K C Mahesh, Thongpan Ilada, Bhattarai Namita, Chen Phylip, Chamblee Michelle, Hsu Cheng Chih, Peeples Mark E, Li Jianrong
Center for Microbe and Immunity Research, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH, USA.
Infectious Disease Institute, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2025;2948:59-71. doi: 10.1007/978-1-0716-4666-3_4.
The addition of marker protein genes to respiratory syncytial virus (RSV) has enabled studies of the spread of RSV in different types of cell cultures and quantification of viral replication in those cultures. Genetic deletion of individual RSV genes from RSV genome has been used to determine their importance in virus infection and the differences between infection of cultured cells lines and of primary well-differentiated human bronchial epithelial (HBE) cultures. Modifications of individual viral proteins can identify the importance of a particular glycosylation, cleavage, or antigenic sites or reveal sites with these functions. However, the standard recombinant systems for the RSV genome, based on natural and inserted restriction sites, have been difficult to use, slow to accomplish, and frequently not successful. Here, we describe a yeast-based cDNA assembly system that streamlines both the construction of a cDNA clone of an RSV strain as well as any modification of an RSV cDNA, thereby enabling the rapid generation of an RSV mutant virus.
向呼吸道合胞病毒（RSV）中添加标记蛋白基因，使得对RSV在不同类型细胞培养物中的传播以及这些培养物中病毒复制的定量研究成为可能。从RSV基因组中对单个RSV基因进行基因缺失，已被用于确定它们在病毒感染中的重要性，以及培养细胞系感染与原代充分分化的人支气管上皮（HBE）培养物感染之间的差异。对单个病毒蛋白的修饰可以确定特定糖基化、切割或抗原位点的重要性，或揭示具有这些功能的位点。然而，基于天然和插入限制位点的RSV基因组标准重组系统使用困难、完成速度慢且成功率低。在此，我们描述了一种基于酵母的cDNA组装系统，该系统简化了RSV毒株cDNA克隆的构建以及RSV cDNA的任何修饰，从而能够快速产生RSV突变病毒。
