The effect of favipiravir on interferon related gene expression in human alveolar epithelial cells

Abstract

Favipiravir is a nucleoside analog that is a selective and potent inhibitor of influenza virus RNA-dependent RNA polymerase (RdRp). This drug is considered to be a potential inhibitor of RNA viruses other than influenza due to its effect on the RdRp. In this study, we evaluated the effects of favipiravir on expression of interferon-related genes in alveolar epithelial cells (A549 and BEAS-2B) which produce various types of chemokines. The cytotoxicity and anti-RdRp activity of favipiravir were examined by MTT and virus minireplicon assays, respectively. Favipiravir strongly inhibited human (A/WSN/33-H1N1) and avian type of influenza (A/duck/Pennsylvania-H5N2) virus RdRp at nontoxic doses. The gene transcripts in the cells treated with different doses of favipiravir were analyzed by qPCR and immunoblotting. While CCL2, CAV1 and PRKCZ genes were among the most downregulated genes; IFNB1, IFNE and IL10 genes were the most upregulated in favipiravir-treated cells. The CCL2 and INFB1 transcripts were the most significantly differentiated by favipiravir treatment. The BEAS-2B cells were less sensitive to favipiravir-treatment compared to A549. From this result, it was concluded that favipiravir, while primarily inhibiting the viral RdRp, may also affect cellular interferon defense mechanisms and interfere with virus replication.

Keywords

• Favipiravir
• interferon
• CCL2
• minireplicon

Favipiravir'in İnsan Alveolar Epitel Hücrelerinde İnterferonla İlişkili Gen Ekspresyonuna Etkisi

Abstract

Favipiravir, influenza virüsü RNA bağımlı RNA polimerazının (RdRp) seçici ve güçlü inhibitörü olan bir nükleozid analoğudur. Bu ilacın RdRp üzerindeki etkisi nedeniyle influenza dışındaki diğer RNA virüslerinin de potansiyel bir inhibitörü olduğu düşünülmektedir. Bu çalışmada favipiravirin çeşitli kemokinleri üreten alveolar epitel hücrelerinde (A549 ve BEAS-2B) interferon ile ilişkili genlerin ekspresyonuna etkileri değerlendirildi. Favipiravirin sitotoksisitesi MTT testi ile, anti-RdRp aktivitesi ise influenza A virüsü minireplikon modeli ile değerlendirildi. Favipiravir, toksik olmayan dozlarda (200 µg/ml’ye kadar) insan (A/WSN/33-H1N1) ve kuş tipi influenza (A/duck/Pennsylvania-H5N2) virüsü RdRp'yi güçlü bir şekilde inhibe etti. Farklı dozlarda favipiravir ile muamele edilen hücrelerdeki gen transkriptleri kantitatif polimeraz zincir reaksiyonu (qPZR) ve Western melezleme teknikleri ile analiz edildi. Favipiravir etkisinde kalan hücrelerde CCL2, CAV1 ve PRKCZ ekspresyonu en çok azalan genler arasında yer alırken; IFNB1, IFNE ve IL10 ekspresyonu en çok artan genler arasında yer almışlardır. CCL2 ve INFB1 transkriptleri favipiravir etkisi ile en belirgin şekilde farklılaşan genler olmuştur. BEAS-2B hücreleri A549'a kıyasla favipiravire daha az duyarlı bulunmuştur. Elde edilen bu verilerden favipiravirin öncelikle viral RdRp'yi inhibe ettiği, bununla birlikte hücrelerin interferon savunma mekanizmaları aracılığıyla da virüs replikasyonunu etkileyebileceği sonucuna varılmıştır.

