Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis
Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis
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DOI：
10.1038/7914
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发表时间：
1999-04-01
影响因子：
46.9
通讯作者：
Koszinowski, UH
中科院分区：
文献类型：
--
作者：
Brune, W;Ménard, C;Koszinowski, UH
疱疹病毒是动物和人类的重要病原体。几种疱疹病毒的大DNA基因组已经测序，但大多数推定基因的功能是难以捉摸的。确定哪些基因是其复制所必需的是重要的，以确定潜在的化疗靶点，设计疱疹病毒载体，并产生减毒疫苗。为此，我们最近报道，疱疹病毒基因组可以保持感染性细菌人工染色体（BAC）在大肠杆菌。在这里，我们描述了一个一步程序的随机插入诱变的疱疹病毒BAC使用Tn1721为基础的转座子系统。通过直接测序确定转座子插入位点，并通过用突变体基因组转染培养细胞来回收感染性病毒。通过将含有非感染性基因组的细胞与相应的野生型亚基因组片段连接来挽救致命突变。我们还通过细菌中的等位基因交换构建了回复突变体基因组。这些方法，这是普遍适用于任何克隆疱疹病毒基因组，将有助于分析该病毒家族的基因功能。
Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.