PA-E18G substitution in influenza A virus confers resistance to ZX-7101, a cap-dependent endonuclease inhibitor

Dan Luo; Qing Ye; Rui-Ting Li; Hang-Yu Zhou; Jing-Jing Guo; Suo-Qun Zhao; Sen Zhang; Tao Jiang; Yong-Qiang Deng; Cheng-Feng Qin

doi:10.1016/j.virs.2023.06.002

August 2023

Citation: Dan Luo, Qing Ye, Rui-Ting Li, Hang-Yu Zhou, Jing-Jing Guo, Suo-Qun Zhao, Sen Zhang, Tao Jiang, Yong-Qiang Deng, Cheng-Feng Qin. PA-E18G substitution in influenza A virus confers resistance to ZX-7101, a cap-dependent endonuclease inhibitor .VIROLOGICA SINICA, 2023, 38(4) : 559-567. http://dx.doi.org/10.1016/j.virs.2023.06.002

PA-E18G substitution in influenza A virus confers resistance to ZX-7101, a cap-dependent endonuclease inhibitor

Dan Luo ^a ,
Qing Ye ^a ,
Rui-Ting Li ^a ,
Hang-Yu Zhou ^b ,
Jing-Jing Guo ^a ,
Suo-Qun Zhao ^a ,
Sen Zhang ^a ,
Tao Jiang ^a ,
Yong-Qiang Deng ^{a
,,} ,
Cheng-Feng Qin ^{a
,,}

a. State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China;
b. State Key Laboratory of Medical Molecular Biology, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou 215123, China

Corresponding author: Yong-Qiang Deng, dengyq1977@126.com
Cheng-Feng Qin, qincf@bmi.ac.cn
Received Date: 26 January 2023
Accepted Date: 05 June 2023
Available online: 07 June 2023

Abstract

Cap-dependent endonuclease (CEN) in the polymerase acidic protein (PA) of influenza A virus (IAV) represents a promising drug target due to its critical role in viral gene transcription. The CEN inhibitor, baloxavir marboxil (BXM), was approved in Japan and the US in 2018 and several other countries subsequently. Along with the clinical use of BXM, the emergence and spread of IAV variants with reduced susceptibility to BXM have aroused serious concern. Herein, we comprehensively characterized the in vitro and in vivo antiviral activities of ZX-7101A, an analogue of BXM. The active form of prodrug ZX-7101 showed broad-spectrum antiviral potency against various IAV subtypes, including pH1N1, H3N2, H7N9 and H9N2, in MDCK cells, and the 50% effective concentration (EC₅₀) was calculated to nanomole level and comparable to that of baloxavir acid (BXA), the active form of BXM. Furthermore, in vivo assays showed that administration of ZX-7101A conferred significant protection against lethal pH1N1 challenge in mice, with reduced viral RNA loads and alleviated pulmonary damage. Importantly, serial passaging of H1N1 virus in MDCK cells under selection pressure of ZX-7101 led to a resistant variant at the 15th passage. Reverse genetic and sequencing analysis demonstrated that a single E18G substitution in the PA subunit contributed to the reduced susceptibility to both ZX-7101 and BXA. Taken together, our results not only characterized a new CEN inhibitor of IAV but also identified a novel amino acid substitution responsible for CEN inhibitor resistance, which provides critical clues for future drug development and drug resistance surveillance.
- Influenza virus
- , Cap-dependent endonuclease (CEN)
- , Baloxavir marboxil (BXM)
- , Drug resistance

