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INITIATION OF HSV DNA REPLICATION--UL9 INTERACTIONS

HSV DNA 复制的启动--UL9 相互作用

基本信息

批准号：
6525501
负责人：
Deborah S. Parris
金额：
$ 28.43万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-08-01 至 2004-07-31
项目状态：
已结题

项目摘要

Herpes simplex virus (HSV) provides a powerful genetic model for understanding eukaryotic DNA replication because of its relative simplicity compared to eukaryotic cells and because its haploid genome renders it more genetically tractable. HSV encodes 7 proteins directly involved in and required for viral DNA synthesis: a ss DNA binding protein (ICP8), a processive DNA polymerase (pol/UL42 heterodimer), a heterotrimeric helicase/primase complex (UL5, UL52, and UL8), and a protein mechanism of initiation of DNA replication of native viral DNA genomes in infected cells. Initiation of viral DNA synthesis is likely to be dependent upon the ability of UL9 to form an open complex at ori's present in the input viral DNA. A multiplicity of protein-protein interactions among the HSV DNA replication proteins has been described, through the functions of very few have been elucidated. UL9 most likely plays a central role in assembling the other proteins at ori's since UL9 interacts with a member of each of the complexes described above (ICP8, UL8, and UL42). We hypothesize that UL42 acts as an adapter protein which facilitates the entry of the processive pol (perhaps together with ICP8 and helicase/primase) into an activated ori occupied by UL9. We will combine biochemical and immunologic approaches to better understand the function of the UL9-UL42 interaction on the known UL9 activities. Three specific aims are proposed: 1) To measure the affinity of UL9 for UL42, pol, and pol/UL42 complex using the BIAcore 2000 and solution competition experiments with fusion proteins. The ability of UL9 to displace pol when it is complexed to UL42 will be determined as well as the stoichiometry of proteins in stable complexes. 2) To determine the ability and affinity to bind to different DNA substrates which vary in their structure and ori content, and the effect of UL42 on these properties. 3) To determine the functional significance of UL9-UL42 interaction by examining enzymatic mechanisms using pre-steady state and steady-state kinetic analysis. The role of UL42 in enhancing ATPase and helicase activities will be studied to differentiate specific from non-specific mechanisms of action. Effects of UL42 on load, initial rate constants, and processivity of helicase activities will be distinguished. The knowledge we gain in understanding the importance of interactions among DNA replciation proteins will help in defining an in vitro ori-dependent DNA replication system and in defining targets for the development of novel anti-viral compounds designed to disrupt the viral DNA replication complex.

单纯疱疹病毒（HSV）提供了一个强大的遗传模型，了解真核细胞的DNA复制，因为它相对简单的真核细胞相比，因为它的单倍体基因组使它更容易遗传。HSV编码7种直接参与病毒DNA合成并为病毒DNA合成所需的蛋白质：ss DNA结合蛋白（ICP 8）、进行性DNA聚合酶（pol/UL 42异二聚体）、异三聚体解旋酶/引发酶复合物（UL 5、UL 52和UL 8）以及感染细胞中天然病毒DNA基因组DNA复制起始的蛋白质机制。病毒DNA合成的起始可能依赖于UL 9在输入病毒DNA中存在的ori处形成开放复合物的能力。HSV DNA复制蛋白之间的蛋白质-蛋白质相互作用的多样性已被描述，通过很少的功能已被阐明。由于UL 9与上述复合物（ICP 8、UL 8和UL 42）中的每一个的成员相互作用，因此UL 9最可能在ori处组装其他蛋白质中起中心作用。我们假设UL 42作为一个衔接蛋白，它有助于进行性pol（可能与ICP 8和解旋酶/引发酶一起）进入由UL 9占据的活化ori。我们将结合联合收割机生物化学和免疫学的方法，以更好地了解功能的UL 9-UL 42相互作用的已知UL 9活动。提出了三个具体目标：1）使用BIAcore 2000和融合蛋白的溶液竞争实验来测量UL 9对UL 42、pol和pol/UL 42复合物的亲和力。将测定UL 9与UL 42复合时置换pol的能力以及稳定复合物中蛋白质的化学计量。2)确定与不同结构和ori含量的DNA底物结合的能力和亲和力，以及UL 42对这些性质的影响。3)通过使用预稳态和稳态动力学分析检查酶机制来确定UL 9-UL 42相互作用的功能意义。将研究UL 42在增强ATP酶和解旋酶活性中的作用，以区分特异性和非特异性作用机制。将区分UL 42对负载、初始速率常数和解旋酶活性的持续合成能力的影响。我们在理解DNA复制蛋白之间相互作用的重要性方面获得的知识将有助于定义体外依赖性DNA复制系统，并定义用于开发旨在破坏病毒DNA复制复合物的新型抗病毒化合物的靶点。