SARS-CoV-2 3CLpro inhibits the replication of influenza viruses through the cleavage of NP and PA

Liubing Du; Xiaoyu Cai; Ziliang Peng; Mengxuan Wang; Huijie Li; Xue Liu; Zhihui Zhang; Manqi Cao; Shuchun Zhang; Zhenyu He; Zhanjia Zhang; Yu Yan; Qitong Lin; Yushan Xue; Deyin Guo; Ji-An Pan; Yao-Qing Chen; Xiaoxue Peng

doi:10.1016/j.virs.2026.08.006

August 2026

. doi: 10.1016/j.virs.2026.08.006

Citation: Liubing Du, Xiaoyu Cai, Ziliang Peng, Mengxuan Wang, Huijie Li, Xue Liu, Zhihui Zhang, Manqi Cao, Shuchun Zhang, Zhenyu He, Zhanjia Zhang, Yu Yan, Qitong Lin, Yushan Xue, Deyin Guo, Ji-An Pan, Yao-Qing Chen, Xiaoxue Peng. SARS-CoV-2 3CLpro inhibits the replication of influenza viruses through the cleavage of NP and PA .VIROLOGICA SINICA, 2026, 41(4) : 842-854. http://dx.doi.org/10.1016/j.virs.2026.08.006

新型冠状病毒3CL蛋白酶通过切割NP和PA蛋白进而抑制流感病毒复制的研究

杜柳冰 ^a,b ,
蔡晓宇 ^c ,
彭梓良 ^a ,
王孟轩 ^a ,
李慧捷 ^a ,
刘学 ^d ,
张志辉 ^a ,
曹曼琦 ^a ,
张舒淳 ^a ,
何震宇 ^a ,
张展嘉 ^a ,
颜语 ^a ,
林绮桐 ^a ,
薛雨珊 ^a ,
郭德银 ^b ,
潘纪安 ^a,d,, ,
陈耀庆 ^c,, ,
彭小雪 ^a,d,e,,

a 中山大学中山医学院分子肿瘤研究中心;
b 广州国家实验室;
c 中山大学公共卫生学院(深圳);
d 中山大学中山医学院感染免疫研究中心;
e 中山大学中山医学院深圳市炎症性疾病系统医学重点实验室

通讯作者： 潘纪安, pengxx9@mail.sysu.edu.cn; 陈耀庆, chenyaoqing@mail.sysu.edu.cn; 彭小雪, panjan@mail.sysu.edu.cn
收稿日期： 2026-04-01
录用日期： 2026-08-06

摘要

多种病毒的共同流行可导致不同的病理结果，但高频传播的SARS-CoV-2如何影响其他病毒感染仍知之甚少。在本研究中，我们探究了SARS-CoV-2 3C样蛋白酶（3CLpro）的蛋白水解活性对甲型流感病毒复制的影响。通过计算机模拟分析，我们在多种病毒蛋白中鉴定出大量潜在的3CLpro切割位点；生化实验进一步证实，3CLpro以蛋白酶活性依赖的方式促进流感病毒核蛋白（NP）和聚合酶酸性蛋白（PA）的降解。由于NP和PA对于病毒基因组的包装和转录至关重要，其降解破坏了流感病毒的复制周期并显著抑制了病毒增殖。该效应在外源性3CLpro表达及SARS-CoV-2感染的条件下均得到验证。我们的数据揭示了一种直接的、基于3CLpro酶活性的分子机制，SARS-CoV-2可通过该机制抑制与其共感染的流感病毒的复制。这一发现为COVID-19大流行期间观察到的全球流感活动急剧下降提供了分子层面的解释，并阐明了一种病毒如何利用其酶学武器来遏制竞争病原体。
- 新型冠状病毒
- , 流感病毒
- , 3CL蛋白酶
- , 核蛋白NP
- , RNA聚合酶亚基PA

SARS-CoV-2 3CLpro inhibits the replication of influenza viruses through the cleavage of NP and PA

