Biochemical Mechanism of Poxvirus Replication

痘病毒复制的生化机制

• 批准号: 6954760
• 负责人: MICHAEL E O'DONNELL
• 金额: $ 32.31万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6954760
• 关键词: Biochemical; Mechanism; Poxvirus; Replication

DESCRIPTION (provided by applicant): It is difficult to comprehend the affects of a smallpox outbreak. But the possibility exists, as remote as it may be. One line of control is the use of antiviral compounds. DNA replication enzymes are validated targets of antiviral compounds. This proposal is an intensive biochemical characterization of the proteins involved in replication of the poxvirus genome. The vaccinia virus, a close relative of smallpox, has duplex DNA genome that replicates entirely in the cytoplasm and encodes most or all of its own DNA replication proteins. Among these are the E9 DNA polymerase, A20 polymerase accessory protein, and the D4 uracyl deglycosylase (UDG) polymerase accessory proteins. These three proteins, E9/A20/D4 constitute a processive E9 holoenzyme replicase. In addition the virus encodes the essential D5 NTPase, Bl kinase and a single strand DNA binding protein called 13. We have recently purified recombinant E9 and A20/D4 complex and have constituted the E9 holoenzyme which forms the preliminary data for this proposal. We plan to determine the contact points between these proteins and understand how they provide a tight yet mobile grip of the E9 holoenzyme to DNA. We will also determine if the D5 NTPase is a helicase, and whether it functionally interacts with E9 holoenzyme. Alternatively, D5 may supply some other function to the replicative machinery. We will also examine the role(s) of the 13 SSB in D5 and E9 holoenzyme action. Finally, Rockefeller University's 20,000 chemical compound library will be screened for inhibitors of the E9 holoenzyme as a proof of principle that the proteins and assays developed herein can be useful to a pharmaceutical company with the proper tools and expertise to develop an antiviral compound.

描述（由申请人提供）：很难理解天花爆发的影响。但这种可能性是存在的，尽管可能性很小。一种控制方法是使用抗病毒化合物。 DNA 复制酶是抗病毒化合物的有效靶标。该提案对参与痘病毒基因组复制的蛋白质进行了深入的生化表征。痘苗病毒是天花的近亲，具有完全在细胞质中复制的双链 DNA 基因组，并编码其自身的大部分或全部 DNA 复制蛋白。其中包括 E9 DNA 聚合酶、A20 聚合酶辅助蛋白和 D4 尿酰去糖基酶 (UDG) 聚合酶辅助蛋白。这三种蛋白E9/A20/D4构成了持续性E9全酶复制酶。此外，该病毒还编码必需的 D5 NTPase、Bl 激酶和称为 13 的单链 DNA 结合蛋白。我们最近纯化了重组 E9 和 A20/D4 复合物，并构建了 E9 全酶，这构成了该提案的初步数据。我们计划确定这些蛋白质之间的接触点，并了解它们如何为 E9 全酶提供与 DNA 紧密且可移动的结合。我们还将确定 D5 NTPase 是否是解旋酶，以及它是否与 E9 全酶在功能上相互作用。或者，D5 可以向复制机器提供一些其他功能。我们还将研究 13 SSB 在 D5 和 E9 全酶作用中的作用。最后，洛克菲勒大学的 20,000 种化合物库将筛选 E9 全酶抑制剂，作为原理证明，证明本文开发的蛋白质和测定方法可用于拥有适当工具和专业知识的制药公司开发抗病毒化合物。