INITIATION OF HSV DNA REPLICATION--UL9 INTERACTIONS

单纯疱疹病毒（HSV）为理解真核细胞的DNA复制提供了一个强大的遗传模型，因为它与真核细胞相比相对简单，而且它的单倍体基因组使它在遗传上更容易处理。HSV编码7种直接参与病毒DNA合成所需的蛋白质：ss DNA结合蛋白（ICP8）、过程DNA聚合酶（pol/UL42异源二聚体）、异源三聚体解旋酶/引物酶复合体（UL5、UL52和UL8），以及感染细胞中原生病毒DNA基因组DNA复制起始的蛋白质机制。病毒DNA合成的起始可能取决于UL9在输入病毒DNA中形成开放复合体的能力。已经描述了HSV DNA复制蛋白之间的多种蛋白-蛋白相互作用，通过极少数的功能已经阐明。UL9很可能在ori的其他蛋白质组装中起着核心作用，因为UL9与上述每种复合物（ICP8、UL8和UL42）的一个成员相互作用。我们假设UL42作为一种适配器蛋白，促进进程pol（可能与ICP8和解旋酶/引物酶一起）进入由UL9占据的活化序列。我们将结合生化和免疫学方法来更好地了解UL9- ul42相互作用对已知UL9活性的作用。本文提出了三个具体目标：1)使用BIAcore 2000和融合蛋白溶液竞争实验来测量UL9对UL42、pol和pol/UL42复合物的亲和力。当UL9与UL42络合时，它取代pol的能力将被确定，以及稳定络合物中蛋白质的化学计量学。2)确定UL42与不同结构和含量的DNA底物结合的能力和亲和力，以及UL42对这些性质的影响。3)通过前稳态和稳态动力学分析考察UL9-UL42相互作用的酶促机制，确定其功能意义。UL42在提高atp酶和解旋酶活性中的作用将被研究，以区分特异性和非特异性的作用机制。UL42对负载、初始速率常数和解旋酶活性的影响将被区分。我们在理解DNA复制蛋白之间相互作用的重要性方面所获得的知识将有助于定义体外体外依赖的DNA复制系统，并为开发旨在破坏病毒DNA复制复合体的新型抗病毒化合物确定靶标。

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.