Liubing Du ^a,b ,
Xiaoyu Cai ^c ,
Ziliang Peng ^a ,
Mengxuan Wang ^a ,
Huijie Li ^a ,
Xue Liu ^d ,
Zhihui Zhang ^a ,
Manqi Cao ^a ,
Shuchun Zhang ^a ,
Zhenyu He ^a ,
Zhanjia Zhang ^a ,
Yu Yan ^a ,
Qitong Lin ^a ,
Yushan Xue ^a ,
Deyin Guo ^b ,
Ji-An Pan ^a,d,, ,
Yao-Qing Chen ^c,, ,
Xiaoxue Peng ^a,d,e,,

a. The Molecular Cancer Research Center, Zhongshan School of Medicine, Sun Yat-sen University Shenzhen Campus, Shenzhen 518107, China;
b. The Division of Basic Research, Guangzhou National Laboratory, Bio-island, Guangzhou 510320, China;
c. School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen 518107, China;
d. The Center for Infection and Immunity Study, Zhongshan School of Medicine, Sun Yat-sen University Shenzhen Campus, Shenzhen 518107, China;
e. Shenzhen Key Laboratory for Systems Medicine in Inflammatory Diseases, Shenzhen Campus of Sun Yat-sen University, Shenzhen 518107, China

Corresponding author: Ji-An Pan, pengxx9@mail.sysu.edu.cn Yao-Qing Chen, chenyaoqing@mail.sysu.edu.cn Xiaoxue Peng, panjan@mail.sysu.edu.cn
Received Date: 01 April 2026
Accepted Date: 06 August 2026

Abstract

The co-circulation of multiple viruses can lead to distinct pathological outcomes, yet how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection influences other viral infections remains poorly understood, despite its documented high frequency during the pandemic. In this study, we investigated how the proteolytic activity of SARS-CoV-2 3C-like protease (3CLpro) influences the replication of influenza A virus. In silico analysis identified candidate 3CLpro cleavage sites across numerous viral proteins, and biochemical assays confirmed that 3CLpro catalyzes the degradation of influenza virus nucleoprotein (NP) and polymerase acidic protein (PA) in a manner requiring its protease activity. This degradation of NP and PA, which are essential for viral genome packaging and transcription, disrupted the influenza replicative cycle and suppressed viral replication, both upon ectopic 3CLpro expression and during SARS-CoV-2 infection. Our data uncover a direct, enzyme-based mechanism by which SARS-CoV-2 can suppress influenza virus replication during coinfection. We provide a molecular explanation for the sharp, global decline in influenza activity observed during the COVID-19 pandemic and illustrate how enzymatic weapons of one virus can be repurposed to restrain a competing pathogen.
- SARS-CoV-2
- , Influenza virus
- , 3C-like protease (3CLpro)
- , Nucleoprotein (NP)
- , Polymerase acidic protein (PA)

