Coronavirus M Protein Hijacks Toll-Interacting Protein (TOLLIP) to Suppress NF-κB Signaling and Promote Immune Evasion

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) employs sophisticated strategies to subvert host innate immunity, a critical determinant of viral establishment and dissemination. Nevertheless, the immunomodulatory functions of coronavirus structural proteins remain incompletely understood. Here, we identify the evolutionarily conserved membrane (M) protein of SARS-CoV-2 as an innate immune antagonist that suppresses nuclear factor kappa-B (NF-κB) activation. Functional assays revealed that the M protein markedly inhibited NF-κB activation and reduced proinflammatory cytokine production in vitro. In lung epithelial M-expressing mouse, M significantly attenuated LPS-induced inflammation. Mechanistically, M protein from diverse coronaviruses directly interacts with host Toll-interacting protein (TOLLIP), stabilizing TOLLIP-IRAK1 complex, preventing IRAK1 activation, thereby suppressing downstream NF-κB signaling and creating a permissive cellular microenvironment for viral replication. We mapped a conserved linker region within the M protein as the core motif mediating this interaction. This binding is highly conserved across coronaviruses, highlighting the fundamental role of the M–TOLLIP axis in viral immune evasion. Our findings reveal a conserved pan-coronavirus immune evasion strategy by which coronaviruses target TOLLIP to subvert Toll-like receptor (TLR)–NF-κB signaling. The conserved M-linker region thus represents a potential broad-spectrum antiviral target, providing a structural framework for developing next-generation antivirals against current and emerging coronavirus threats.

Keywords

coronavirus / immune evasion / innate immune response / M protein / Toll-interacting protein

References

[1] T. Greenhalgh, M. Sivan, A. Perlowski, and J. Z. Nikolich, “Long COVID: A Clinical Update,” Lancet 404, no. 10453 (2024): 707–724.

[2] A. C. Sherman, G. E. Gray, B. Cao, et al., “Acute SARS-CoV-2 Infection,” Nature Reviews Disease Primers 11, no. 1 (2025): 75.

[3] A. M. Carabelli, T. P. Peacock, L. G. Thorne, et al., “SARS-CoV-2 Variant Biology: Immune Escape, Transmission and Fitness,” Nature Reviews Microbiology 21, no. 3 (2023): 162–177.

[4] W. Tian, N. Zhang, and R. Jin, “Immune Suppression in the Early Stage of COVID-19 Disease,” Nature Communications 11, no. 1 (2020): 5859.

[5] L. B. Giron, H. Dweep, X. Yin, et al., “Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients,” Frontiers in Immunology 12 (2021): 686240.

[6] J. Wu, Y. Shi, X. Pan, et al., “SARS-CoV-2 ORF9b Inhibits RIG-I-MAVS Antiviral Signaling by Interrupting K63-Linked Ubiquitination of NEMO,” Cell Reports 34, no. 7 (2021): 108761.

[7] F. Feng, J. Chen, R. Li, et al., “DAZAP2 Functions as a Pan-Coronavirus Restriction Factor by Inhibiting Viral Entry and Genomic Replication,” MBio 16, no. 9 (2025): e0038525.

[8] A. Wahl, L. E. Gralinski, C. E. Johnson, et al., “SARS-CoV-2 Infection is Effectively Treated and Prevented by EIDD-2801,” Nature 591, no. 7850 (2021): 451–457.

[9] M. H. Choi, E. Y. F. Wan, I. C. K. Wong, et al., “Comparative Effectiveness of Combination Therapy With Nirmatrelvir-Ritonavir and Remdesivir Versus Monotherapy With Remdesivir or Nirmatrelvir-Ritonavir in Patients Hospitalised With COVID-19: A Target Trial Emulation Study,” The Lancet Infectious Diseases 24, no. 11 (2024): 1213–1224.

[10] E. V. Koonin, V. V. Dolja, and M. Krupovic, “The Logic of Virus Evolution,” Cell Host & Microbe 30, no. 7 (2022): 917–929.

