HERPES SIMPLEX VIRUS TERMINASES

这项建议的长期目标是通过以下方式了解这些机制 哪种单纯疱疹病毒(HSV)裂解并包装基因组长度的病毒 DNA进入预制衣壳。包装HSV DNA所需的蛋白质将 有望特异性地结合和切割DNA Pac序列以形成 基因组末端和水解三磷酸腺苷以转运DNA。单纯疱疹病毒基因 可能实施这些步骤的产品包括Ul15和Ul36编码 蛋白质。数据表明，ul15基因是DNA切割所必需的 编码两种蛋白质，表观分子量分别为35,000和75,000。这个 HSV-1 U136编码VP1蛋白是因为它被证明是特异性的 结合HSV-1 Pac序列。具体目标是：1.确定 UL15编码的蛋白质的来源。要区分 35,000-M蛋白通过蛋白水解物表达的可能性 切割，一种新的启动子，还是选择性剪接，我们将(I)确定 35000m蛋白质的N-末端氨基酸序列或(Ii)执行 含有表位标记UL15基因的病毒的免疫学分析。 2.确定UL15基因产物在DNA切割和转录中的作用 包装。感染细胞DNA裂解和包装的检测 表达空基因的病毒突变体和单个UL15蛋白将 指出每个基因产物的功能。蛋白质和个体 在异源系统中表达的UL15蛋白将被测试ATP和 DNA结合。引入携带突变的基因来消融这些 病毒基因组的活性和DNA裂解和DNA裂解的检查 在感染了这些病毒突变体的细胞中包装将表明这种作用 U115蛋白DNA结合和ATPase活性在病毒DNA裂解和 包装。3.确定其他蛋白质在DNA包装中的作用。VPI 在异源系统中表达的蛋白质将进行DNA结合测试 和ATPase活性。如果蛋白质不能结合Pac位点 DNA特别是在没有其他蛋白质的情况下，将采取措施 鉴定之前显示的表达140,000 M蛋白的基因 与副总统合作。突变的VP1基因将在异源植物中表达 系统，并将测试DNA结合和ATPase活性以 确定这些活动所需的序列。对细胞的分析 感染了表达Null和突变VPI蛋白的病毒突变体 能够表达这些活动将表明VPI和VP1的功能- DNA切割和包装中的相关活动。研究也在进行 提议对UL6进行识别和定性。UL25和UL33编码 感染细胞中的蛋白质，并测试各自的蛋白质的DNA结合 和ATPase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.