Electronic Supplementary Material

10.1016j.virs.2023.06.002-ESM.docx
References
1. Area, E., Martín-Benito, J., Gastaminza, P., Torreira, E., Valpuesta, J.M., Carrascosa, J.L., Ortín, J., 2004. 3D structure of the influenza virus polymerase complex:localization of subunit domains. Proceedings of the National Academy of Sciences of the United States of America 101, 308-313.
2. Baker, J., Block, S.L., Matharu, B., Burleigh Macutkiewicz, L., Wildum, S., Dimonaco, S., Collinson, N., Clinch, B., Piedra, P.A., 2020. Baloxavir Marboxil Single-dose Treatment in Influenza-infected Children:A Randomized, Double-blind, Active Controlled Phase 3 Safety and Efficacy Trial (miniSTONE-2). The Pediatric infectious disease journal 39, 700-705.
3. Bhat, S., James, J., Sadeyen, J.R., Mahmood, S., Everest, H.J., Chang, P., Walsh, S.K., Byrne, A.M.P., Mollett, B., Lean, F., Sealy, J.E., Shelton, H., Slomka, M.J., Brookes, S.M., Iqbal, M., 2022. Coinfection of Chickens with H9N2 and H7N9 Avian Influenza Viruses Leads to Emergence of Reassortant H9N9 Virus with Increased Fitness for Poultry and a Zoonotic Potential. Journal of virology 96(5):e0185621.
4. Credille, C.V., Morrison, C.N., Stokes, R.W., Dick, B.L., Feng, Y., Sun, J., Chen, Y., Cohen, S.M., 2019. SAR Exploration of Tight-Binding Inhibitors of Influenza Virus PA Endonuclease. Journal of medicinal chemistry 62(21):9438-9449.
5. Dias, A., Bouvier, D., Crépin, T., McCarthy, A.A., Hart, D.J., Baudin, F., Cusack, S., Ruigrok, R.W., 2009. The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914-918.
6. Engelhardt, O.G., Fodor, E., 2006. Functional association between viral and cellular transcription during influenza virus infection. Reviews in medical virology 16(5):329-345.
7. He, Y., Xu, K., Keiner, B., Zhou, J., Czudai, V., Li, T., Chen, Z., Liu, J., Klenk, H.D., Shu, Y.L., Sun, B., 2010. Influenza A virus replication induces cell cycle arrest in G0/G1 phase. Journal of virology 84(24):12832-12840.
8. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proceedings of the National Academy of Sciences of the United States of America 97, 6108-6113.
9. Hossain, M.G., Akter, S., Dhole, P., Saha, S., Kazi, T., Majbauddin, A., Islam, M.S., 2021. Analysis of the Genetic Diversity Associated With the Drug Resistance and Pathogenicity of Influenza A Virus Isolated in Bangladesh From 2002 to 2019. Front Microbiol 12, 735305.
10. Huang, Y., Owino, S.O., Crevar, C.J., Carter, D.M., Ross, T.M., 2020. N-Linked Glycans and K147 Residue on Hemagglutinin Synergize To Elicit Broadly Reactive H1N1 Influenza Virus Antibodies. Journal of virology 94(6):e01432-19.
11. Ince, W.L., Smith, F.B., O'Rear, J.J., Thomson, M., 2020. Treatment-Emergent Influenza Virus Polymerase Acidic Substitutions Independent of Those at I38 Associated With Reduced Baloxavir Susceptibility and Virus Rebound in Trials of Baloxavir Marboxil. The Journal of infectious diseases 222, 957-961.
12. Ison, M.G., Portsmouth, S., Yoshida, Y., Shishido, T., Mitchener, M., Tsuchiya, K., Uehara, T., Hayden, F.G., 2020. Early treatment with baloxavir marboxil in high-risk adolescent and adult outpatients with uncomplicated influenza (CAPSTONE-2):a randomised, placebo-controlled, phase 3 trial. The Lancet. Infectious diseases 20, 1204-1214.
13. Jiang, W., Wang, Q., Chen, S., Gao, S., Song, L., Liu, P., Huang, W., 2013. Influenza A virus NS1 induces G0/G1 cell cycle arrest by inhibiting the expression and activity of RhoA protein. Journal of virology 87, 3039-3052.
14. Jones, J.C., Kumar, G., Barman, S., Najera, I., White, S.W., Webby, R.J., Govorkova, E.A., 2018. Identification of the I38T PA Substitution as a Resistance Marker for Next-Generation Influenza Virus Endonuclease Inhibitors. mBio 9(2):e00430-18.
15. Jones, J.C., Marathe, B.M., Lerner, C., Kreis, L., Gasser, R., Pascua, P.N., Najera, I., Govorkova, E.A., 2016. A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro. Antimicrobial agents and chemotherapy 60, 5504-5514.