[11] M. S. Diamond and T. D. Kanneganti, “Innate Immunity: The First Line of Defense Against SARS-CoV-2,” Nature Immunology 23, no. 2 (2022): 165–176.

[12] Q. Guo, Y. Jin, X. Chen, et al., “NF-kappaB in Biology and Targeted Therapy: New Insights and Translational Implications,” Signal Transduction and Targeted Therapy 9, no. 1 (2024): 53.

[13] N. Maihemuti, Y. Shi, K. Zhang, et al., “Toll-Like Receptors in the Immunotherapy Era: Dual-Edged Swords of Tumor Immunity and Clinical Translation,” MedComm (2020) 6, no. 8 (2025): e70308.

[14] S. Mantovani, B. Oliviero, S. Varchetta, A. Renieri, and M. U. Mondelli, “TLRs: Innate Immune Sentries Against SARS-CoV-2 Infection,” International Journal of Molecular Sciences 24, no. 9 (2023): 8065.

[15] K. A. Fitzgerald and J. C. Kagan, “Toll-Like Receptors and the Control of Immunity,” Cell 180, no. 6 (2020): 1044–1066.

[16] K. M. Kim, N. H. Hwang, J. S. Hyun, and D. Shin, “Recent Advances in IRAK1: Pharmacological and Therapeutic Aspects,” Molecules 29, no. 10 (2024): 2226.

[17] D. De Nardo, K. R. Balka, Y. Cardona Gloria, V. R. Rao, E. Latz, and S. L. Masters, “Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) Plays a Dual Role in Myddosome Formation and Toll-Like Receptor Signaling,” Journal of Biological Chemistry 293, no. 39 (2018): 15195–15207.

[18] W. W. Li, X. X. Fan, Z. S. Xu, et al., “BLK Positively Regulates TLR/IL-1R Signaling by Catalyzing TOLLIP Phosphorylation,” Journal of Cell Biology 223, no. 2 (2024): e202302081.

[19] L. C. Su, W. D. Xu, and A. F. Huang, “IRAK Family in Inflammatory Autoimmune Diseases,” Autoimmunity Reviews 19, no. 3 (2020): 102461.

[20] E. J. A. Kowalski and L. Li, “Toll-Interacting Protein in Resolving and Non-Resolving Inflammation,” Frontiers in Immunology 8 (2017): 511.

[21] M. Pereira and R. T. Gazzinelli, “Regulation of Innate Immune Signaling by IRAK Proteins,” Frontiers in Immunology 14 (2023): 1133354.

[22] Z. Y. Low, N. Z. Zabidi, A. J. W. Yip, A. Puniyamurti, V. T. K. Chow, and S. K. Lal, “SARS-CoV-2 Non-Structural Proteins and Their Roles in Host Immune Evasion,” Viruses 14, no. 9 (2022): 1991.

[23] M. Zandi, M. Shafaati, D. Kalantar-Neyestanaki, et al., “The Role of SARS-CoV-2 Accessory Proteins in Immune Evasion,” Biomedicine & Pharmacotherapy 156 (2022): 113889.

[24] J. M. Minkoff and B. tenOever, “Innate Immune Evasion Strategies of SARS-CoV-2,” Nature Reviews Microbiology 21, no. 3 (2023): 178–194.

[25] Y. Zhao, M. Kuang, J. Li, et al., “SARS-CoV-2 Spike Protein Interacts With and Activates TLR41,” Cell Research 31, no. 7 (2021): 818–820.

[26] M. Zheng, R. Karki, E. P. Williams, et al., “TLR2 Senses the SARS-CoV-2 Envelope Protein to Produce Inflammatory Cytokines,” Nature Immunology 22, no. 7 (2021): 829–838.

[27] X. Guo, S. Yang, Z. Cai, et al., “SARS-CoV-2 Specific Adaptations in N Protein Inhibit NF-kappaB Activation and Alter Pathogenesis,” Journal of Cell Biology 224, no. 1 (2025): e202404131.

