IDENTIFICATION OF NOVEL HERPES-SIMPLEX VIRUS REPLICATIVE INTERMEDIATES BY FIELD-INVERSION GEL-ELECTROPHORESIS - IMPLICATIONS FOR VIRAL-DNA AMPLIFICATION STRATEGIES
IDENTIFICATION OF NOVEL HERPES-SIMPLEX VIRUS REPLICATIVE INTERMEDIATES BY FIELD-INVERSION GEL-ELECTROPHORESIS - IMPLICATIONS FOR VIRAL-DNA AMPLIFICATION STRATEGIES
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DOI：
10.1006/viro.1994.1375
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发表时间：
1994-08-01
期刊：
影响因子：
3.7
通讯作者：
SIMMONS, A
中科院分区：
文献类型：
--
作者：
ZHANG, X;EFSTATHIOU, S;SIMMONS, A
单纯疱疹病毒（HSV）DNA复制的许多方面还不清楚，我们的知识进展取决于高分子量复制中间体的准确表征。在目前的工作中，我们已经使用了改进的场反转凝胶电泳（FIGE）来分析感染的细胞DNA。将感染的Vero细胞包封并在琼脂糖微珠中操作，从而在电泳去除152-kb线性病毒基因组后，可以容易地从FIGE凝胶的威尔斯孔中回收完整的复制中间体。用Spat消化复制中间体，其切割病毒基因组一次，产生两个新的DNA片段（186和118 kb），除了预期的单位长度片段（152 kb），预测产生于由滚环复制产生的头到尾多联体。SpeI片段是先前未鉴定的多联体的产物，所述多联体含有相对于病毒基因组的长片段的方向的不同HSV异构体的头-尾排列。这种多联体在DNA合成的早期阶段非常突出，当时复制的DNA似乎仍处于环状构型，这提高了病毒基因组异构化与DNA复制的初始轮密切相关的可能性。此外，高分子量复制中间体仅侧接病毒基因组的长片段，表明在HSV DNA复制和病毒成熟期间独特的起始/终止或切割/包装机制，（C）1994 Academic Press，Inc.
Many facets of herpes simplex virus (HSV) DNA replication are not understood and advances in our knowledge depend on accurate characterization of high-molecular-weight replicative intermediates. In the present work, we have used a refinement of field-inversion gel electrophoresis (FIGE) to analyze infected-cell DNA. Infected Vero cells were encapsulated and manipulated in agarose microbeads, allowing intact replicative intermediates to be recovered easily from the wells of FIGE gels after electrophoretic removal of 152-kb linear viral genomes. Digestion of replicative intermediates with Spat, which cuts the viral genome once, generated two novel DNA fragments (186 and 118 kb), in addition to the expected unit-length fragment (152 kb) predicted to arise from head to tail concatemers generated by rolling-circle replication. The Spel fragments are the products of previously unidentified concatemers containing a head to tail arrangement of different HSV isomers, with respect to the orientation of the long segment of the viral genome. Such concatemers were prominent at an early stage of DNA synthesis when replicating DNA appeared still to be in a circular configuration, raising the possibility that isomerization of the viral genome is intimately linked to the initial round of DNA replication. Moreover, high-molecular-weight replicative intermediates were flanked exclusively by the long segment of the viral genome, indicating a unique initiation/termination or cleavage/packaging mechanism during HSV DNA replication and viral maturation, (C) 1994 Academic Press, Inc.