GENETICS OF HSV DNA REPLICATION

单纯疱疹病毒在人群中流行，并与 治疗各种临床疾病，其中一些会危及生命 尤其是在免疫功能受损的个体或新生儿中。疱疹 单纯疱疹病毒1型(HSV-1)基因组编码7个病毒基因，分别是 细菌中依赖于起源的质粒扩增的充要条件 瞬时转染法：UL30和UL42，它们指定两个亚基 DNA聚合酶；UL29，单链DNA结合蛋白；UL5，UL8和 UL52，它们共同组成一个三蛋白解旋酶-Primase复合体和 UL9，它指定HSV起源结合蛋白。UL5、8、9和52 在复制中，哪些是最不丰富和最不被研究的 蛋白质被选为这项研究的对象。这项提议的目的是 就是对这四个方面进行了详细的结构功能分析 蛋白质为了绘制负责催化活性的结构域， DNA结合(特定的和非特定的)，相互作用和 分子内相互作用、核苷酸结合和信号 细胞内定位。 第一个目标是构建必要的试剂来进行所有 后续目标；包括每种蛋白质表达的构建物 在各种表达系统中具体的表达和表征 抗血清。目标2是分离大量的点、插入和 每个基因的缺失突变。第三和第四个目标是实现 Aim-2突变体的体外和体内特性研究 对于执行DNA复制的总体能力和特定的 特定和非特定DNA结合、NTP结合、 依赖DNA的ATPase、解旋酶和底物酶。核子定位 每个蛋白质上的信号将不仅通过观察定位来映射 每种蛋白质的野生型和突变型，以及基因融合 位于细胞质中的蛋白质。第五个目标是定位上的区域 UL5、UL8和UL52和UL9负责蛋白质之间的相互作用 使用遗传和生化方法。潜在的相互作用 这些蛋白质和复制复合体的其他成员也将 被追捕。遗传方法包括：易显性干扰 突变，使用一种新的双杂交系统来鉴定相互作用 蛋白质，分离第二位点抑制突变和 免疫荧光共定位。生化检测方法 相互作用包括：基于亲和力的方法、免疫共沉淀和 物理方法，如甘油梯度和凝胶过滤。它是 预计这四个方面的详细结构功能分析 复制蛋白不仅有助于我们理解 它们在复制复合体内的作用机制，但也可以 导致了抗病毒治疗新策略的发展。

Herpes simplex viruses are endemic in the population and are responsible for a variety of clinical diseases some of which are life threatening especially in immunocompromised individuals or in newborns. The herpes simplex virus type-1 (HSV-1) genome encodes seven viral genes that are necessary and sufficient for origin-dependent plasmid amplification in a transient transfection assay: UL30 and UL42 which specify a two subunit DNA polymerase; UL29, the single strand DNA binding protein; UL5, UL8 and UL52 which together make up a three protein helicase-primase complex and UL9 which specifies the HSV origin binding protein. UL5, 8, 9, and 52 which are the least abundant and least well studied of the replication proteins have been chosen for this study. The objective of this proposal is to carry out a detailed structure-function analysis of these four proteins in order to map domains responsible for catalytic activities, DNA binding (specific and nonspecific), sites for inter- and intramolecular interactions, nucleotide binding, and signals for intracellular localization. The first aim is to construct the necessary reagents to carry out all subsequent aims; these include constructs for expression of each protein in a variety of expression systems and the characterization of specific antisera. Aim 2 is to isolate a large number of point, insertion and deletion mutations in each gene. The third and fourth aims are to carry out in vivo and in vitro characterization of mutants isolated in aim 2 for overall ability to carry out DNA replication and for specific activities such as specific and non specific DNA binding, NTP binding, DNA-dependent ATPase, helicase and primase. The nuclear localization signals on each protein will be mapped by observing localization not only of wild-type and mutant forms of each protein but also of gene fusions to cytoplasmically located proteins. The fifth aim is to locate regions on UL5, UL8 and UL52 and UL9 responsible for protein-protein interactions using genetic and biochemical approaches. Potential interactions between these proteins and other members of the replication complex will also be pursued. Genetic methods include: interference by transdominant mutations, use of a novel two-hybrid system to identify interacting proteins, isolation of second site suppressor mutations and colocalization by immunofluorescence. Biochemical methods for detecting interactions include: affinity-based methods, coimmunoprecipitation and physical methods such as glycerol gradients and gel filtration. It is anticipated that a detailed structure-function analysis of these four replication proteins will not only facilitate our understanding of the mechanisms of their action within the replication complex but may also lead to the development of novel strategies for antiviral therapy.