INITIATION OF HSV DNA REPLICATION--UL9 INTERACTIONS

单纯疱疹病毒(HSV)与真核细胞相比相对简单，而且其单倍体基因组使其更易于遗传处理，为理解真核细胞的DNA复制提供了强大的遗传模型。HSV编码直接参与病毒DNA合成和病毒DNA合成所需的7种蛋白质：单链DNA结合蛋白(ICP8)、过程性DNA聚合酶(POL/UL42异二聚体)、异源三聚体解旋酶/引物酶复合体(UL5、UL52和UL8)，以及在感染细胞中启动固有病毒DNA基因组DNA复制的蛋白质机制。病毒DNA合成的启动可能依赖于UL9在输入的病毒DNA中形成开放复合体的能力。HSV DNA复制蛋白之间的多种蛋白质-蛋白质相互作用已被描述，通过极少数的功能已被阐明。由于UL9与上述每个复合体(ICP8、UL8和UL42)中的一个成员相互作用，UL9很可能在ORI‘s组装其他蛋白质的过程中发挥核心作用。我们推测，UL42作为一种适配蛋白，促进了POL(可能与ICP8和解旋酶/启动酶一起)进入UL9所占据的激活ORI。我们将结合生化和免疫学方法，更好地了解UL9-UL42相互作用对已知UL9活性的作用。提出了三个具体的目标：1)利用Biacore 2000和与融合蛋白的溶液竞争实验来测量UL9对UL42、POL和POL/UL42复合体的亲和力。UL9与UL42络合时取代Poll的能力以及稳定络合物中蛋白质的化学计量比将被确定。2)测定不同结构和ORI含量的DNA底物的结合能力和亲和力，以及UL42对这些性质的影响。3)通过稳态前动力学分析和稳态动力学分析研究UL9-UL42相互作用的功能意义。将研究UL42在增强ATPase和解旋酶活性中的作用，以区分特定和非特定的作用机制。UL42对解旋酶活性的负荷、初始速率常数和加工性的影响将被区分开来。我们在了解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一个依赖于Ori的体外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点。

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.