[28] B. W. Neuman, G. Kiss, A. H. Kunding, et al., “A Structural Analysis of M Protein in Coronavirus Assembly and Morphology,” Journal of Structural Biology 174, no. 1 (2011): 11–22.

[29] W. Pezeshkian, F. Grunewald, O. Narykov, et al., “Molecular Architecture and Dynamics of SARS-CoV-2 Envelope by Integrative Modeling,” Structure 31, no. 4 (2023): 492–503 e7.

[30] M. Rodwell and S. Chtarbanova, “STING-NF-kappaB Signaling: Viral Infection Drives Gut Aging Effects,” Current Biology 34, no. 13 (2024): R618–R620.

[31] F. Liu, Y. Zhao, Y. Pei, F. Lian, and H. Lin, “Role of the NF-kB Signalling Pathway in Heterotopic Ossification: Biological and Therapeutic Significance,” Cell Communication and Signaling 22, no. 1 (2024): 159.

[32] C. A. Dinarello, “Overview of the IL-1 Family in Innate Inflammation and Acquired Immunity,” Immunological Reviews 281, no. 1 (2018): 8–27.

[33] Y. Li, X. Tan, J. Deng, et al., “An Optimized High-Throughput SARS-CoV-2 Dual Reporter Trans-Complementation System for Antiviral Screening in Vitro and in Vivo,” Virologica Sinica 39, no. 3 (2024): 447–458.

[34] X. Huang, Y. Li, J. Deng, et al., “A Biosafe Mouse Model for SARS-CoV-2 Infection That More Realistically Simulates COVID-19 Symptoms,” Signal Transduction and Targeted Therapy 11, no. 1 (2026): 97.

[35] X. Hui, L. Zhang, L. Cao, et al., “SARS-CoV-2 Promote Autophagy to Suppress Type I Interferon Response,” Signal Transduction and Targeted Therapy 6, no. 1 (2021): 180.

[36] Y. Qian, M. Commane, J. Ninomiya-Tsuji, K. Matsumoto, and X. Li, “IRAK-Mediated Translocation of TRAF6 and TAB2 in the Interleukin-1-Induced Activation of NFkappa B,” Journal of Biological Chemistry 276, no. 45 (2001): 41661–41667.

[37] M. Yamamoto, J. Gohda, T. Akiyama, and J. I. Inoue, “TNF Receptor-Associated Factor 6 (TRAF6) Plays Crucial Roles in Multiple Biological Systems Through Polyubiquitination-Mediated NF-kappaB Activation,” Proceedings of the Japan Academy, Series B, Physical and Biological Sciences 97, no. 4 (2021): 145–160.

[38] X. Li, G. C. Goobie, and Y. Zhang, “Toll-Interacting Protein Impacts on Inflammation, Autophagy, and Vacuole Trafficking in Human Disease,” Journal of Molecular Medicine (Berlin) 99, no. 1 (2021): 21–31.

[39] B. A. Williams, C. H. Jones, V. Welch, and J. M. True, “Outlook of Pandemic Preparedness in a Post-COVID-19 World,” NPJ Vaccines 8, no. 1 (2023): 178.

[40] T. J. Loftus, R. Ungaro, M. Dirain, et al., “Overlapping but Disparate Inflammatory and Immunosuppressive Responses to SARS-CoV-2 and Bacterial Sepsis: an Immunological Time Course Analysis,” Frontiers in Immunology 12 (2021): 792448.

[41] E. J. Leacy, J. W. Teh, and A. M. O'Rourke, “Effect of Immunosuppression on the Immune Response to SARS-CoV-2 Infection and Vaccination,” International Journal of Molecular Sciences 25, no. 10 (2024): 5239.

[42] F. Rashid, Z. Xie, M. Suleman, A. Shah, S. Khan, and S. Luo, “Roles and Functions of SARS-CoV-2 Proteins in Host Immune Evasion,” Frontiers in Immunology 13 (2022): 940756.

