Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research

Rui Yu; Xiaohong Li; Peng Zhang; Minghao Xu; Jitong Zhao; Jingjing Yan; Chenli Qiu; Jiayi Shu; Shuo Zhang; Miaomiao Kang; Xiaoyan Zhang; Jianqing Xu; Shuye Zhang

doi:10.1016/j.virs.2024.03.004

June 2024

Rui Yu, Xiaohong Li, Peng Zhang, Minghao Xu, Jitong Zhao, Jingjing Yan, Chenli Qiu, Jiayi Shu, Shuo Zhang, Miaomiao Kang, Xiaoyan Zhang, Jianqing Xu and Shuye Zhang. Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research[J]. Virologica Sinica, 2024, 39(3): 422-433. doi: 10.1016/j.virs.2024.03.004

Citation: Rui Yu, Xiaohong Li, Peng Zhang, Minghao Xu, Jitong Zhao, Jingjing Yan, Chenli Qiu, Jiayi Shu, Shuo Zhang, Miaomiao Kang, Xiaoyan Zhang, Jianqing Xu, Shuye Zhang. Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research .VIROLOGICA SINICA, 2024, 39(3) : 422-433. http://dx.doi.org/10.1016/j.virs.2024.03.004

将HiBiT整合到肠道病毒中：推进肠道病毒学研究的通用工具

喻锐 ^a ,
李肖宏 ^a ,
张鹏 ^c ,
徐明昊 ^d ,
赵纪曈 ^d ,
闫静静 ^g ,
邱趁丽 ^b,e ,
束佳熠 ^f ,
张硕 ^b ,
康苗苗 ^b ,
张晓燕 ^b,, ,
徐建青 ^b,, ,
章树业 ^a,b,,

cstr: 32224.14.j.virs.2024.03.004

a. 上海大学医学院;
b. 复旦大学附属中山医院生物治疗临床中心;
c. 中国科学院广州生物医药与健康研究院;
d. 复旦大学生物医学研究院;
e. 上海市老年医学中心;
f. 复旦大学附属中山医院/厦门中山医院生物治疗临床中心;
g. 复旦大学附属上海市公共卫生临床中心

通讯作者： 张晓燕, zhangxiaoyan@fudan.edu.cn; 徐建青, xujianqing@fudan.edu.cn; 章树业, shuye_zhang@fudan.edu.cn
收稿日期： 2023-11-22
录用日期： 2024-03-11

摘要

报道基因工程化的肠道病毒能够促进我们对肠道病毒生物学及其应用的理解，从而能够制定有效的治疗和预防策略。在这项研究中，我们最初尝试将NanoLuc 荧光素酶 (NLuc) 报告基因引入肠道病毒基因组中，但未能成功拯救可存活的后代。我们推测插入标签的大小可能是拯救病毒的决定因素。因此，我们将11个氨基酸的HiBiT标签插入到肠道病毒A71（EV-A71）、柯萨奇病毒A10（CVA10）、柯萨奇病毒A7（CVA7）、柯萨奇病毒A16（CVA16）的基因组中，即EV-A71-HiBiT， CVA16-HiBiT、CVA10-HiBiT、CVA7-HiBiT，观察到带有 HiBiT 标签的病毒表现出非常高的拯救效率。值得注意的是，HiBiT 标记的肠道病毒表现出与野生型病毒相当的特征。CVA16-NLuc 和 CVA16-HiBiT 重组病毒之间的直接比较表明，小分子量的 HiBiT 插入对病毒感染性和复制动力学的影响很小。此外，这些 HiBiT 标记的肠道病毒在不同细胞系中多次传代后表现出高度的遗传稳定性。带有HiBiT标签的病毒在抗病毒药物测定中得到了成功测试，并且病毒对药物的敏感性不受HiBiT标签的影响。最终，我们的研究结果提供了明确的证据，证明 HiBiT 与肠道病毒的整合为推进肠道病毒病毒学领域的研究提供了一种通用、方便且重要的方法。此外，HiBiT 标记的肠道病毒在多种应用中表现出巨大的潜力，包括抗病毒药物的开发和病毒感染机制的阐明。
- 肠道病毒
- , 感染性克隆
- , 报告基因
- , 荧光素酶
- , 药物筛选