Keywords

• Favipiravir
• interferon
• CCL2
• minireplikon

References

1. L. Delang, R. Abdelnabi, J. Neyts, Favipiravir as a potential countermeasure against neglected and emerging RNA viruses, Antiviral Res. 153., (2018) 85-94.
2. S. Yash, K. Sarika, Favipiravir: An effective RNA polymerase modulating anti-influenza drug, Biosci. Biotech. Res. Asia, 20 (2023) 465-475.
3. Q. Peng, R. Peng, B. Yuan, M. Wang, J. Zhao, L. Fu, J. Qi, Y. Shi, Structural basis of SARS-CoV-2 polymerase inhibition by favipiravir, Innovation (Camb), 2 (2021) 100080.
4. Y. Furuta, K. Takahashi, K. Shiraki, K. Sakamoto, D.F. Smee, D.L. Barnard, B.B. Gowen, J.G. Julander, J.D. Morrey, T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections, Antiviral. Res., 82 (2009) 95-102.
5. G. Lingas, K. Rosenke, D. Safronetz, J. Guedj, Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin, PLoS Comput. Biol., 17 (2021) e1008535.
6. L. Oestereich, A. Ludtke, S. Wurr, T. Rieger, C. Munoz-Fontela, S. Gunther, Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model, Antiviral Res., 105 (2014) 17-21.
7. C.P. Pires de Mello, X. Tao, T.H. Kim, J.B. Bulitta, J.L. Rodriquez, J.J. Pomeroy, A.N. Brown, Zika Virus replication is substantially inhibited by novel favipiravir and interferon alpha combination regimens, Antimicrob. Agents Chemother., 62 (2018).
8. L. Qiu, S.E. Patterson, L.F. Bonnac, R.J. Geraghty, Nucleobases and corresponding nucleosides display potent antiviral activities against dengue virus possibly through viral lethal mutagenesis, PLoS Negl. Trop. Dis., 12 (2018) e0006421.

