Regulation of Papillomavirus DNA Replication

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DESCRIPTION (provided by applicant): Cancer of the uterine cervix is one of the few human cancers known to be caused by a specific virus, the human papillomavirus. Despite its known etiology, there are no effective antiviral agents or vaccines to prevent or halt an infection and its progression to cervical cancer. A firm understanding of papillomavirus biology is essential in order to understand specific mechanisms of cancer induction, find methods to prevent infections, and gain insights into the biology of normal and cancer cells. The major objective of the proposed research is to understand mechanisms used by papillomaviruses to regulate their DNA replication. This proposal focuses on the activity of the major viral replication protein, El, and the role of phosphorylation in regulating El function. To fulfill this primary objective, two specific aims will be addressed: one, determine the replication phenotype of phosphorylation defective El mutant viral genomes; and two, analyze purified wild-type and mutant El proteins for biochemical activities and defects. To accomplish these research objectives, a combination of genetic and biochemical approaches will be used. Mutations will be introduced at sites that are phosphorylated on the El protein, and El mutant viral genomes will be analyzed for their replication phenotype in mammalian cells. El proteins that result in replication defects will be expressed in mammalian or insect cells and purified. Wild-type and mutant El proteins will be analyzed for specific El functions, including replication origin DNA binding, helicase enzyme activity, and binding to cellular DNA polymerase. This work will contribute significant new information to the fields of papillomavirus biology and DNA replication, and offer new insights into the unique properties of viral latency.