REGULATION OF BPV E1 PROTEIN ACTIVITY BY PHOSPHORYLATION

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Papillomaviruses are the causative agents of cutaneous and genital warts in the human population. they are also the likely cause of the majority of cases of human cervical cancer. Viral DNA in cells transformed by these viruses replicates as a stable episome in the nucleus. Papillomavirus DNA replication is tightly regulated, and takes place in synchrony with the host cell DNA. While we now know in some detail the steps leading to viral replication initiation, very little is understood about how papillomavirus DNA replication is regulated. The best characterized papillomavirus system is bovine papillomavirus type-1 (BPV- 1 or BPV). Two viral proteins, E1 and E2, are necessary and sufficient for BPV viral DNA replication. these proteins are likely to function by initiating the assembly of a functional replication complex at the viral replication origin, and by participating in the initial unwinding of the viral DNA. All other enzymatic activities of the replication process are carried out by host cell proteins. E1 is a phosphoprotein that recognizes and binds specifically to the BPV replication origin. In addition to its DNA binding activity, E1 forms a tight complex with the E2 protein, and has both ATPase and helicase activity. The role of the E1/E2 complex in DNA replication is not clear. E2 may alter the conformation of E1 to stimulate specific DNA binding activity. I propose to examine the role phosphorylation of the E1 protein plays in regulating E1 protein and DNA replication activity. We will answer the following questions: 1.) Which amino acids on the E1 protein are phosphorylated? To answer this question, we will use proteolytic digestion and peptide analysis of in vivo labeled E1 protein. 2.) What is the function of specific phosphoamino acids for E1 protein activity? Mutations will be made at phosphorylation sites and the effect of these mutations on the DNA binding, E2 protein binding, enzyme activity and replication activity of the E1 proteins will be determined. 3.) Are there changes in the pattern of E1 phosphorylation in different stages of the cell cycle? Our approach will be to isolate E1 protein from cells in specific stages of the cell cycle and analyze the phosphorylation pattern of these proteins. 4.) Which host cell enzymes control the phosphorylation state of the E1 protein? Antibodies and inhibitors to specific kinases and phosphatases will be used to identify enzymes active on the E1 protein. Host cell activities are known to be required for the control of BPV DNA replication. From our results, we therefore expect to learn something about the regulation of cellular, as well as viral, DNA replication, and mechanisms by which this control may be lost in cancer cells. These results may ultimately lead to new ideas for ways to inhibit the growth of papillomaviruses.