PROTEINS INVOLVED IN REPLICATION & EXPRESSION OF HSV DNA

感染1型和2型单纯疱疹病毒(HSV-1和HSV-2) 在美国构成了一个主要的健康问题。这项提议旨在 提纯和鉴定HSV感染细胞中存在的蛋白质 参与病毒DNA的复制和表达。我们会 继续我们在主要HSV DNA结合(ICP8)方面的工作。亲和常数 对于ICP8的相互作用，将获得结合的化学计量 由单链和双链核酸组成。我们还将确定 这种蛋白质在核酸之间的交换速率。各种不同的 ICP8的功能结构域将通过特定的修饰来绘制 单个氨基酸残基和ICP8性质的研究 在不同位置含有温度敏感型损伤的分子。 ICP8的存在对同源和异源(HSV)的影响 将测定(哺乳动物、酵母和原核生物)DNA聚合酶。一个 已在HSV感染细胞中确定的DNA启动酶活性将 与世隔绝，独树一帜。将使用纯化的Primase活性 以尝试重建HSV复制设备。Z-DNA结合 HSV系统中的蛋白质和Z-DNA序列将被识别和 与世隔绝。将测试Z-DNA结合蛋白的结合特性- 市使用克隆的HSV限制性内切酶片段。Z-DNA序列 存在于HSV DNA中的病毒将使用氯仿进行增强活性测试- 苯尼考转乙酰基酶(CAT)测定。使用的方法包括： 荧光光谱、滤膜结合、分子克隆、DNA琼脂糖凝胶 纤维素层析法和Z-DNA免疫亲和力测定。

Infections with Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) constitute a major health problem in the United States. This proposal aims to purify and characterize proteins present in HSV infected cells which are involved in the replication and expression of the viral DNA. We will continue our work on the major HSV DNA binding (ICP8). Affinity constants and stoichiometries of binding will be obtained for the interaction of ICP8 with single and double-stranded nucleic acids. We will also determine the rate of exchange of this protein between nucleic acids. The various functional domains of ICP8 will be mapped by means of specific modification of individual amino acid residues and by studies on the properties of ICP8 molecules containing temperature sensitive lesions in various locations. The effect of the presence of ICP8 on homologous (HSV) and heterologous (mammalian, yeast and procaryotic) DNA poloymerases will be determined. A DNA primase activity which has been identified in HSV-infected cells will be isolated and characterized. The purified primase activity will be used in an attempt to reconstruct the HSV replication apparatus. Z-DNA binding proteins and Z-DNA suequences in the HSV system will be identified and isolated. The Z-DNA binding proteins will be tested for binding specifi- city using cloned HSV restriction enzyme fragments. Z-DNA sequences present in HSV DNA will be tested for enhancer activity using a chloram- phenicol transacetylase (CAT) assay. Methodology used will include: fluorescence spectroscopy, filter binding, molecular cloning, DNA agarose/ cellulose chromatography and Z-DNA immunoaffinity assays.