Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction
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...
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2025-01-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/17/1/128 |
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| author | Miaomiao Kang Xiangyi Li Xiaohong Li Rui Yu Shuo Zhang Jingjing Yan Xiaoyan Zhang Jianqing Xu Buyong Ma Shuye Zhang |
| author_facet | Miaomiao Kang Xiangyi Li Xiaohong Li Rui Yu Shuo Zhang Jingjing Yan Xiaoyan Zhang Jianqing Xu Buyong Ma Shuye Zhang |
| author_sort | Miaomiao Kang |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-30037b9aa1f146e693b386221a090b6d |
| institution | Kabale University |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj-art-30037b9aa1f146e693b386221a090b6d2025-01-24T13:52:40ZengMDPI AGViruses1999-49152025-01-0117112810.3390/v17010128Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus ConstructionMiaomiao Kang0Xiangyi Li1Xiaohong Li2Rui Yu3Shuo Zhang4Jingjing Yan5Xiaoyan Zhang6Jianqing Xu7Buyong Ma8Shuye Zhang9Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, ChinaEngineering Research Center of Cell & Therapeutic Antibody (MOE), School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, ChinaClinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, ChinaPatronus Biotech Co., Ltd., Guangzhou 510715, ChinaClinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, ChinaShanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaShanghai Public Health Clinical Center, Fudan University, Shanghai 201508, ChinaClinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, ChinaEngineering Research Center of Cell & Therapeutic Antibody (MOE), School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, ChinaClinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai 200433, ChinaThis 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.https://www.mdpi.com/1999-4915/17/1/128Enterovirus A71capsidstags |
| spellingShingle | Miaomiao Kang Xiangyi Li Xiaohong Li Rui Yu Shuo Zhang Jingjing Yan Xiaoyan Zhang Jianqing Xu Buyong Ma Shuye Zhang Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction Viruses Enterovirus A71 capsids tags |
| title | Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction |
| title_full | Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction |
| title_fullStr | Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction |
| title_full_unstemmed | Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction |
| title_short | Screening of Insertion Sites and Tags on EV-A71 VP1 Protein for Recombinant Virus Construction |
| title_sort | screening of insertion sites and tags on ev a71 vp1 protein for recombinant virus construction |
| topic | Enterovirus A71 capsids tags |
| url | https://www.mdpi.com/1999-4915/17/1/128 |
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