An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening <i>in vitro</i> and <i>in vivo</i>

Yingjian Li; Xue Tan; Jikai Deng; Xuemei Liu; Qianyun Liu; Zhen Zhang; Xiaoya Huang; Chao Shen; Ke Xu; Li Zhou; Yu Chen

doi:10.1016/j.virs.2024.03.009

June 2024

Yingjian Li, Xue Tan, Jikai Deng, Xuemei Liu, Qianyun Liu, Zhen Zhang, Xiaoya Huang, Chao Shen, Ke Xu, Li Zhou and Yu Chen. An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo[J]. Virologica Sinica, 2024, 39(3): 447-458. doi: 10.1016/j.virs.2024.03.009

Citation: Yingjian Li, Xue Tan, Jikai Deng, Xuemei Liu, Qianyun Liu, Zhen Zhang, Xiaoya Huang, Chao Shen, Ke Xu, Li Zhou, Yu Chen. An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo .VIROLOGICA SINICA, 2024, 39(3) : 447-458. http://dx.doi.org/10.1016/j.virs.2024.03.009

一种可用于体内外抗病毒筛选的优化高通量SARS-CoV-2双报告基因反式互补系统

cstr: 32224.14.j.virs.2024.03.009

a. 武汉大学生命科学学院, 病毒学国家重点实验室;
b. 武汉大学动物实验中心/ABSL-III实验室/疫苗研究院

通讯作者： 陈宇, chenyu@whu.edu.cn
收稿日期： 2023-12-04
录用日期： 2024-03-21

摘要

新型冠状病毒 (SARS-CoV-2)造成了全球新冠疫情，目前涉及SARS-CoV-2的操作需要在生物安全三级实验室(ABSL-III)进行，严重阻碍了对SARS-CoV-2的研究。在本研究中，我们构建了一种GFP-HiBiT双报告基因的SARS-CoV-2 DN反式互补复制子递送颗粒(RDP)系统，双报告基因和反式互补细胞在优化后其稳定性得到了提升，同时双报告基因在抗病毒药物筛选和评价上具有易检测和高通量的优势。通过慢病毒载体将N基因递送到K18-hACE2 KI小鼠体内表达，我们成功构建了一种支持RDP感染的小鼠模型。RDP能够模拟SARS-CoV-2的感染和复制，在体内和体外的抗病毒药物评估中表现出和SARS-CoV-2活病毒接近的结果。综上所述，RDP及其动物感染模型可以作为一种研究SARS-CoV-2以及抗病毒药物评估的平台。
- 新型冠状病毒
- , 反式互补复制子递送颗粒
- , 抗病毒药物评估
- , 小鼠模型
- , BSL-2

An optimized high-throughput SARS-CoV-2 dual reporter trans-complementation system for antiviral screening in vitro and in vivo

a. State Key Laboratory of Virology, RNA Institute, College of Life Sciences and Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, 430072, China;
b. Institute for Vaccine Research at Animal Bio-safety Level Ⅲ Laboratory, Wuhan University School of Medicine, Wuhan, 430071, China

Corresponding author: Yu Chen, chenyu@whu.edu.cn
Received Date: 04 December 2023
Accepted Date: 21 March 2024

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements.
- SARS-CoV-2
- , RDP
- , Antiviral evaluation
- , Mouse model
- , BSL-2

