The objective of this work is to gain a better understanding of the functional role of DNA topoisomerases in central events of herpes simplex virus (HSV-1) replication. Preliminary experiments are presented which strongly indicate that a viral topoiosomerase activity is associated with a delayed early gene product of 65,000 daltons. The proposal is based upon the development of assay systems which make it possible to measure the HSV-1 topoisomerase activity selectively against a background of cognate host enzymes. In addition, well characterized monospecific antibody directed against p65 will be used in mapping the gene, identifying the mRNA encoding p65 and establishing the direction of transcription. More precise mapping of the transcript will be performed with the purpose of identifying 5' and 3' termini. Finally, the protein coding as well as up and downstream regions will be analyzed by DNA sequencing. The next step of the project is to use the information derived at the gene level to rigorously test the hypothesis that the product of the gene encoding p65 is a topoisomerase. This goal will be achieved by constructing mutations in the gene and evaluating the resulting gene product using the topoisomerase assays which are specific the p65 associated enzyme. Finally, a major effort will be devoted toward establishing if p65 is an essential or non-essential gene in the viral replication cycle in vitro. The primary significance of this research is to advance our knowledge of the role of topoisomerases in essential genetic processes that accompany the replication of HSV-1. Topoisomerases cleave and covalently bind viral DNA, therefore studies of these unique DNA binding proteins are important since new avenues for antiviral chemotherapy will be opened.
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