References
1. Babawale, P.I.,Guerrero-Plata, A., 2024. Respiratory Viral Coinfections: Insights into Epidemiology, Immune Response, Pathology, and Clinical Outcomes. Pathogens, 13.
2. Biswas, S.K., Boutz, P.L.,Nayak, D.P., 1998. Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. J Virol, 72, 5493-5501.
3. Bouvier, N.M.,Palese, P., 2008. The biology of influenza viruses. Vaccine, 26 Suppl 4, D49-53.
4. Chen, J., Lin, S., Tang, J., Gao, M., Liu, Q., Du, J., Tan, C., Gong, Z., Liang, L., Zhu, T.,Liu, G., 2026. Cross-utilization of viral polymerase: parainfluenza virus hijacks the RdRp of porcine sapelovirus to facilitate its replication during co-infection. mBio, 17, e0381725.
5. Chen, Z., Tsui, J.L., Gutierrez, B., Busch Moreno, S., Du Plessis, L., Deng, X., Cai, J., Bajaj, S., Suchard, M.A., Pybus, O.G., Lemey, P., Kraemer, M.U.G.,Yu, H., 2024. COVID-19 pandemic interventions reshaped the global dispersal of seasonal influenza viruses. Science, 386, eadq3003.
6. Chin, T., Foxman, E.F., Watkins, T.A.,Lipsitch, M., 2024. Considerations for viral co-infection studies in human populations. mBio, 15, e0065824.
7. Colman, P.M., Varghese, J.N.,Laver, W.G., 1983. Structure of the catalytic and antigenic sites in influenza virus neuraminidase. Nature, 303, 41-44.
8. Dai, W., Zhang, B., Jiang, X.M., Su, H., Li, J., Zhao, Y., Xie, X., Jin, Z., Peng, J., Liu, F., Li, C., Li, Y., Bai, F., Wang, H., Cheng, X., Cen, X., Hu, S., Yang, X., Wang, J., Liu, X., Xiao, G., Jiang, H., Rao, Z., Zhang, L.K., Xu, Y., Yang, H., Liu, H., 2020. Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease. Science, 368, 1331-1335.
9. Dowdle, W.R., Davenport, F.M., Fukumi, H., Schild, G.C., Tumova, B., Webster, R.G.,Zakstelskaja, L.Y., 1975. Orthomyxoviridae. Intervirology, 5, 245-251.
10. Du, L., Xie, Y., Zheng, K., Wang, N., Gao, M., Yu, T., Cao, L., Shao, Q., Zou, Y., Xia, W., Fang, Q., Zhao, B., Guo, D., Peng, X.,Pan, J.A., 2021. Oxidative stress transforms 3CLpro into an insoluble and more active form to promote SARS-CoV-2 replication. Redox Biol, 48, 102199.
11. Fodor, E., 2013. The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication. Acta Virol, 57, 113-122.
12. Gabriel, G., Herwig, A.,Klenk, H.D., 2008. Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog, 4, e11.
13. Garnsey, M.R., Robinson, M.C., Nguyen, L.T., Cardin, R, Tillotson, J, Mashalidis, E., Yu, A., Aschenbrenner, L., Balesano, A., Behzadi, A., Boras B., Chang, J.S., Eng, H, Ephron, A., Foley, T., Ford, K.K., Frick, J.M., Gibson, S., Hao, L., Hurst, B., Kalgutkar, A.S., Korczynska, M., Lengyel-Zhand, Z., Gao, L, Meredith, H.R., Patel, N.C., Polivkova, J., Rai, D., Rose, C.R., Rothan, H., Sakata, S.K., Vargo, T.R., Qi, W., Wu, H., Liu, Y., Yurgelonis, I., Zhang, J., Zhu, Y., Zhang, L., Lee, A.A., 2024. Discovery of SARS-CoV-2 papain-like protease (PL(pro)) inhibitors with efficacy in a murine infection model. Sci Adv, 10, eado4288.
14. Godinho, D.P., Rodrigues, L.R., Lefevre, S., Magalhaes, S.,Duncan, A.B., 2024. Coinfection accelerates transmission to new hosts despite no effects on virulence and parasite growth. Philos Trans R Soc Lond B Biol Sci, 379, 20230139.
15. Guo, J., Lai, Y., Yang, Z., Song, W., Zhou, J., Li, Z., Su, W., Xiao, S.,Fang, L., 2024. Coinfection and nonrandom recombination drive the evolution of swine enteric coronaviruses. Emerg Microbes Infect, 13, 2332653.
16. Hoffmann, M., Kleine-Weber, H., Schroeder, S., Kruger, N., Herrler, T., Erichsen, S., Schiergens, T.S., Herrler, G., Wu, N.H., Nitsche, A., Muller, M.A., Drosten, C.,Pohlmann, S., 2020. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 181, 271-280 e278.