References
1. Almazan, F., Gonzalez, J.M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J.,Enjuanes, L., 2000. Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. U. S. A., 97, 5516-5521.
2. Bai, Z., Cao, Y., Liu, W.,Li, J., 2021. The SARS-CoV-2 nucleocapsid protein and its role in viral structure, biological functions, and a potential target for drug or vaccine mitigation. Viruses, 13.
3. Bao, L., Deng, W., Huang, B., Gao, H., Liu, J., Ren, L., Wei, Q., Yu, P., Xu, Y., Qi, F., Qu, Y., Li, F., Lv, Q., Wang, W., Xue, J., Gong, S., Liu, M., Wang, G., Wang, S., Song, Z., Zhao, L., Liu, P., Zhao, L., Ye, F., Wang, H., Zhou, W., Zhu, N., Zhen, W., Yu, H., Zhang, X., Guo, L., Chen, L., Wang, C., Wang, Y., Wang, X., Xiao, Y., Sun, Q., Liu, H., Zhu, F., Ma, C., Yan, L., Yang, M., Han, J., Xu, W., Tan, W., Peng, X., Jin, Q., Wu, G.,Qin, C., 2020. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature, 583, 830-833.
4. Cai, H.-L.,Huang, Y.-W., 2023. Reverse genetics systems for SARS-CoV-2: development and applications. Virol. Sin., 38, 837-850.
5. Chen, L., Guan, W.J., Qiu, Z.E., Xu, J.B., Bai, X., Hou, X.C., Sun, J., Qu, S., Huang, Z.X., Lei, T.L., Huang, Z.Y., Zhao, J., Zhu, Y.X., Ye, K.N., Lun, Z.R., Zhou, W.L., Zhong, N.S.,Zhang, Y.L., 2022. SARS-CoV-2 nucleocapsid protein triggers hyperinflammation via protein-protein interaction-mediated intracellular Cl(-) accumulation in respiratory epithelium. Signal Transduct. Target. Ther., 7, 255.
6. Chen, Y., Liu, Q.,Guo, D., 2020. Emerging coronaviruses: genome structure, replication, and pathogenesis. J. Med. Virol., 92, 418-423.
7. Cheung, P.H., Ye, Z.W., Lui, W.Y., Ong, C.P., Chan, P., Lee, T.T., Tang, T.T., Yuen, T.L., Fung, S.Y., Cheng, Y., Chan, C.P., Chan, C.P.,Jin, D.Y., 2022. Production of single-cycle infectious SARS-CoV-2 through a trans-complemented replicon. J. Med. Virol., 94, 6078-6090.
8. Feng, X., Zhang, X., Jiang, S., Tang, Y., Cheng, C., Krishna, P.A., Wang, X., Dai, J., Zeng, J., Xia, T.,Zhao, D., 2022. A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis. Comput. Struct. Biotechnol. J., 20, 5193-5202.
9. Hassan, A.O., Case, J.B., Winkler, E.S., Thackray, L.B., Kafai, N.M., Bailey, A.L., Mccune, B.T., Fox, J.M., Chen, R.E., Alsoussi, W.B., Turner, J.S., Schmitz, A.J., Lei, T., Shrihari, S., Keeler, S.P., Fremont, D.H., Greco, S., Mccray, P.B., Jr., Perlman, S., Holtzman, M.J., Ellebedy, A.H.,Diamond, M.S., 2020. A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies. Cell, 182, 744-753.e744.
10. He, X., Quan, S., Xu, M., Rodriguez, S., Goh, S.L., Wei, J., Fridman, A., Koeplinger, K.A., Carroll, S.S., Grobler, J.A., Espeseth, A.S., Olsen, D.B., Hazuda, D.J.,Wang, D., 2021. Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing. Proc. Natl. Acad. Sci. U. S. A., 118.
11. Hou, Y.J., Okuda, K., Edwards, C.E., Martinez, D.R., Asakura, T., Dinnon, K.H., 3rd, Kato, T., Lee, R.E., Yount, B.L., Mascenik, T.M., Chen, G., Olivier, K.N., Ghio, A., Tse, L.V., Leist, S.R., Gralinski, L.E., Schafer, A., Dang, H., Gilmore, R., Nakano, S., Sun, L., Fulcher, M.L., Livraghi-Butrico, A., Nicely, N.I., Cameron, M., Cameron, C., Kelvin, D.J., De Silva, A., Margolis, D.M., Markmann, A., Bartelt, L., Zumwalt, R., Martinez, F.J., Salvatore, S.P., Borczuk, A., Tata, P.R., Sontake, V., Kimple, A., Jaspers, I., O'neal, W.K., Randell, S.H., Boucher, R.C.,Baric, R.S., 2020. SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell, 182, 429-446.e414.
