Vaccinia DNA Replication

描述(由申请人提供)：可以说，遗传物质的忠实复制是所有生物过程中最基本的。对于DNA病毒和细胞来说，一系列复杂的蛋白质调节DNA合成，并通过重组和修复来协调复制。在这项提案中，我们提出了继续研究痘苗病毒--典型的痘病毒--如何完成基因组复制的计划。痘病毒是仅有的几种感染周期仅限于受感染细胞胞浆的DNA病毒之一。这种独立于宿主核的自主性带来了不同寻常的挑战，因为病毒必须编码一个完整的复制装置，并建立专门的细胞质复制部位。痘病毒除了具有重要的生物医学意义外，还代表了有趣的模型系统。病毒复制蛋白作为开发合理的抗病毒疗法的靶点的重要性进一步加强了我们研究的影响。在下一个资助期间，我们将利用遗传学、生化和细胞生物学的方法，通过以下三个目标进一步阐明痘苗病毒的复制：目的I：分析E9/A20/UDG聚合酶全酶在复制和修复中的作用。我们将继续我们对DNA聚合酶复合体的研究，该复合体由E9DNA POL和由A20和D4蛋白组成的异源二聚体组成。我们将重点了解UDG/A20赋予POL程序的机制。由于D4是一种活性的尿嘧啶DNA糖基酶(UDG)，我们将研究复制相关的碱基切除修复是如何由病毒和细胞蛋白完成的。目的II：分析DNA复制的机制，研究D5Primase/Helicase、A50连接酶和G5核酸酶在DNA复制过程中的作用。我们将回顾痘病毒复制的机制，并评估领先和滞后链合成的可能参与。在这种情况下，我们将继续我们的分析基本的D5 ATPase，它可能既有底物酶又有解旋酶活性。我们还将研究病毒DNA连接酶和与FEN1核酸酶同源的G5蛋白在病毒基因组复制和成熟中的作用。目的三：分析病毒复制工厂的组装和结构。病毒复制发生在称为复制工厂的专用细胞质区域。这些工厂由来自内质网(ER)的膜分隔，并与之相关。我们将检验我们的假设，即丰富的H5蛋白在工厂内充当脚手架。此外，我们将使用靶向siRNA文库来鉴定有助于内质网重组的细胞膜转运蛋白，以用于复制工厂组装。

DESCRIPTION (provided by applicant): The faithful duplication of the genetic material is arguably the most fundamental of all biological processes. For DNA viruses as for cells, a complex repertoire of proteins mediates DNA synthesis and coordinates replication with recombination and repair. In this proposal, we present our plan for continued investigation of how vaccinia virus, the prototypic poxvirus, accomplishes genome replication. Poxviruses are among the only DNA viruses whose infectious cycle is restricted to the cytoplasm of infected cells. This autonomy from the host nucleus poses unusual challenges, in that the virus must encode a complete replication apparatus and establish dedicated cytoplasmic sites for replication. Poxviruses represent intriguing model systems in addition to being of significant biomedical importance. The impact of our studies is further strengthened by the importance of the viral replication proteins as targets for the development of rational antiviral therapies. During the next funding period, we will utilize genetic, biochemical and cell biological approaches to further elucidate the replication of vaccinia virus by focusing on the following three aims: AIM I: Analysis of the E9/A20/UDG polymerase holoenzyme in replication and repair. We will continue our studies of the DNA polymerase complex, which is comprised of the E9 DNA Pol and a heterodimeric processivity factor consisting of the A20 and D4 proteins. We will focus on understanding the mechanism whereby UDG/A20 confers processivity on Pol. Because D4 is an active uracil DNA glycosylase (UDG), we will investigate how replication-associated base excision repair is accomplished by viral and cellular proteins. AIM II: Analysis of the mechanism of DNA replication, and investigation of the contributions of the D5 primase/helicase, the A50 ligase, and the G5 nuclease to this process. We will revisit the mechanism of poxvirus replication and assess the possible involvement of both leading and lagging strand synthesis. In this context, we will continue our analysis of the essential D5 ATPase, which is likely to have both primase and helicase activity. We will also investigate the roles of the viral DNA ligase and the G5 protein, which has homology to FEN1 nucleases, in the replication and maturation of the viral genome. AIM III: Analysis of the assembly and structure of viral replication factories. Viral replication takes place in dedicated cytoplasmic regions known as replication factories. These factories are delimited by, and associated with, membranes derived from the endoplasmic reticulum (ER). We will test our hypothesis that the abundant H5 protein serves as a scaffold within the factories. Furthermore, we will use a targeted siRNA library to identify cellular membrane-trafficking proteins that contribute to the reorganization of the ER for the purpose of replication factory assembly.