Integration of HiBiT into enteroviruses: A universal tool for advancing enterovirus virology research

Rui Yu ^a ,
Xiaohong Li ^a ,
Peng Zhang ^c ,
Minghao Xu ^d ,
Jitong Zhao ^d ,
Jingjing Yan ^g ,
Chenli Qiu ^b,e ,
Jiayi Shu ^f ,
Shuo Zhang ^b ,
Miaomiao Kang ^b ,
Xiaoyan Zhang ^b,, ,
Jianqing Xu ^b,, ,
Shuye Zhang ^a,b,,

a. School of Medicine, Shanghai University, Shanghai, 200444, China;
b. Clinical Center for Biotherapy, Zhongshan Hospital, Fudan University, Shanghai, 200433, China;
c. Guangzhou Institutes of Biomedicine and Health, The Chinese Academy of Sciences, Guangzhou, 510530, China;
d. Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China;
e. Shanghai Geriatric Medical Center, Shanghai, 201104, China;
f. Clinical Center for Biotherapy, Zhongshan Hospital/Zhongshan Hospital (Xiamen), Fudan University, 361015, China;
g. Shanghai Public Health Clinical Center, Fudan University, Shanghai, 201508, China

Corresponding author: Xiaoyan Zhang, zhangxiaoyan@fudan.edu.cn Jianqing Xu, xujianqing@fudan.edu.cn Shuye Zhang, shuye_zhang@fudan.edu.cn
Received Date: 22 November 2023
Accepted Date: 11 March 2024

Abstract

The utilization of enteroviruses engineered with reporter genes serves as a valuable tool for advancing our understanding of enterovirus biology and its applications, enabling the development of effective therapeutic and preventive strategies. In this study, our initial attempts to introduce a NanoLuc luciferase (NLuc) reporter gene into recombinant enteroviruses were unsuccessful in rescuing viable progenies. We hypothesized that the size of the inserted tag might be a determining factor in the rescue of the virus. Therefore, we inserted the 11-amino-acid HiBiT tag into the genomes of enterovirus A71 (EV-A71), coxsackievirus A10 (CVA10), coxsackievirus A7 (CVA7), coxsackievirus A16 (CVA16), namely EV-A71-HiBiT, CVA16-HiBiT, CVA10-HiBiT, CVA7-HiBiT, and observed that the HiBiT-tagged viruses exhibited remarkably high rescue efficiency. Notably, the HiBiT-tagged enteroviruses displayed comparable characteristics to the wild-type viruses. A direct comparison between CVA16-NLuc and CVA16-HiBiT recombinant viruses revealed that the tiny HiBiT insertion had minimal impact on virus infectivity and replication kinetics. Moreover, these HiBiT-tagged enteroviruses demonstrated high genetic stability in different cell lines over multiple passages. In addition, the HiBiT-tagged viruses were successfully tested in antiviral drug assays, and the sensitivity of the viruses to drugs was not affected by the HiBiT tag. Ultimately, our findings provide definitive evidence that the integration of HiBiT into enteroviruses presents a universal, convenient, and invaluable method for advancing research in the realm of enterovirus virology. Furthermore, HiBiT-tagged enteroviruses exhibit great potential for diverse applications, including the development of antivirals and the elucidation of viral infection mechanisms.
- Enterovirus
- , Infectious clones
- , Reporter genes
- , Luciferase
- , Drug screening