12. Hu, B., Guo, H., Zhou, P.,Shi, Z.L., 2021. Characteristics of SARS-CoV-2 and COVID-19. Nat. Rev. Microbiol., 19, 141-154.
13. Jiang, R.D., Liu, M.Q., Chen, Y., Shan, C., Zhou, Y.W., Shen, X.R., Li, Q., Zhang, L., Zhu, Y., Si, H.R., Wang, Q., Min, J., Wang, X., Zhang, W., Li, B., Zhang, H.J., Baric, R.S., Zhou, P., Yang, X.L.,Shi, Z.L., 2020. Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin-converting enzyme 2. Cell, 182, 50-58.e58.
14. Jin, Y.Y., Lin, H., Cao, L., Wu, W.C., Ji, Y., Du, L., Jiang, Y., Xie, Y., Tong, K., Xing, F., Zheng, F., Shi, M., Pan, J.A., Peng, X.,Guo, D., 2021. A convenient and biosafe replicon with accessory genes of SARS-CoV-2 and its potential application in antiviral drug discovery. Virol. Sin., 36, 913-923.
15. Ju, X., Zhu, Y., Wang, Y., Li, J., Zhang, J., Gong, M., Ren, W., Li, S., Zhong, J., Zhang, L., Zhang, Q.C., Zhang, R.,Ding, Q., 2021. A novel cell culture system modeling the SARS-CoV-2 life cycle. PLoS Pathog., 17, e1009439.
16. Kouprina, N.,Larionov, V., 2006. TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat. Rev. Genet., 7, 805-812.
17. Liu, Q., Zhao, H., Li, Z., Zhang, Z., Huang, R., Gu, M., Zhuang, K., Xiong, Q., Chen, X., Yu, W., Qian, S., Zhang, Y., Tan, X., Zhang, M., Yu, F., Guo, M., Huang, Z., Wang, X., Xiang, W., Wu, B., Mei, F., Cai, K., Zhou, L., Zhou, L., Wu, Y., Yan, H., Cao, S., Lan, K.,Chen, Y., 2023. Broadly neutralizing antibodies derived from the earliest COVID-19 convalescents protect mice from SARS-CoV-2 variants challenge. Signal Transduct. Target. Ther., 8, 347.
18. Malicoat, J., Manivasagam, S., Zuniga, S., Sola, I., Mccabe, D., Rong, L., Perlman, S., Enjuanes, L.,Manicassamy, B., 2022. Development of a single-cycle infectious SARS-CoV-2 virus replicon particle system for use in biosafety level 2 laboratories. J. Virol., 96, e0183721.
19. Nguyen, H.T., Falzarano, D., Gerdts, V.,Liu, Q., 2021. Construction of a noninfectious SARS-CoV-2 replicon for antiviral-drug testing and gene function studies. J. Virol., 95, e0068721.
20. Park, S.B., Khan, M., Chiliveri, S.C., Hu, X., Irvin, P., Leek, M., Grieshaber, A., Hu, Z., Jang, E.S., Bax, A.,Liang, T.J., 2023. SARS-CoV-2 omicron variants harbor spike protein mutations responsible for their attenuated fusogenic phenotype. Commun. Biol., 6, 556.
21. Ricardo-Lax, I., Luna, J.M., Thao, T.T.N., Le Pen, J., Yu, Y., Hoffmann, H.H., Schneider, W.M., Razooky, B.S., Fernandez-Martinez, J., Schmidt, F., Weisblum, Y., Trueb, B.S., Berenguer Veiga, I., Schmied, K., Ebert, N., Michailidis, E., Peace, A., Sanchez-Rivera, F.J., Lowe, S.W., Rout, M.P., Hatziioannou, T., Bieniasz, P.D., Poirier, J.T., Macdonald, M.R., Thiel, V.,Rice, C.M., 2021. Replication and single-cycle delivery of SARS-CoV-2 replicons. Science, 374, 1099-1106.
