Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes
Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes
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DOI：
10.3390/v6010354
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发表时间：
2014-01-01
期刊：
影响因子：
4.7
通讯作者：
Borst, Eva Maria
中科院分区：
文献类型：
--
作者：
Elbasani, Endrit;Gabaev, Ildar;Borst, Eva Maria
人巨细胞病毒（HCMV）基因组全长240 kb，可编码700多种基因产物，其中许多尚未鉴定。细菌人工染色体（BAC）克隆的CMV基因组的诱变极大地促进了病毒基因功能的分析。然而，必需蛋白质的作用往往仍然特别难以捉摸，因为他们的调查需要繁琐的建立合适的互补系统。在这里，我们表明，HCMV基因组可以引入到细胞中前所未有的效率，通过应用基于复制缺陷，灭活腺病毒颗粒（adenofection）的转染协议。在腺转染的几个允许的细胞类型与HCMV基因组携带突变的必需基因，转染率高达60%，观察到所有动力学类的病毒蛋白质的表达。这使得能够通过标准生物化学技术进一步分析转染的细胞。值得注意的是，HCMV基因组缺乏病毒DNA复制所必需的元件，如复制的裂解起点，仍然表达几种晚期蛋白。总之，腺转染允许在BAC转染的细胞中直接研究必需的HCMV基因，而不需要复杂的互补策略。
Human cytomegalovirus (HCMV) has a large 240 kb genome that may encode more than 700 gene products with many of them remaining uncharacterized. Mutagenesis of bacterial artificial chromosome (BAC)-cloned CMV genomes has greatly facilitated the analysis of viral gene functions. However, the roles of essential proteins often remain particularly elusive because their investigation requires the cumbersome establishment of suitable complementation systems. Here, we show that HCMV genomes can be introduced into cells with unprecedented efficiency by applying a transfection protocol based on replication-defective, inactivated adenovirus particles (adenofection). Upon adenofection of several permissive cell types with HCMV genomes carrying mutations in essential genes, transfection rates of up to 60% were observed and viral proteins of all kinetic classes were found expressed. This enabled further analyses of the transfected cells by standard biochemical techniques. Remarkably, HCMV genomes lacking elements essential for viral DNA replication, such as the lytic origin of replication, still expressed several late proteins. In conclusion, adenofection allows the study of essential HCMV genes directly in BAC-transfected cells without the need for sophisticated complementation strategies.