REGULATION OF BPV E1 PROTEIN ACTIVITY BY PHOSPHORYLATION

乳头瘤病毒是皮肤和生殖器疣的病原体。 在人类人口中。他们也是大多数人的可能原因 人类宫颈癌病例的比例。转化细胞中的病毒DNA 这些病毒以稳定的Episome形式在细胞核内复制。 乳头瘤病毒DNA复制受到严格控制，发生在 与宿主细胞DNA同步。虽然我们现在更详细地了解了 导致病毒复制启动的步骤，人们了解的很少 关于乳头瘤病毒DNA复制是如何被调控的。最好的 特征的乳头瘤病毒系统是牛乳头瘤病毒1型(BPV-1 1或BPV)。两种病毒蛋白，E1和E2是必要和充分的 用于BPV病毒DNA复制。这些蛋白质可能通过以下途径发挥作用 在病毒处启动功能复制复合体的组装 复制起源，并通过参与最初的 病毒DNA。复制过程的所有其他酶活性是 由宿主细胞蛋白质执行。E1是一种磷蛋白，它可以 识别并特异性地结合BPV复制起始点。在……里面 除了它的DNA结合活性外，E1还与 E2蛋白，并同时具有ATPase和解旋酶活性。美国政府的角色 E1/E2复合体在DNA复制中的作用尚不清楚。雌二醇可能会改变 构象的E1刺激特异的DNA结合活性。我建议 检测E1蛋白的磷酸化在调节中的作用 E1蛋白和DNA复制活性。我们将回答以下问题 问题：1.)E1蛋白上的哪些氨基酸是磷酸化的？ 为了回答这个问题，我们将使用蛋白质分解消化和多肽 体内标记的E1蛋白的分析。2.)的功能是什么？ E1蛋白活性的特定磷酸氨基酸？突变将是 在磷酸化位点上发生的突变以及这些突变对 DNA结合、E2蛋白结合、酶活性和复制活性 将会测定出其中的。3.)是否发生了变化？ 细胞周期不同阶段的E1磷酸化模式？我们的 方法是从处于特定阶段的细胞中分离出E1蛋白 细胞周期和分析这些蛋白的磷酸化模式。 4.)哪些宿主细胞酶控制着E1的磷酸化状态 蛋白?特定激酶和磷酸酶的抗体和抑制物 将被用来鉴定在E1蛋白上活性的酶。宿主细胞 已知控制BPV DNA所需的活动 复制。因此，我们希望从我们的结果中学到一些东西 关于细胞和病毒DNA复制的调控，以及 癌细胞可能失去这种控制的机制。这些 结果可能最终导致抑制生长的方法的新想法 乳头瘤病毒。

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.