References
1. Arnold, G.F., Resnick, D.A., Smith, A.D., Geisler, S.C., Holmes, A.K.,Arnold, E., 1996. Chimeric rhinoviruses as tools for vaccine development and characterization of protein epitopes. Intervirology, 39, 72-78.
2. Cao, J.M., Qu, M., Liu, H.T., Wan, X., Li, F., Hou, A., Zhou, Y., Sun, B., Cai, L.J., Su, W.H.,Jiang, C.L., 2020. Myristoylation of EV71 VP4 is essential for infectivity and interaction with membrane structure. Virol. Sin., 35, 599-613.
3. Chen, Y.K., Li, X.H., Wang, M., Li, Y., Fan, J., Yan, J.J., Zhang, S.Y., Lu, L.,Zou, P., 2023. A cysteine protease inhibitor GC376 displays potent antiviral activity against coxsackievirus infection. Curr. Res. Microb. Sci., 5, 100203.
4. Chiu, W.Y., Lo, Y.H.,Yeh, T.C., 2016. Coxsackievirus associated hand, foot and mouth disease in an adult. QJM-an International Journal of Medicine, 109, 823-824.
5. Chou, A.H., Liu, C.C., Chang, J.Y., Jiang, R.N., Hsieh, Y.C., Tsao, A., Wu, C.L., Huang, J.L., Fung, C.P., Hsieh, S.M., Wang, Y.F., Wang, J.R., Hu, M.H., Chiang, J.R., Su, I.J.,Chong, P.C.S., 2013. Formalin-inactivated EV71 vaccine candidate induced cross-neutralizing antibody against subgenotypes B1, B4, B5 and C4A in adult volunteers. PLoS One, 8: e79783.
6. Chu, J.Q., Lee, Y.J., Park, J.N., Kim, S.M., Lee, K.N., Ko, Y.J., Lee, H.S., Cho, I.S., Kim, B.,Park, J.H., 2013. Construction of a bovine enterovirus-based vector expressing a foot-and-mouth disease virus epitope. J. Virol Methods, 189, 101-104.
7. Chua, K.B., Ng, Q., Meng, T.,Jia, Q., 2022. Development of stable, cold-adapted, temperature-sensitive/conditional lethal chimeric enterovirus A71 and coxsackievirus A16. Virol. Sin., 37, 769-773.
8. Deng, C.L., Li, X.D., Liu, S.Q., Xu, L.L., Ye, H.Q., Qin, C.F.,Zhang, B., 2015. Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone. Virol. Sin., 30, 269-276.
9. Gao, Q.Q., Yuan, S.L., Zhang, C., Wang, Y., Wang, Y.Z., He, G.M., Zhang, S.Y., Altmeyer, R.,Zou, G., 2015. Discovery of itraconazole with broad-spectrum, antienterovirus activity that targets nonstructural protein 3A. Antimicrob. Agents Chemother., 59, 2654-2665.
10. Gaspar, N., Zambito, G., Dautzenberg, I.J.C., Cramer, S.J., Hoeben, R.C., Lowik, C., Walker, J.R., Kirkland, T.A., Smith, T.P., Van Weerden, W.M., De Vrij, J.,Mezzanotte, L., 2020. NanoBiT system and hydrofurimazine for optimized detection of viral infection in mice-a novel in vivo imaging platform. Int. J. Mol. Sci., 21, 5863.
11. Hashem, S.M., Gad, M.K., Anwar, H.M., Saleh, N.M., Shamma, R.N.,Elsherif, N.I., 2023. Itraconazole-loaded ufasomes: evaluation, characterization, and anti-fungal activity against. Pharmaceutics, 15, 26.
12. Jubelt, B.,Lipton, H.L., 2014. Enterovirus/picornavirus infections. Handb. Clin. Neurol., 123, 379-416.
13. Kim, C., Kang, H., Kim, D.E., Song, J.H., Choi, M., Kang, M., Lee, K., Kim, H.S., Shin, J.S., Jeong, H., Jung, S., Han, S.B., Kim, J.H., Ko, H.J., Lee, C.K., Kim, M.,Cho, S., 2016. Antiviral activity of micafungin against enterovirus 71. Virol. J., 13, 99.
