regulation.; Virus strains carrying alanine, glutamate and threonine substitutions at Ser411 were found to be unaffected in proliferation. The strains were also found to support wild type levels of immediate early gene activation, regardless of whether protein expression was allowed in infection or not. The over-all phosphorylation of VP16 from these strains was comparable to wild type. It was concluded that Ser411 was not an essential site for VP16 function in infection, regardless of the phosphorylation state of the site. |
