GENETICS OF HSV DNA REPLICATION

单纯疱疹病毒在人群中流行， 用于多种临床疾病，其中一些是危及生命的 特别是在免疫功能低下的个体或新生儿中。 疱疹 单纯病毒1型（HSV-1）基因组编码7个病毒基因， 在一个特定的细胞中， 瞬时转染测定：UL 30和UL 42，其指定两个亚基 DNA聚合酶; UL 29，单链DNA结合蛋白; UL 5，UL 8和UL 10; UL 52，它们一起构成三蛋白解旋酶-引发酶复合物， UL 9，其指定HSV起源结合蛋白。 UL 5、8、9和52 这是最不丰富的和最不好的研究复制 蛋白质被选择用于这项研究。 本提案的目的 是对这四种基因进行详细的结构-功能分析 蛋白质以绘制负责催化活性的结构域， DNA结合（特异性和非特异性），相互作用位点和 分子内相互作用，核苷酸结合，以及 细胞内定位 第一个目标是构建必要的试剂， 随后的目标;这些包括用于表达每种蛋白质的构建体 在多种表达系统中， 抗血清 目标2是分离大量的点、插入点和 每个基因的缺失突变。 第三和第四个目标是 在aim 2中分离的突变体的体外和体内表征 进行DNA复制的整体能力和特定的 活性如特异性和非特异性DNA结合，NTP结合， 依赖DNA的ATP酶、解旋酶和引发酶。 核定位 每个蛋白质上的信号将通过观察定位来绘制，不仅 每种蛋白质的野生型和突变体形式，以及基因融合， 位于细胞质中的蛋白质。 第五个目标是定位区域 UL 5、UL 8、UL 52和UL 9负责蛋白质-蛋白质相互作用 使用遗传学和生物化学方法。 之间的潜在相互作用 这些蛋白质和复制复合物的其它成员也将被 追求。 遗传学方法包括：反显性干扰 突变，使用一种新的双杂交系统，以确定相互作用 第二位点抑制突变的分离， 通过免疫荧光共定位。 生化检测方法 相互作用包括：基于亲和力的方法，共免疫沉淀和 物理方法如甘油梯度和凝胶过滤。 是 预计这四个的详细结构-功能分析 复制蛋白不仅有助于我们理解 它们在复制复合体中的作用机制，但也可能 导致抗病毒治疗的新策略的发展。

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.