MOLECULAR BIOLOGY OF HERPES SIMPLEX VIRUS

我们以前已经表明，有缺陷的单纯疱疹病毒（HSV）基因组 其在连续传代的病毒储液中积累， 标准病毒DNA有限子集的头-尾重复 序列的 串联包装的缺陷基因组可以通过以下方法获得： 用辅助病毒DNA和克隆的单体种子共转染细胞 含有复制起点和切割/包装的（扩增子） 信号了 HSV扩增子也可用作导入载体 将外源DNA序列导入有缺陷的病毒基因组， 在连续传代的病毒原液中繁殖。 使用扩增子检测 我们已经在标准病毒中定位了三个复制起点 DNA：发现两个相同的起源（ori-1和ori-1 '）在DNA中定位。 S分量的反向重复。 第三复制起点（ori-2） 映射在L分量的独特序列内。 的DNA序列 ori-2在细菌克隆过程中被删除，但被有效修复 在与辅助病毒DNA共转染期间。 我们建议继续 我们对ori-2的研究，并探讨其作用机制， 删除修复 这些和其他拟议的研究应有助于 了解HSV DNA复制的机制。 缺陷基因组存在于一系列繁殖的未稀释传代， 发现HSV-1株Justin含有大的重复单元， 包括编码糖蛋白D（gD）和E（E）的US病毒DNA序列， （gE）。 发现用缺陷病毒原液感染的细胞 过量产生gE的前体。 然而，没有增加gD合成， 知道了 构建含有多个gD的缺陷基因组的分析 基因模板同样揭示了gD的量的调节， 在病毒感染期间产生。 我们现建议进一步调查 调节这种重要的病毒糖蛋白，它在 诱导宿主免疫反应。

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.