22. Rihn, S.J., Merits, A., Bakshi, S., Turnbull, M.L., Wickenhagen, A., Alexander, A.J.T., Baillie, C., Brennan, B., Brown, F., Brunker, K., Bryden, S.R., Burness, K.A., Carmichael, S., Cole, S.J., Cowton, V.M., Davies, P., Davis, C., De Lorenzo, G., Donald, C.L., Dorward, M., Dunlop, J.I., Elliott, M., Fares, M., Da Silva Filipe, A., Freitas, J.R., Furnon, W., Gestuveo, R.J., Geyer, A., Giesel, D., Goldfarb, D.M., Goodman, N., Gunson, R., Hastie, C.J., Herder, V., Hughes, J., Johnson, C., Johnson, N., Kohl, A., Kerr, K., Leech, H., Lello, L.S., Li, K., Lieber, G., Liu, X., Lingala, R., Loney, C., Mair, D., Mcelwee, M.J., Mcfarlane, S., Nichols, J., Nomikou, K., Orr, A., Orton, R.J., Palmarini, M., Parr, Y.A., Pinto, R.M., Raggett, S., Reid, E., Robertson, D.L., Royle, J., Cameron-Ruiz, N., Shepherd, J.G., Smollett, K., Stewart, D.G., Stewart, M., Sugrue, E., Szemiel, A.M., Taggart, A., Thomson, E.C., Tong, L., Torrie, L.S., Toth, R., Varjak, M., Wang, S., Wilkinson, S.G., Wyatt, P.G., Zusinaite, E., Alessi, D.R., Patel, A.H., Zaid, A., Wilson, S.J.,Mahalingam, S., 2021. A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biol., 19, e3001091.
23. Sun, C.P., Jan, J.T., Wang, I.H., Ma, H.H., Ko, H.Y., Wu, P.Y., Kuo, T.J., Liao, H.N., Lan, Y.H., Sie, Z.L., Chen, Y.H., Ko, Y.A., Liao, C.C., Chen, L.Y., Lee, I.J., Tsung, S.I., Lai, Y.J., Chiang, M.T., Liang, J.J., Liu, W.C., Wang, J.R., Yuan, J.P., Lin, Y.S., Tsai, Y.C., Hsieh, S.L., Li, C.W., Wu, H.C., Ko, T.M., Lin, Y.L.,Tao, M.H., 2021. Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19. PLoS Pathog., 17, e1009758.
24. Takashita, E., Yamayoshi, S., Halfmann, P., Wilson, N., Ries, H., Richardson, A., Bobholz, M., Vuyk, W., Maddox, R., Baker, D.A., Friedrich, T.C., O'connor, D.H., Uraki, R., Ito, M., Sakai-Tagawa, Y., Adachi, E., Saito, M., Koga, M., Tsutsumi, T., Iwatsuki-Horimoto, K., Kiso, M., Yotsuyanagi, H., Watanabe, S., Hasegawa, H., Imai, M.,Kawaoka, Y., 2022. In vitro efficacy of antiviral agents against omicron subvariant BA.4.6. N. Engl. J. Med., 387, 2094-2097.
25. Tanaka, T., Saito, A., Suzuki, T., Miyamoto, Y., Takayama, K., Okamoto, T.,Moriishi, K., 2022. Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening. Antiviral Res., 199, 105268.
26. Tang, G., Liu, Z.,Chen, D., 2022. Human coronaviruses: origin, host and receptor. J. Clin. Virol., 155, 105246.
27. Thi Nhu Thao, T., Labroussaa, F., Ebert, N., V'kovski, P., Stalder, H., Portmann, J., Kelly, J., Steiner, S., Holwerda, M., Kratzel, A., Gultom, M., Schmied, K., Laloli, L., Husser, L., Wider, M., Pfaender, S., Hirt, D., Cippa, V., Crespo-Pomar, S., Schroder, S., Muth, D., Niemeyer, D., Corman, V.M., Muller, M.A., Drosten, C., Dijkman, R., Jores, J.,Thiel, V., 2020. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature, 582, 561-565.
28. Thorne, L.G., Bouhaddou, M., Reuschl, A.K., Zuliani-Alvarez, L., Polacco, B., Pelin, A., Batra, J., Whelan, M.V.X., Hosmillo, M., Fossati, A., Ragazzini, R., Jungreis, I., Ummadi, M., Rojc, A., Turner, J., Bischof, M.L., Obernier, K., Braberg, H., Soucheray, M., Richards, A., Chen, K.H., Harjai, B., Memon, D., Hiatt, J., Rosales, R., Mcgovern, B.L., Jahun, A., Fabius, J.M., White, K., Goodfellow, I.G., Takeuchi, Y., Bonfanti, P., Shokat, K., Jura, N., Verba, K., Noursadeghi, M., Beltrao, P., Kellis, M., Swaney, D.L., Garcia-Sastre, A., Jolly, C., Towers, G.J.,Krogan, N.J., 2022. Evolution of enhanced innate immune evasion by SARS-CoV-2. Nature, 602, 487-495.
