REPLICATION AND EXPRESSION OF HERPES SIMPLEX VIRUS DNA

此前已发现单纯疱疹病毒(HSV)基因组有缺陷 由多个首尾重复的有限区域组成 标准病毒DNA。此外，与缺陷和缺陷共同感染的细胞 辅助病毒已被证明过度产生病毒多肽，这种病毒多肽 编码在有缺陷的基因组重复单元中。这项研究建议 在这一应用中涉及到利用这种有缺陷的HSV基因组来研究 单纯疱疹病毒感染细胞中病毒DNA复制和病毒基因表达。 具体地说，这些研究是基于我们之前的观察结果 用“寄养”辅助病毒DNA和“种子”共转染细胞 单纯疱疹病毒缺陷基因组的单体重复单位导致 串联缺陷病毒DNA分子的再生 包装成单纯疱疹病毒病毒粒子。我们建议利用这项计划 缺陷基因组再生研究单纯疱疹病毒顺式复制功能 DNA，包括复制起点，被识别为切割的序列 串联复制中间体，以及指定 将病毒DNA包装成核衣壳。此外，我们计划 构建含细菌的单纯疱疹病毒克隆扩增载体 复制来源和抗药性标记使其能够生长和 在细菌中选择适合插入的限制性内切酶位点 添加的DNA序列，以及一组顺式复制功能 允许将其用作种子重复单位。该HSV载体将用于克隆 并扩增标准HSV DNA的特定区域，包括该区域 跨度坐标0.37-0.44，已知包含一簇DNA 复制功能。通过使用新再生的有缺陷的HSV 基因组及其衍生的删除的衍生品，我们建议定义 特定病毒基因的结构域，并研究假定的识别位点 它们决定了HSV中病毒基因表达的调控方案 被感染的细胞。

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.