Biochemical Mechanism of Poxvirus Replication

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复制酶是经验证的抗病毒化合物的靶标。该建议是痘病毒基因组复制中涉及的蛋白质的密集的生物化学表征。牛痘病毒是天花病毒的近亲，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尿嘧啶酰去糖基化酶（UDG）聚合酶辅助蛋白。E9/A20/D4这三种蛋白质组成了一个进行性E9全酶复制酶。此外，该病毒编码必需的D5 NTR、B1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，这为该提议提供了初步数据。我们计划确定这些蛋白质之间的接触点，并了解它们如何提供E9全酶对DNA的紧密但移动的抓地力。我们还将确定D5 NTR是否是解旋酶，以及它是否在功能上与E9全酶相互作用。或者，D5可能为复制机制提供其他功能。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种化合物库将被筛选E9全酶的抑制剂，作为本文开发的蛋白质和测定法可用于具有适当工具和专业知识的制药公司开发抗病毒化合物的原理证明。

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.