14. Kimmis, B.D., Downing, C.,Tyring, S., 2018. Hand-foot-and-mouth disease caused by coxsackievirus A6 on the rise. Cutis, 102, 353-356.
15. Liang, X.Y., Zhu, Q.C., Liang, J.Q., Liu, S.Y., Liu, D.X.,Fung, T.S., 2020. Development of HiBiT-tagged recombinant infectious bronchitis coronavirus for efficient and viral quantification. Front. Microbiol., 11, 2100.
16. Lyu, K., Wang, G.C., He, Y.L., Han, J.F., Ye, Q., Qin, C.F.,Chen, R., 2015. Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design. J. Biol. Chem., 290, 3198-3208.
17. Miyakawa, K., Jeremiah, S.S., Ohtake, N., Matsunaga, S., Yamaoka, Y., Nishi, M., Morita, T., Saji, R., Nishii, M., Kimura, H., Hasegawa, H., Takeuchi, I.,Ryo, A., 2020. Rapid quantitative screening assay for SARS-CoV-2 neutralizing antibodies using HiBiT-tagged virus-like particles. J. Mol. Cell Biol., 12, 987-990.
18. Nagashima, S., Primadharsini, P.P., Nishiyama, T., Takahashi, M., Murata, K.,Okamoto, H., 2023. Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform. J. Virol., 97, e0050823.
19. Ren, P.J., Zheng, Y.M., Wang, W.Q., Hong, L.P., Delpeyroux, F., Arenzana-Seisdedos, F.,Altmeyer, R., 2017. Suramin interacts with the positively charged region surrounding the 5-fold axis of the EV-A71 capsid and inhibits multiple enterovirus A. Sci. Rep., 7, 42902.
20. Ren, P.J., Zou, G., Bailly, B., Xu, S.S., Zeng, M., Chen, X.S., Shen, L., Zhang, Y., Guillon, P., Arenzana-Seisdedos, F., Buchy, P., Li, J., Von Itzstein, M., Li, Q.H.,Altmeyer, R., 2014. The approved pediatric drug suramin identified as a clinical candidate for the treatment of EV71 infection-suramin inhibits EV71 infection in vitro and in vivo. Emerg. Microbes Infect., 3: e62.
21. Rezaei, R., Surendran, A., Singaravelu, R., Jamieson, T.R., Taklifi, P., Poutou, J., Azad, T.,Ilkow, C.S., 2021. Detection of SARS-CoV-2 receptor-binding domain antibody using a HiBiT-based bioreporter. J. Vis. Exp., 174: e62488, https://doi.org/10.3791/62488.
22. Sarma, N., 2013. Hand, foot, and mouth disease: current scenario and Indian perspective. Indian J. Dermatol. Venereol. Leprol., 79, 165-175.
23. Shang, B.D., Deng, C.L., Ye, H.G., Xu, W.B., Yuan, Z.M., Shi, P.Y.,Zhang, B., 2013. Development and characterization of a stable eGFP enterovirus 71 for antiviral screening. Antivir. Res., 97, 198-205.
24. Shribman, A.J.,Hanning, C.D., 1986. Hyperbaric bupivacaine and hyperbaric cinchocaine: a comparison of their use for spinal anaesthesia. Eur. J. Anaesthesiol., 3, 103-110.
25. Simmonds, P., Gorbalenya, A.E., Harvala, H., Hovi, T., Knowles, N.J., Lindberg, A.M., Oberste, M.S., Palmenberg, A.C., Reuter, G., Skern, T., Tapparel, C., Wolthers, K.C., Woo, P.C.Y.,Zell, R., 2020. Recommendations for the nomenclature of enteroviruses and rhinoviruses. Arch. Virol., 165, 793-797.
26. Sinclair, W.,Omar, M., 2023. Enterovirus. StatPearls. Treasure Island (FL).
27. Strating, J.R.P.M., Van Der Linden, L., Albulescu, L., Bigay, J., Arita, M., Delang, L., Leyssen, P., Van Der Schaar, H.M., Lanke, K.H.W., Thibaut, H.J., Ulferts, R., Drin, G., Schlinck, N., Wubbolts, R.W., Sever, N., Head, S.A., Liu, J.O., Beachy, P.A., De Matteis, M.A., Shair, M.D., Olkkonen, V.M., Neyts, J.,Van Kuppeveld, F.J.M., 2015. Itraconazole inhibits enterovirus replication by targeting the oxysterol-binding protein. Cell Rep., 10, 600-615.
