FUNCTIONAL DOMAINS OF THE HSV-1 UL42 PROTEIN

单纯疱疹病毒(HSV)为人类免疫缺陷病毒提供了一种易于处理的遗传模型系统。 真核DNA复制，因为它至少编码7种蛋白质，这些蛋白质 直接参与病毒DNA的合成，所有这些都是 对病毒复制至关重要。这些蛋白质中有两种负责 用于延长DNA链--DNA聚合酶(Poll)催化核心 及其辅助蛋白UL42。这些蛋白质在体内形成稳定的复合体。 在体外和一起进行DNA合成。长期目标 该项目的主要目的是了解UL42在DNA复制和 它是如何与Poll相互作用进行DNA复制的。它是 假设对UL42络合能力的干扰 POL将阻止病毒DNA合成。因此，域的标识 对它们的交互至关重要的UL42和POL提供了目标 用于开发新型抗病毒化合物。由于结构上的原因 高度分化物种间POL辅助蛋白的保守性 定义这个综合体的结构可能会导致 更广泛的方法来合理设计能够 抑制肿瘤细胞的DNA合成。《公约》的具体目标 项目有1)确定UL42对DNA作用机制的影响 聚合反应的暂态动力学分析；2)使用酵母两个- 能够识别UL42和POL亚域的混合系统 活体内的物理相互作用；3)选择非相互作用的POL和 酵母双杂交系统中UL42突变体和基因外抑制因子的研究 在使用聚合酶链式反应或寡核苷酸诱变进行随机突变后 改变带电残基；4)测量野生型和 利用GST-融合蛋白体外突变蛋白并对其进行评价 它们与酵母中表现出的亲和力之间的关系 双杂交系统；5)构建HSV-1和杆状病毒重组体； 分析它们以进行依赖于ORI的复制，并将其作为筛选 干扰突变；6)确定蛋白质的层次结构：蛋白质 无DNA条件下蛋白质组装过程中的相互作用 用免疫荧光和共聚焦显微镜合成。

Herpes simplex virus (HSV) provides a tractable genetic model system for eukaryotic DNA replication since it encodes at least 7 proteins which are involved directly in the synthesis of viral DNA, all of which are essential for virus replication. Two of these proteins are responsible for elongation of the DNA chain--the DNA polymerase (pol) catalytic core and its accessory protein UL42. These proteins form a stable complex in vitro and together perform processive DNA synthesis. The long term goal of the project is to understand the role of UL42 in DNA replication and how it interacts with pol for processive DNA replication. It is hypothesized that interference with the ability of UL42 to complex with pol will block viral DNA synthesis. Thus, identification of the domains of UL42 and pol which are critical for their interaction provides a target for the development of novel anti-viral compounds. Due to the structural conservation of pol accessory proteins among highly divergent species, it is possible that defining the structure of this complex will lead to an even broader approach for the rational design of compounds capable of inhibiting DNA synthesis in neoplastic cells. The specific aims of the project are 1) to determine the effect of UL42 on the mechanism of DNA polymerization using transient kinetic analysis; 2) to use the yeast two- hybrid system to identify the subdomains of UL42 and pol capable of physical interaction in vivo; 3) to select for non-interacting pol and UL42 mutants and extragenic suppressors in the yeast two-hybrid system following random mutagenesis using PCR or oligo-directed mutagenesis to alter charged residues; 4) to measure the affinities of wild-type and mutant proteins in vitro using GST-fusion proteins and to evaluate the relationship between these and the affinities demonstrated in the yeast two-hybrid system; 5) to construct HSV-1 and baculovirus recombinants and to analyze them for ori-dependent replication and as a means to screen for interference mutations; 6) to determine the hierarchy of protein:protein interactions during the assembly of proteins in the absence of DNA synthesis using immunofluorescence and confocal microscopy.