HERPES SIMPLEX VIRUS TERMINASES

本建议的长期目标是通过以下方式了解这些机制： 其中单纯疱疹病毒（HSV）切割并包装基因组长度的病毒 DNA进入预先形成的衣壳。 包装HSV DNA所需的蛋白质 预期特异性结合和切割DNA Pac序列， 基因组末端和水解ATP以易位DNA。 HSV基因 可能实现这些步骤的产品包括编码的UL 15和UL 36 proteins. 数据表明，U15基因是DNA切割所必需的 包装，并编码两种蛋白质的表观M35，000和75，000。 的 HSV-1 U136编码蛋白VP1，因为它已被证明特异性地 结合HSV-1 Pac序列。 具体目标是：1. 确定 由UL15编码的蛋白质的来源。 区分 35，000-M蛋白通过蛋白水解表达的可能性 切割，一个新的启动子，或选择性剪接，我们将（i）确定 35，000m蛋白的N-末端氨基酸序列，或（ii）执行 含有表位标记的UL15基因的病毒的免疫学分析。 2. 确定UL15基因产物在DNA切割中的作用， 包装. 检查感染细胞中的DNA切割和包装 表达无效基因和单个UL15蛋白的病毒突变体将 说明每个基因产物的功能。 蛋白质和个体 将测试在异源系统中表达的UL15蛋白的ATP和 DNA结合。 引入携带突变的基因， 活性进入病毒基因组和检查DNA切割， 在感染这些病毒突变体的细胞中的包装将表明 U115蛋白DNA结合和ATP酶活性在病毒DNA切割和 包装. 3. 确定其他蛋白质在DNA包装中的作用。 VPI 将测试在异源系统中表达的蛋白质的DNA结合 ATP酶活性。 如果蛋白质不能结合Pac位点， 特别是在没有其他蛋白质的情况下，将采取步骤 鉴定表达140，000 M蛋白质的基因，该蛋白质先前显示为 与VP共同纯化。 突变体VP1基因将在异源细胞中表达。 系统，并将测试DNA结合和ATP酶活性， 确定这些活动所需的顺序。 分析细胞 感染表达无效和突变VPI蛋白的病毒突变体， 能够表达这些活性将指示VPI和VP1的功能- DNA切割和包装的相关活动。 研究也 建议识别和表征UL6。UL25和UL33编码 感染细胞中的蛋白质，并测试相应蛋白质的DNA结合 ATP酶活性。

The long term objective of this proposal is to understand the mechanisms by which herpes simplex virus (HSV) cleaves and packages genomic length viral DNA into preformed capsids. Proteins required for packaging HSV DNA would be expected to specifically bind and cleave DNA Pac sequences to form genomic termini and hydrolyze ATP for translocating DNA. The HSV gene products likely to implement these steps include the Ul15 and Ul36 encoded proteins. Data indicate that the Ul15 gene is required for DNA cleavage packaging, and encodes two proteins of apparent M 35,000 and 75,000. The HSV-1 U136 encoded protein VP1 because it has been shown to specifically bind the HSV-1 Pac sequences. The specific objectives are: 1. Determine the origin of the proteins encoded by UL15. To distinguish the possibilities that the 35,000-M, protein is expressed by proteolytic cleavage, a novel promoter, or alternative splicing we will (i) determine the N-terminal amino acid sequence of the 35,000 m protein or (ii) perform immunologic analyses of viruses containing epitopically tagged UL15 genes. 2. Determine the role of the UL15 gene products in DNA cleavage and packaging. Examination of DNA cleavage and packaging in cells infected with viral mutants expressing null genes and single UL15 proteins will indicate the function of each gene product. Both proteins and individual UL15 proteins expressed in heterologous systems will be tested for ATP and DNA binding. Introduction of genes bearing mutations ablating these activities into the viral genome and examination of DNA cleavage and packaging in cells infected with these viral mutants will indicate the role of U115 protein DNA binding and ATPase activities in viral DNA cleavage and packaging. 3. Determine the role of other proteins in DNA packaging. VPI proteins expressed in heterologous systems will be tested for DNA binding and ATPase activities. If the protein is not capable of binding Pac site DNA specifically in the absence of other proteins, steps will be taken to identify the gene expressing a 140,000 M, protein previously shown to copurify with VP. Mutant VP1 genes will be expressed in heterologous systems and will be tested for DNA binding and ATPase activities to identify sequences required for these activities. Analysis of cells infected with viral mutants expressing null and mutant VPI proteins not able to express these activities will indicate the function of VPI and VP1- associated activities in DNA cleavage and packaging. Studies are also proposed to identify and characterize the UL6. UL25, and UL33 encoded proteins in infected cells and test the respective proteins for DNA binding and ATPase activities.