28. Sumiyadorj, A., Murai, K., Shimakami, T., Kukroki, K., Nishikawa, T., Kakuya, M., Yamada, A., Wang, Y., Ishida, A., Shirasaki, T., Kawase, S., Li, Y.Y., Okada, H., Nio, K., Kawaguchi, K., Yamashita, T., Sakai, Y., Duger, D., Mizukoshi, E., Honda, M.,Kaneko, S., 2022. A single hepatitis B virus genome with a reporter allows the entire viral life cycle to be monitored in primary human hepatocytes. Hepatol. Commun., 6, 2441-2454.
29. Ulferts, R., De Boer, S.M., Van Der Linden, L., Bauer, L., Lyoo, H.R., Mate, M.J., Lichiere, J., Canard, B., Lelieveld, D., Omta, W., Egan, D., Coutard, B.,Van Kuppeveld, F.J.M., 2016. Screening of a library of FDA-approved drugs identifies several enterovirus replication inhibitors that target viral protein 2C. Antimicrob. Agents Chemother., 60, 2627-2638.
30. Van Der Schaar, H.M., Melia, C.E., Van Bruggen, J.a.C., Strating, J.R.P.M., Van Geenen, M.E.D., Koster, A.J., Barcena, M.,Van Kuppeveld, M., 2016. Illuminating the sites of enterovirus replication in living cells by using a split-GFP-tagged viral protein. mSphere, 1, e00104-e00116.
31. Wang, M., Yan, J., Zhu, L., Wang, M., Liu, L., Yu, R., Chen, M., Xun, J., Zhang, Y., Yi, Z.,Zhang, S., 2020. The establishment of infectious clone and single round infectious particles for coxsackievirus A10. Virol. Sin., 35, 426-435.
32. Xu, L.L., Shan, C., Deng, C.L., Li, X.D., Shang, B.D., Ye, H.Q., Liu, S.Q., Yuan, Z.M., Wang, Q.Y., Shi, P.Y.,Zhang, B., 2015. Development of a stable luciferase enterovirus 71 reporter virus. J. Virol. Methods, 219, 62-66.
33. Yu, R., Wang, M., Liu, L., Yan, J., Fan, J., Li, X., Kang, M., Xu, J., Zhang, X.,Zhang, S., 2022. The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene. Front. Microbiol., 13, 1101850.
34. Yun, S.I., Song, B.H., Woolley, M.E., Frank, J.C., Julander, J.G.,Lee, Y.M., 2020. Development, characterization, and application of two reporter-expressing recombinant Zika viruses. Viruses-Basel, 12, 572.
35. Zhang, C., Zhang, G., Zhang, Y., Lin, X., Zhao, X., Cui, Q., Rong, L.,Du, R., 2023. Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform. J. Med. Virol., 95, e28345.
36. Zhou, Y., Li, J.X., Jin, P.F., Wang, Y.X.,Zhu, F.C., 2016. Enterovirus 71: a whole virion inactivated enterovirus 71 vaccine. Expert Rev. Vaccines, 15, 803-813.
37. Zhu, F.C., Meng, F.Y., Li, J.X., Li, X.L., Mao, Q.Y., Tao, H., Zhang, Y.T., Yao, X., Chu, K., Chen, Q.H., Hu, Y.M., Wu, X., Liu, P., Zhu, L.Y., Gao, F., Jin, H., Chen, Y.J., Dong, Y.Y., Liang, Y.C., Shi, N.M., Ge, H.M., Liu, L., Chen, S.G., Ai, X., Zhang, Z.Y., Ji, Y.G., Luo, F.J., Chen, X.Q., Zhang, Y., Zhu, L.W., Liang, Z.L.,Shen, X.L., 2013. Efficacy, safety, and immunology of an inactivated alum-adjuvant enterovirus 71 vaccine in children in China: a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial. Lancet, 381, 2024-2032.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2024.03.004-ESM.docx