16. Jones, J.C., Marathe, B.M., Vogel, P., Gasser, R., Najera, I., Govorkova, E.A., 2017. The PA Endonuclease Inhibitor RO-7 Protects Mice from Lethal Challenge with Influenza A or B Viruses. Antimicrobial agents and chemotherapy 61(5):e02460-16.
17. Jones, J.C., Zagribelnyy, B., Pascua, P.N.Q., Bezrukov, D.S., Barman, S., Okda, F., Webby, R.J., Ivanenkov, Y.A., Govorkova, E.A., 2022. Influenza A virus polymerase acidic protein E23G/K substitutions weaken key baloxavir drug-binding contacts with minimal impact on replication and transmission. PLoS Pathog 18, e1010698.
18. Koszalka, P., George, A., Dhanasekaran, V., Hurt, A.C., Subbarao, K., 2022. Effect of Baloxavir and Oseltamivir in Combination on Infection with Influenza Viruses with PA/I38T or PA/E23K Substitutions in the Ferret Model. mBio 13, e0105622.
19. Mifsud, E.J., Hayden, F.G., Hurt, A.C., 2019. Antivirals targeting the polymerase complex of influenza viruses. Antiviral research 169, 104545.
20. Music, N., Reber, A.J., Kim, J.H., York, I.A., 2016. Peripheral Leukocyte Migration in Ferrets in Response to Infection with Seasonal Influenza Virus. PloS one 11, e0157903.
21. Nian, Q.G., Jiang, T., Zhang, Y., Deng, Y.Q., Li, J., Qin, E.D., Qin, C.F., 2016. High thermostability of the newly emerged influenza A (H7N9) virus. The Journal of infection 72, 393-394.
22. Noshi, T., Kitano, M., Taniguchi, K., Yamamoto, A., Omoto, S., Baba, K., Hashimoto, T., Ishida, K., Kushima, Y., Hattori, K., Kawai, M., Yoshida, R., Kobayashi, M., Yoshinaga, T., Sato, A., Okamatsu, M., Sakoda, Y., Kida, H., Shishido, T., Naito, A., 2018. In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit. Antiviral research 160, 109-117.
23. Park, J.H., Kim, B., Antigua, K.J.C., Jeong, J.H., Kim, C.I., Choi, W.S., Oh, S., Kim, C.H., Kim, E.G., Choi, Y.K., Baek, Y.H., Song, M.S., 2021. Baloxavir-oseltamivir combination therapy inhibits the emergence of resistant substitutions in influenza A virus PA gene in a mouse model. Antiviral research 193, 105126.
24. Quan, C., Shi, W., Yang, Y., Yang, Y., Liu, X., Xu, W., Li, H., Li, J., Wang, Q., Tong, Z., Wong, G., Zhang, C., Ma, S., Ma, Z., Fu, G., Zhang, Z., Huang, Y., Song, H., Yang, L., Liu, W.J., Liu, Y., Liu, W., Gao, G.F., Bi, Y., 2018. New Threats from H7N9 Influenza Virus:Spread and Evolution of High- and Low-Pathogenicity Variants with High Genomic Diversity in Wave Five. Journal of virology 92(11):e00301-18.
25. Reich, S., Guilligay, D., Pflug, A., Malet, H., Berger, I., Crépin, T., Hart, D., Lunardi, T., Nanao, M., Ruigrok, R.W., Cusack, S., 2014. Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516, 361-366.
26. Takashita, E., Abe, T., Morita, H., Nagata, S., Fujisaki, S., Miura, H., Shirakura, M., Kishida, N., Nakamura, K., Kuwahara, T., Mitamura, K., Ichikawa, M., Yamazaki, M., Watanabe, S., Hasegawa, H., 2020a. Influenza A(H1N1)pdm09 virus exhibiting reduced susceptibility to baloxavir due to a PA E23K substitution detected from a child without baloxavir treatment. Antiviral research 180, 104828.
27. Takashita, E., Daniels, R.S., Fujisaki, S., Gregory, V., Gubareva, L.V., Huang, W., Hurt, A.C., Lackenby, A., Nguyen, H.T., Pereyaslov, D., Roe, M., Samaan, M., Subbarao, K., Tse, H., Wang, D., Yen, H.L., Zhang, W., Meijer, A., 2020b. Global update on the susceptibilities of human influenza viruses to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir, 2017-2018. Antiviral research 175, 104718.
28. Yogaratnam, J., Rito, J., Kakuda, T.N., Fennema, H., Gupta, K., Jekle, C.A., Mitchell, T., Boyce, M., Sahgal, O., Balaratnam, G., Chanda, S., Van Remoortere, P., Symons, J.A., Fry, J., 2019. Antiviral Activity, Safety, and Pharmacokinetics of AL-794, a Novel Oral Influenza Endonuclease Inhibitor:Results of an Influenza Human Challenge Study. The Journal of infectious diseases 219, 177-185.
29. Yuan, P., Bartlam, M., Lou, Z., Chen, S., Zhou, J., He, X., Lv, Z., Ge, R., Li, X., Deng, T., Fodor, E., Rao, Z., Liu, Y., 2009. Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458, 909-913.
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