Kang Miaomiao, Li Xiangyi, Li Xiaohong, Yu Rui, Zhang Shuo, Yan Jingjing, Zhang Xiaoyan, Xu Jianqing, Ma Buyong, Zhang Shuye
Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, China.
Engineering Research Center of Cell & Therapeutic Antibody (MOE), School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Viruses. 2025 Jan 17;17(1):128. doi: 10.3390/v17010128.
This study aimed to create a new recombinant virus by modifying the EV-A71 capsid protein, serving as a useful tool and model for studying human Enteroviruses. We developed a new screening method using EV-A71 pseudovirus particles to systematically identify suitable insertion sites and tag types in the VP1 capsid protein. The pseudovirus's infectivity and replication can be assessed by measuring postinfection luciferase signals. We reported that the site after the 100th amino acid within the VP1 BC loop of EV-A71 is particularly permissive for the insertion of various tags. Notably, the introduction of S and V5 tags at this position had minimal effect on the fitness of the tagged pseudovirus. Furthermore, recombinant infectious EV-A71 strains tagged with S and V5 epitopes were successfully rescued, and the stability of these tags was verified. Computational analysis suggested that viable insertions should be compatible with capsid assembly and receptor binding, whereas non-viable insertions could potentially disrupt the capsid's binding with heparan sulfate. We expect the tagged recombinant EV-A71 to be a useful tool for studying the various stages of the enterovirus life cycle and for virus purification, immunoprecipitation, and research in immunology and vaccine development. Furthermore, this study serves as a proof of principle and may help develop similar tags in enteroviruses, for which there are fewer available tools.
本研究旨在通过修饰肠道病毒A71（EV-A71）衣壳蛋白来创建一种新的重组病毒，作为研究人类肠道病毒的有用工具和模型。我们开发了一种利用EV-A71假病毒颗粒的新筛选方法，以系统地鉴定VP1衣壳蛋白中合适的插入位点和标签类型。假病毒的感染性和复制可通过测量感染后荧光素酶信号来评估。我们报告称，EV-A71的VP1 BC环内第100个氨基酸之后的位点对各种标签的插入特别宽容。值得注意的是，在此位置引入S和V5标签对标记假病毒的适应性影响最小。此外，成功拯救了带有S和V5表位标签的重组感染性EV-A71毒株，并验证了这些标签的稳定性。计算分析表明，可行的插入应与衣壳组装和受体结合兼容，而非可行的插入可能会破坏衣壳与硫酸乙酰肝素的结合。我们期望标记的重组EV-A71成为研究肠道病毒生命周期各个阶段以及用于病毒纯化、免疫沉淀以及免疫学和疫苗开发研究的有用工具。此外，本研究作为原理验证，可能有助于在肠道病毒中开发类似的标签，目前针对肠道病毒的可用工具较少。
