Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome
Cloning and mutagenesis of the murine gammaherpesvirus 68 genome as an infectious bacterial artificial chromosome
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DOI：
10.1128/jvi.74.15.6964-6974.2000
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发表时间：
2000-08-01
影响因子：
5.4
通讯作者：
Koszinowski, UH
中科院分区：
文献类型：
--
作者：
Adler, H;Messerle, M;Koszinowski, UH
伽马疱疹病毒对人类造成重要的感染，特别是在免疫功能低下的患者中。近年来，小鼠伽马疱疹病毒68(MHV-68)感染小鼠被发展为伽马疱疹病毒致病的小动物模型。MHV-68突变体的高效产生将有助于理解病毒基因在病毒-宿主相互作用中的功能，从而进一步增强该模型的潜力。为此，我们在大肠杆菌中克隆了MHV-68基因组作为细菌人工染色体(BAC)。在大肠杆菌中繁殖过程中，偶尔会观察到克隆的MHV-68基因组内部和末端重复序列中的自发重组事件，影响重复序列的拷贝数。绿色荧光蛋白基因被整合到克隆的BAC中，用于鉴定感染细胞。BAC载体序列的两侧有loxP位点，以允许使用重组酶Cre来切除这些序列，并允许产生具有野生型基因组特性的重组病毒。传染性病毒是从BAG克隆的MHV-68重组而来的。在细胞培养中生长的袋源病毒与野生型MHV-68没有什么区别。为了评价克隆的MHV-68基因组突变的可行性，我们构建了一个缺失开放阅读框4的突变病毒。转基因MHV-68现在可以在功能修饰的小鼠品系中进行分析，以评估伽马疱疹病毒基因在病毒-宿主相互作用和发病机制中的作用。
Gammaherpesviruses cause important infections of humans, in particular in immunocompromised patients. Recently, murine gammaherpesvirus 68 (MHV-68) infection of mice has been developed as a small animal model of gammaherpesvirus pathogenesis. Efficient generation of mutants of MHV-68 would significantly contribute to the understanding of viral gene functions in virus-host interaction, thereby further enhancing the potential of this model. To this end, we cloned the MHV-68 genome as a bacterial artificial chromosome (BAC) in Escherichia coli. During propagation in E. coli, spontaneous recombination events within the internal and terminal repeats of the cloned MHV-68 genome, affecting the copy number of the repeats, were occasionally observed. The gene for the green fluorescent protein was incorporated into the cloned BAC for identification of infected cells. BAC vector sequences were flanked by loxP sites to allow the excision of these sequences using recombinase Cre and to allow the generation of recombinant viruses with wild-type genome properties. Infectious virus was reconstituted from the BAG-cloned MHV-68. Grow th of the BAG-derived virus in cell culture was indistinguishable from that of wild-type MHV-68. To assess the feasibility of mutagenesis of the cloned MHV-68 genome, a mutant virus with a deletion of open reading frame 4 was generated. Genetically modified MHV-68 can now be analyzed in functionally modified mouse strains to assess the role of gammaherpesvirus genes in virus-host interaction and pathogenesis.