17. Hou, Z., Wang, J., Tan, B.,Zhang, S., 2025. A Systematic Study of Bovine Viral Diarrhoea Virus Co-Infection with Other Pathogens. Viruses, 17.
18. Iuliano, A.D., Roguski, K.M., Chang, H.H., Muscatello, D.J., Palekar, R., Tempia, S., Cohen, C., Gran, J.M., Schanzer, D., Cowling, B.J., Wu, P., Kyncl, J., Ang, L.W., Park, M., Redlberger-Fritz, M., Yu, H., Espenhain, L., Krishnan, A., Emukule, G., van Asten, L., Pereira da Silva, S., Aungkulanon, S., Buchholz, U., Widdowson, M.A., Bresee, J.S., 2018. Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet, 391, 1285-1300.
19. Jiang, Y., Tong, K., Yao, R., Zhou, Y., Lin, H., Du, L., Jin, Y., Cao, L., Tan, J., Zhang, X.D., Guo, D., Pan, J.A.,Peng, X., 2021. Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication. Cell Biosci, 11, 140.
20. 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.
21. Jin, Z., Zhao, Y., Sun, Y., Zhang, B., Wang, H., Wu, Y., Zhu, Y., Zhu, C., Hu, T., Du, X., et al., 2020. Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug carmofur. Nat Struct Mol Biol, 27, 529-532.
22. Kao, R.Y., Yang, D., Lau, L.S., Tsui, W.H., Hu, L., Dai, J., Chan, M.P., Chan, C.M., Wang, P., Zheng, B.J., Sun, J., Huang, J.D., Madar, J., Chen, G., Chen, H., Guan, Y.,Yuen, K.Y., 2010. Identification of influenza A nucleoprotein as an antiviral target. Nat Biotechnol, 28, 600-605.
23. Krammer, F., Smith, G.J.D., Fouchier, R.a.M., Peiris, M., Kedzierska, K., Doherty, P.C., Palese, P., Shaw, M.L., Treanor, J., Webster, R.G.,Garcia-Sastre, A., 2018. Influenza. Nat Rev Dis Primers, 4, 3.
24. Lansbury, L., Lim, B., Baskaran, V.,Lim, W.S., 2020. Co-infections in people with COVID-19: a systematic review and meta-analysis. J Infect, 81, 266-275.
25. Matrosovich, M.N., Matrosovich, T.Y., Gray, T., Roberts, N.A.,Klenk, H.D., 2004. Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A, 101, 4620-4624.
26. Medina, R.A.,Garcia-Sastre, A., 2011. Influenza A viruses: new research developments. Nat Rev Microbiol, 9, 590-603.
27. Nayak, D.P., Hui, E.K.,Barman, S., 2004. Assembly and budding of influenza virus. Virus Res, 106, 147-165.
28. Nicholson, K.G., Wood, J.M.,Zambon, M., 2003. Influenza. Lancet, 362, 1733-1745.
29. Olsen, S.J., Azziz-Baumgartner, E., Budd, A.P., Brammer, L., Sullivan, S., Pineda, R.F., Cohen, C.,Fry, A.M., 2020. Decreased Influenza Activity During the COVID-19 Pandemic - United States, Australia, Chile, and South Africa, 2020. MMWR Morb Mortal Wkly Rep, 69, 1305-1309.
30. Olsen, S.J., Winn, A.K., Budd, A.P., Prill, M.M., Steel, J., Midgley, C.M., Kniss, K., Burns, E., Rowe, T., Foust, A., et al., 2021. Changes in Influenza and Other Respiratory Virus Activity During the COVID-19 Pandemic - United States, 2020-2021. MMWR Morb Mortal Wkly Rep, 70, 1013-1019.
31. Omar, S.,Woodman, Z.L., 2024. The evolution of envelope function during coinfection with phylogenetically distinct human immunodeficiency virus. BMC Infect Dis, 24, 934.
32. Pan, J.A., Sun, Y., Jiang, Y.P., Bott, A.J., Jaber, N., Dou, Z., Yang, B., Chen, J.S., Catanzaro, J.M., Du, C., Ding, W.X., Diaz-Meco, M.T., Moscat, J., Ozato, K., Lin, R.Z.,Zong, W.X., 2016. TRIM21 Ubiquitylates SQSTM1/p62 and Suppresses Protein Sequestration to Regulate Redox Homeostasis. Mol Cell, 61, 720-733.
33. Pan, J.A., Ullman, E., Dou, Z.,Zong, W.X., 2011. Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes. Mol Cell Biol, 31, 3158-3170.
34. Perez, D.R.,Donis, R.O., 2001. Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity. J Virol, 75, 8127-8136.
35. Pflug, A., Guilligay, D., Reich, S.,Cusack, S., 2014. Structure of influenza A polymerase bound to the viral RNA promoter. Nature, 516, 355-360.
