Department of Virology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Viruses. 2014 Jan 23;6(1):354-70. doi: 10.3390/v6010354.
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.
人巨细胞病毒 (HCMV) 具有一个 240kb 的大型基因组，可能编码超过 700 种基因产物，其中许多尚未被描述。对细菌人工染色体 (BAC) 克隆的 CMV 基因组进行诱变极大地促进了病毒基因功能的分析。然而，由于需要建立繁琐的合适互补系统，基本蛋白质的作用通常仍然难以捉摸。在这里，我们通过应用基于复制缺陷、失活的腺病毒颗粒 (腺转染) 的转染方案，展示了 HCMV 基因组可以以前所未有的效率引入细胞。在用携带必需基因突变的 HCMV 基因组转染几种允许的细胞类型后，观察到高达 60%的转染率，并且发现表达了所有动力学类别的病毒蛋白。这使得可以通过标准生化技术进一步分析转染的细胞。值得注意的是，缺失病毒 DNA 复制所必需元件的 HCMV 基因组，例如裂解复制起点，仍然表达了几种晚期蛋白。总之，腺转染允许在 BAC 转染细胞中直接研究必需的 HCMV 基因，而无需复杂的互补策略。