9. V. Madelain, A. Duthey, F. Mentre, F. Jacquot, C. Solas, B. Lacarelle, A. Vallve, S. Barron, L. Barrot, S. Mundweiler, D. Thomas, C. Carbonnelle, H. Raoul, X. de Lamballerie, J. Guedj, Ribavirin does not potentiate favipiravir antiviral activity against Ebola virus in non-human primates, Antiviral Res., 177 (2020) 104758.
10. J. Guedj, G. Piorkowski, F. Jacquot, V. Madelain, T.H.T. Nguyen, A. Rodallec, S. Gunther, C. Carbonnelle, F. Mentre, H. Raoul, X. de Lamballerie, Antiviral efficacy of favipiravir against Ebola virus: A translational study in cynomolgus macaques, PLoS Med., 15 (2018) e1002535.
11. J.A. Kim, R.K. Seong, M. Kumar, O.S. Shin, Favipiravir and ribavirin inhibit replication of asian and african strains of Zika virus in different cell models, Viruses, 10 (2018) 72.
12. K. Matz, J. Emanuel, J. Callison, D. Gardner, R. Rosenke, R. Mercado-Hernandez, B.N. Williamson, H. Feldmann, A. Marzi, Favipiravir (T-705) protects IFNAR(-/-) mice against lethal Zika virus infection in a sex-dependent manner, Microorganisms, 9 (2021) 1178.
13. R. Marlin, D. Desjardins, V. Contreras, G. Lingas, C. Solas, P. Roques, T. Naninck, Q. Pascal, S. Behillil, P. Maisonnasse, J. Lemaitre, N. Kahlaoui, B. Delache, A. Pizzorno, A. Nougairede, C. Ludot, O. Terrier, N. Dereuddre-Bosquet, F. Relouzat, C. Chapon, R. Ho Tsong Fang, S. van der Werf, M. Rosa Calatrava, D. Malvy, X. de Lamballerie, J. Guedj, R. Le Grand, Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates, Nat. Commun., 13 (2022) 5108.
14. M. Hassaniazad, H. Farshidi, A. Gharibzadeh, A. Bazram, E. Khalili, A. Noormandi, M. Fathalipour, Efficacy and safety of favipiravir plus interferon-beta versus lopinavir/ritonavir plus interferon-beta in moderately ill patients with COVID-19: A randomized clinical trial, J. Med. Virol., 94 (2022) 3184-3191.
15. S.H. Lan, C.C. Lai, S.P. Chang, L.C. Lu, S.H. Hung, W.T. Lin, Favipiravir-based treatment for outcomes of patients with COVID-19: a systematic review and meta-analysis of randomized controlled trials, Expert. Rev. Clin. Pharmacol., 15 (2022) 759-766.
16. K. Itoh, I. Sakamaki, T. Hirota, H. Iwasaki, Evaluation of minocycline combined with favipiravir therapy in coronavirus disease 2019 patients: A case-series study, J. Infect. Chemother., 28 (2022) 124-127.
17. W. Zhang, H. Deng, Y. Liu, S. Chen, Y. Liu, Y. Zhao, Antiviral effectivity of favipiravir against peste des petits ruminants virus is mediated by the JAK/STAT and PI3K/AKT pathways, Front. Vet. Sci., 8 (2021) 722840.
18. Y. Lin, M. Zhang, P.F. Barnes, Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis, Infect. Immun., 66 (1998) 1121-1126.
19. K. Misaki, H. Takano, H. Kanazawa, K.I. Inoue, Biological response-enhancing activity with antigens in A549 cells exposed to representative polycyclic aromatic hydrocarbons, ACS Omega, 6 (2021) 22224-22232.
20. T. Fujisawa, Y. Kato, J. Atsuta, A. Terada, K. Iguchi, H. Kamiya, H. Yamada, T. Nakajima, M. Miyamasu, K. Hirai, Chemokine production by the BEAS-2B human bronchial epithelial cells: differential regulation of eotaxin, IL-8, and RANTES by TH2- and TH1-derived cytokines, J. Allergy Clin. Immunol., 105 (2000) 126-133.
21. P.T.V. Pham, K. Turan, K. Nagata, A. Kawaguchi, Biochemical characterization of avian influenza viral polymerase containing PA or PB2 subunit from human influenza A virus, Microbes Infect., 20 (2018) 353-359.
22. K. Turan, M. Mibayashi, K. Sugiyama, S. Saito, A. Numajiri, K. Nagata, Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome, Nucleic Acids Res., 32 (2004) 643-652.
23. H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene, 108 (1991) 193-199.
24. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method, Methods, 25 (2001) 402-408.
25. X. Li, J. Li, Q. Zhao, L. Qiao, L. Wang, C. Yu, Physiological, biochemical, and genomic elucidation of the Ensifer adhaerens M8 strain with simultaneous arsenic oxidation and chromium reduction, J. Hazard. Mater., 441 (2023) 129862.
26. X. Ji, J. Tang, J. Zhang, effects of salt stress on the morphology, growth and physiological parameters of Juglansmicrocarpa L. seedlings, Plants (Basel), 11 (2022).
27. D. Jochmans, S. van Nieuwkoop, S.L. Smits, J. Neyts, R.A. Fouchier, B.G. van den Hoogen, Antiviral activity of favipiravir (T-705) against a broad range of Paramyxoviruses in vitro and against human Metapneumovirus in hamsters, Antimicrob. Agents Chemother., 60 (2016) 4620-4629.
28. B. Lawson, S. Suppiah, P.A. Rota, C.J. Hickman, D.R. Latner, in vitro inhibition of mumps virus replication by favipiravir (T-705), Antiviral Res., 180 (2020) 104849.
29. Y. Furuta, T. Komeno, T. Nakamura, Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci., 93 (2017) 449-463.
30. S. Cuesta, R. Kireev, K. Forman, C. Garcia, G. Escames, C. Ariznavarreta, E. Vara, J.A. Tresguerres, Melatonin improves inflammation processes in liver of senescence-accelerated prone male mice (SAMP8), Exp. Gerontol., 45 (2010) 950-9556.
31. T.M.J. Evers, V. Sheikhhassani, M.C. Haks, C. Storm, T.H.M. Ottenhoff, A. Mashaghi, Single-cell analysis reveals chemokine-mediated differential regulation of monocyte mechanics, iScience, 25 (2022) 103555.
32. S. Zhu, M. Liu, S. Bennett, Z. Wang, K.D.G. Pfleger, J. Xu, The molecular structure and role of CCL2 (MCP-1) and C-C chemokine receptor CCR2 in skeletal biology and diseases, J. Cell Physiol., 236 (2021) 7211-7222.
33. D.T. Graves, G. Naguib, H. Lu, C. Leone, H. Hsue, E. Krall, Inflammation is more persistent in type 1 diabetic mice, J. Dent. Res., 84 (2005) 324-328.
34. P.L. Acosta, A.B. Byrne, D.R. Hijano, L.B. Talarico, Human type I Interferon antiviral effects in respiratory and reemerging viral infections, J. Immunol. Res., 2020 (2020) 1372494.
35. P. Hillyer, R. Shepard, M. Uehling, M. Krenz, F. Sheikh, K.R. Thayer, L. Huang, L. Yan, D. Panda, C. Luongo, U.J. Buchholz, P.L. Collins, R.P. Donnelly, R.L. Rabin, Differential responses by human respiratory epithelial cell lines to Respiratory Syncytial Virus reflect distinct patterns of infection control, J. Virol., 92 (2018) e02202-17.