36. Pinto, L.H., Holsinger, L.J.,Lamb, R.A., 1992. Influenza virus M2 protein has ion channel activity. Cell, 69, 517-528.
37. Portela, A.,Digard, P., 2002. The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol, 83, 723-734.
38. Rosario-Ferreira, N., Preto, A.J., Melo, R., Moreira, I.S.,Brito, R.M.M., 2020. The Central Role of Non-Structural Protein 1 (NS1) in Influenza Biology and Infection. Int J Mol Sci, 21.
39. Rossman, J.S.,Lamb, R.A., 2011. Influenza virus assembly and budding. Virology, 411, 229-236.
40. Roy, D.,Chakraborty, N., 2024. Editorial: Impact of viral co-infection on cellular or human health and its clinical outcome. Front Cell Infect Microbiol, 14, 1399184.
41. Skehel, J.J.,Wiley, D.C., 2000. Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem, 69, 531-569.
42. Stempel, H.E., Martin, E.T., Kuypers, J., Englund, J.A.,Zerr, D.M., 2009. Multiple viral respiratory pathogens in children with bronchiolitis. Acta Paediatr, 98, 123-126.
43. Szymanski, K., Cieslak, K., Kowalczyk, D.,Brydak, L.B., 2017. Co-infection with Influenza Viruses and Influenza-Like Virus During the 2015/2016 Epidemic Season. Adv Exp Med Biol, 968, 7-12.
44. Taubenberger, J.K.,Morens, D.M., 2008. The pathology of influenza virus infections. Annu Rev Pathol, 3, 499-522.
45. Vogels, C.B.F., Ruckert, C., Cavany, S.M., Perkins, T.A., Ebel, G.D.,Grubaugh, N.D., 2019. Arbovirus coinfection and co-transmission: A neglected public health concern? PLoS Biol, 17, e3000130.
46. Wagner, R., Matrosovich, M.,Klenk, H.D., 2002. Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol, 12, 159-166.
47. 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.
48. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M.,Kawaoka, Y., 1992. Evolution and ecology of influenza A viruses. Microbiol Rev, 56, 152-179.
49. Wolfel, R., Corman, V.M., Guggemos, W., Seilmaier, M., Zange, S., Muller, M.A., Niemeyer, D., Jones, T.C., Vollmar, P., Rothe, C., Hoelscher, M., Bleicker, T., Brunink, S., Schneider, J., Ehmann, R., Zwirglmaier, K., Drosten, C.,Wendtner, C., 2020. Virological assessment of hospitalized patients with COVID-2019. Nature, 581, 465-469.
50. Yan, L., Ge, J., Zheng, L., Zhang, Y., Gao, Y., Wang, T., Huang, Y., Yang, Y., Gao, S., Li, M., Liu, Z., Wang, H., Li, Y., Chen, Y., Guddat, L.W., Wang, Q., Rao, Z.,Lou, Z., 2021. Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis. Cell, 184, 184-193 e110.
51. Yan, L., Huang, Y., Liu, Y., Ge, J., Gao, S., Tan, L., Liu, L., Liu, Z., Ye, S., Wang, J., Xiong, J., Zhou, Y., Zhao, H., Zhao, X., Guddat, L.W., Gao, Y., Zhu, L., Rao, Z.,Lou, Z., 2025. Structural basis for the concurrence of template recycling and RNA capping in SARS-CoV-2. Cell, 188, 7194-7205 e7110.
52. Yan, T., Zhu, S., Wang, H., Li, C., Diao, Y.,Tang, Y., 2020. Synergistic pathogenicity in sequential coinfection with fowl adenovirus type 4 and avian orthoreovirus. Vet Microbiol, 251, 108880.
53. Zhang, L., Yang, Q., Shao, Y., Ding, S., Guo, J., Gao, G.F.,Deng, T., 2025. Influenza A virus NS2 protein acts on vRNA-resident polymerase to drive the transcription to replication switch. Nucleic Acids Res, 53.
54. Zhao, Y., Du, X., Duan, Y., Pan, X., Sun, Y., You, T., Han, L., Jin, Z., Shang, W., Yu, J., et al., 2021. High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors. Protein Cell, 12, 877-888.
55. Zhou, J., Zhou, D., Du, X., Xue, J., Yang, J., Wang, G.,Cheng, Z., 2022. Interaction between Avian Leukosis Virus Subgroup J Surface Protein and Doublecortin-Like Kinase 1 Accelerates Cell Proliferation and Epithelial-Mesenchymal Transition. J Virol, 96, e0165721.
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