MOLECULAR BIOLOGY OF HERPES SIMPLEX VIRUS

我们之前已经证明，有缺陷的单纯疱疹病毒(HSV)基因组 在连续传代的病毒库中积累的由多个 标准病毒DNA的有限子集的从头到尾的重复 序列。连续包装的有缺陷的基因组可以通过 辅助病毒DNA与克隆单体种子共转染细胞的研究 (扩增片段)含有复制起点和裂解/包装 信号。HSV扩增片段也可以作为载体用于导入 将外来DNA序列插入到有缺陷的病毒基因组中，这些病毒基因组 在连续传代的病毒库中传播。使用扩增片段的测试 传播我们已经在标准病毒中映射了三个复制起点 DNA：发现两个相同的起源(ORI-1和ORI-1‘)被定位在 S成分的反向重复。第三个复制起点(ORI-2) 在L组分的独特序列中被映射。人类基因组DNA序列 ORI-2在细菌克隆过程中被删除，但得到有效修复 在与辅助病毒DNA共转染的过程中。我们建议继续 我们对ORI-2的研究，并探讨了其作用机制 删除-修复。这些研究和其他拟议的研究应有助于 了解单纯疱疹病毒DNA复制的机制。 通过未稀释的传代繁殖的一系列有缺陷的基因组 HSV-1株Justin被发现含有大的重复单位，这是 包括编码糖蛋白D(GD)和E的美国病毒DNA序列 (GE)。感染有缺陷的病毒库的细胞被发现 过度生产通用电气的前身。然而，NO增加了GD的合成 注意到了。构建包含多个GD的缺陷基因组的分析 基因模板也同样揭示了GD量的调节 是在病毒感染期间制造的。我们现在建议进一步调查 对这种重要的病毒糖蛋白的调节，它在 诱导宿主的免疫反应。

We have previously shown that defective herpes simplex virus (HSV) genomes which accumulated in serially passaged virus stocks consisted of multiple head-to-tail reiterations of limited subsets of the standard virus DNA sequences. Concatemeric packaged defective genomes could be derived by cotransfection of cells with helper virus DNA and cloned monomeric seeds (amplicons) containing a replication origin and a cleavage/packaging signal. HSV amplicons could also be used as vectors for the introduction of foreign DNA sequences into defective virus genomes, which were stably propagated in serially passaged virus stocks. Using tests for amplicon propagation we have mapped three replication origins in standard virus DNA: Two identical origins (ori-1 and ori-1') were found to map within the inverted repeat of the S component. The third replication origin (ori-2) mapped within the unique sequences of the L component. DNA sequences of ori-2 are deleted during bacterial cloning but are efficiently repaired during the cotransfection with helper virus DNA. We propose to continue our studies of ori-2 and to investigate the mechanism of the deletion-repair. These and other proposed studies should contribute to the understanding of the mechanism of HSV DNA replication. Defective genomes present in a series propagated by undiluted passaging of HSV-1 strain Justin were found to contain large repeat units, which included the Us viral DNA sequences encoding the glycoproteins D (gD) and E (gE). Cells infected with the defective virus stocks were found to overproduce the precursor to gE. However, no increased gD synthesis was noted. Analyses of defective genomes constructed to contain multiple gD gene templates have similarly revealed the regulation of the amount of gD made during viral infections. We now propose to further investigate the regulation of this important viral glycoprotein which plays a major role in the induction of the host immune response.