REPLICATION AND EXPRESSION OF HERPES SIMPLEX VIRUS DNA

单纯疱疹病毒（HSV）的基因组缺陷先前已被证明 由有限区域的多个头到尾排列组成， 标准病毒DNA 此外，细胞共感染缺陷和 辅助病毒已被证明会过量产生病毒多肽， 都被编码在有缺陷的基因组重复单元中。 建议的研究 在本申请中涉及使用这种缺陷型HSV基因组来研究 HSV感染细胞中的病毒DNA复制和病毒基因表达。 具体来说，这些研究是基于我们以前的观察， 用“培养”辅助病毒DNA和“种子”共转染细胞 HSV缺陷基因组的单体重复单元导致 多联体缺陷病毒DNA分子的再生， 包装成HSV病毒体。 我们建议利用这项计划， 再生缺陷基因组以研究HSV的顺式复制功能 DNA，包括复制起点，被识别用于切割的序列 多联体复制中间体的序列，以及指定多联体复制中间体的序列。 将病毒DNA包装成核衣壳。 此外，我们计划 构建HSV克隆扩增载体，其将含有细菌 复制起点和耐药性标记，使其能够生长， 在细菌中选择适合插入的限制性酶切位点 添加的DNA序列，以及一组顺式复制功能， 使其能够用作种子重复单元。 该HSV载体将用于克隆 并扩增标准HSV DNA的特定区域，包括 跨度坐标0.37-0.44，已知含有一簇DNA 复制功能。 通过使用新再生的缺陷HSV 基因组和删除衍生物从他们，我们建议定义 特定病毒基因的结构域，并研究推定的识别位点 其决定了HSV中病毒基因表达的调节方案 感染细胞

Defective genomes of herpes simplex virus (HSV) have been previously shown to consist of multiple head to tail reiterations of limited regions of the standard virus DNA. Furthermore, cells co-infected with defective and helper viruses have been shown to overproduce the viral polypeptides which are encoded within the defective genome repeat units. The studies proposed in this application involve the use of such defective HSV genomes to study viral DNA replication and viral gene expression in the HSV infected cell. Specifically, these studies are based on our previous observations that cotransfections of cells with "foster" helper virus DNA and "seed" monomeric repeat units of the HSV defective genomes result in the regeneration of concatemeric defective virus DNA molecules which can be packaged into HSV virions. We propose to make use of such a scheme for the regeneration of defective genomes to study cis replication functions of HSV DNA, including replication origins, sequences recognized for the cleavage of concatemeric replicative intermediates, and sequences specifying the packaging of viral DNA into nucleocapsids. Furthermore, we plan to construct an HSV cloning-amplifying vector which will contain a bacterial replication origin and drug resistance markers enabling its growth and selection in bacteria, restriction enzyme sites suitable for the insertion of added DNA sequences, and the set of cis replication functions which will enable its use as seed repeat units. This HSV vector will be used to clone and amplify specific regions of the standard HSV DNA, including the region spanning coordinates 0.37-0.44, known to contain a cluster of DNA replication functions. By use of the newly regenerated defective HSV genomes and deleted derivatives derived from them, we propose to define the domains of specific viral genes, and to study putative recognition sites which determine the regulated scheme of viral gene expression in the HSV infected cell.