Development of methods that facilitate generation of recombinant herpesviruses using BAC system

近年来，一些实验室报道了在大肠杆菌中克隆疱疹病毒基因组作为细菌人工染色体（BACs）以及利用细菌重组机制操纵这些基因组的程序。然而，到目前为止报道的疱疹病毒- bacs要么是复制能力不足，要么是由于BAC载体插入导致一个或多个病毒基因缺失而具有传染性。作为一种用于疱疹病毒研究的多用途克隆，我们试图生成包含HSV-1全基因组且不丢失任何病毒基因的传染性单纯疱疹病毒(HSV) -BACs。我们的结果如下。(我)E。在U_L3和U_L4之间的基因间区插入一个以loxP位点为两侧的BAG，构建了含有HSV-1全长基因组（pYEbac102）的大肠杆菌（YEbac102）。（ii） pYEbac102是一个具有传染性的分子克隆，转染兔皮肤细胞可产生感染性病毒（YK304）。（iii）用YK304和表达Cre重组酶的重组腺病毒AxCANCre共同感染Vero细胞，BAC载体序列几乎可以从重组病毒YK304的基因组中完全切除。（iv）经检测，pYEbac102重组病毒在体内和体外均与野生型病毒无表型分化。因此，病毒在Vero细胞中生长和野生型病毒一样好，并且在小鼠脑内接种时表现出野生型毒力。(v)感染性分子克隆pYEbac102实际上可用于通过细菌遗传学对HSV-1基因组进行诱变，并产生了在αO基因的两个拷贝中携带氨基酸替换的重组病毒。pYEbac102将广泛应用于HSV-1基因工程重组的快速生成、HSV-1的基础研究和HSV载体在人类治疗中的发展。

In recent years, several laboratories have reported on the cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in E. coli and on procedures to manipulate these genomes using the bacterial recombination machinery. However, the herpesvirus-BACs reported so far are either replication-incompetent or infectious with a deletion of one or more viral genes due to the BAC vector insertion. As a multi-purpose clone for use in the research of herpesviruses, we attempted to generate infectious herpes simplex virus (HSV) -BACs containing the full genome of HSV-1 without any loss of^viral genes. Our results were as follows. (i)E. coli (YEbac102) harboring the full-length HSV-1 genome (pYEbac102) in which a BAG, flanked by loxP sites were inserted into the intergenic region between U_L3 and U_L4 was constructed, (ii) pYEbac102 was an infectious molecular clone, given that its transfection into rabbit skin cells resulted in production of infectious virus (YK304). (iii)' The BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus YK304 by co-infection of Vero cells with YK304 and a recombinant adenovirus AxCANCre expressing Cre recombinase. (iv) As far as was examined, the reconstituted viruses from pYEbac102 could not be phenotypically differentiated from wild-type viruses in vitro and in vivo. Thus the viruses grew as well in Vero cells as did the wild-type virus and exhibited wild-type virulency in mice on intracerebral inoculation. (v) The infectious molecular clone pYEbac102 is in fact useful for mutagenesis of the HSV-1 genome by bacterial genetics and a recombinant virus carrying amino acid substitutions in both copies of the αO gene was generated. pYEbac102 will be multi-applicable to the rapid generation of genetically engineered HSV-1 recombinants in basic research into HSV-1 and in the development of HSV vectors in human therapy.