29. Wang, B., Zhang, C., Lei, X., Ren, L., Zhao, Z., Wang, J.,Huang, H., 2021. Construction of non-infectious SARS-CoV-2 replicons and their application in drug evaluation. Virol. Sin., 36, 890-900.
30. Wang, W., Peng, X., Jin, Y., Pan, J.A.,Guo, D., 2022. Reverse genetics systems for SARS-CoV-2. J. Med. Virol., 94, 3017-3031.
31. Winkler, E.S., Bailey, A.L., Kafai, N.M., Nair, S., Mccune, B.T., Yu, J., Fox, J.M., Chen, R.E., Earnest, J.T., Keeler, S.P., Ritter, J.H., Kang, L.I., Dort, S., Robichaud, A., Head, R., Holtzman, M.J.,Diamond, M.S., 2020. SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat. Immunol., 21, 1327-1335.
32. Wu, A., Peng, Y., Huang, B., Ding, X., Wang, X., Niu, P., Meng, J., Zhu, Z., Zhang, Z., Wang, J., Sheng, J., Quan, L., Xia, Z., Tan, W., Cheng, G.,Jiang, T., 2020. Genome composition and divergence of the novel coronavirus (2019-nCoV) originating in China. Cell Host Microbe, 27, 325-328.
33. Xie, X., Muruato, A., Lokugamage, K.G., Narayanan, K., Zhang, X., Zou, J., Liu, J., Schindewolf, C., Bopp, N.E., Aguilar, P.V., Plante, K.S., Weaver, S.C., Makino, S., Leduc, J.W., Menachery, V.D.,Shi, P.Y., 2020. An infectious cDNA clone of SARS-CoV-2. Cell Host Microbe, 27, 841-848.e843.
34. Yang, B., Liu, C., Ju, X., Wu, B., Wang, Z., Dong, F., Yu, Y., Hou, X., Fang, M., Gao, F., Guo, X., Gui, Y., Ding, Q.,Li, W., 2023. A tissue specific-infection mouse model of SARS-CoV-2. Cell Discov., 9, 43.
35. Ye, C., Chiem, K., Park, J.G., Oladunni, F., Platt, R.N., 2nd, Anderson, T., Almazan, F., De La Torre, J.C.,Martinez-Sobrido, L., 2020. Rescue of SARS-CoV-2 from a single bacterial artificial chromosome. mBio, 11.
36. Zhang, H., Fischer, D.K., Shuda, M., Moore, P.S., Gao, S.J., Ambrose, Z.,Guo, H., 2022. Construction and characterization of two SARS-CoV-2 minigenome replicon systems. J. Med. Virol., 94, 2438-2452.
37. Zhang, X., Liu, Y., Liu, J., Bailey, A.L., Plante, K.S., Plante, J.A., Zou, J., Xia, H., Bopp, N.E., Aguilar, P.V., Ren, P., Menachery, V.D., Diamond, M.S., Weaver, S.C., Xie, X.,Shi, P.Y., 2021. A trans-complementation system for SARS-CoV-2 recapitulates authentic viral replication without virulence. Cell, 184, 2229-2238.e2213.
38. Zhang, Y., Song, W., Chen, S., Yuan, Z.,Yi, Z., 2021. A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2. Antiviral Res., 185, 104974.
39. Zhang, Z.Y., Ju, C.Y., Wu, L.Z., Yan, H., Hong, W.B., Chen, H.Z., Yang, P.B., Wang, B.R., Gou, T., Chen, X.Y., Jiang, Z.H., Wang, W.J., Lin, T., Li, F.N.,Wu, Q., 2023. Therapeutic potency of compound RMY-205 for pulmonary fibrosis induced by SARS-CoV-2 nucleocapsid protein. Cell Chem. Biol., 30, 261-277.e268.
40. Zheng, J., Wong, L.R., Li, K., Verma, A.K., Ortiz, M.E., Wohlford-Lenane, C., Leidinger, M.R., Knudson, C.M., Meyerholz, D.K., Mccray, P.B., Jr.,Perlman, S., 2021. COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice. Nature, 589, 603-607.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2024.03.009-ESM.docx