[43] J. Y. Lee, S. Bae, and J. Myoung, “Middle East Respiratory Syndrome Coronavirus-Encoded Accessory Proteins Impair MDA5-and TBK1-Mediated Activation of NF-kappaB,” Journal of Microbiology and Biotechnology 29, no. 8 (2019): 1316–1323.

[44] S. W. Li, C. Y. Wang, Y. J. Jou, et al., “SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway Through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6,” International Journal of Molecular Sciences 17, no. 5 (2016): 678.

[45] Y. Zheng, M. W. Zhuang, L. Han, et al., “Severe Acute respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Membrane (M) Protein Inhibits Type I and III Interferon Production by Targeting RIG-I/MDA-5 Signaling,” Signal Transduction and Targeted Therapy 5, no. 1 (2020): 299.

[46] Y. Z. Fu, S. Y. Wang, Z. Q. Zheng, et al., “SARS-CoV-2 Membrane Glycoprotein M Antagonizes the MAVS-Mediated Innate Antiviral Response,” Cellular & Molecular Immunology 18, no. 3 (2021): 613–620.

[47] P. Zhou, W. Wu, J. Wei, et al., “MARCH6 Suppresses Tembusu Virus Replication by Targeting Viral NS5 Protein for TOLLIP-Mediated Selective Autophagic Degradation,” Journal of Virology 99, no. 7 (2025): e0073525.

[48] B. Zhang, S. Xu, M. Liu, et al., “The Nucleoprotein of Influenza A Virus Inhibits the Innate Immune Response by Inducing Mitophagy,” Autophagy 19, no. 7 (2023): 1916–1933.

[49] Z. Ma, J. Bai, C. Jiang, et al., “Tegument Protein UL21 of Alpha-Herpesvirus Inhibits the Innate Immunity by Triggering CGAS Degradation Through TOLLIP-Mediated Selective Autophagy,” Autophagy 19, no. 5 (2023): 1512–1532.

[50] J. Hao, J. Li, Z. Zhang, et al., “NLRC5 Restricts Dengue Virus Infection by Promoting the Autophagic Degradation of Viral NS3 Through E3 Ligase CUL2 (cullin 2),” Autophagy 19, no. 4 (2023): 1332–1347.

[51] D. Bojkova, K. Klann, B. Koch, et al., “Proteomics of SARS-CoV-2-Infected Host Cells Reveals Therapy Targets,” Nature 583, no. 7816 (2020): 469–472.

[52] B. Baral, V. Saini, M. Kandpal, et al., “The Interplay of Co-Infections in Shaping COVID-19 Severity: Expanding the Scope Beyond SARS-CoV-2,” Journal of Infection and Public Health 17, no. 8 (2024): 102486.

[53] X. Li, G. C. Goobie, A. D. Gregory, D. J. Kass, and Y. Zhang, “Toll-Interacting Protein in Pulmonary Diseases. Abiding by the Goldilocks Principle,” American Journal of Respiratory Cell and Molecular Biology 64, no. 5 (2021): 536–546.

[54] H. R. Nouri, N. Schaunaman, M. Kraft, M. Numata, D. Vercelli, and H. W. Chu, “Tollip Deficiency Enhances Mitophagy and Reduces STING Activation in Influenza A Virus-Infected Mice,” Journal of Immunology 214, no. 7 (2025): 1741–1753.

[55] N. Schaunaman, K. G. Dimasuay, M. Kraft, and H. W. Chu, “Tollip Interaction With STAT3: A Novel Mechanism to Regulate human Airway Epithelial Responses to Type 2 Cytokines,” Respiratory Research 23, no. 1 (2022): 31.

[56] V. Pokatayev, K. Yang, X. Tu, et al., “Homeostatic Regulation of STING Protein at the Resting State by Stabilizer TOLLIP,” Nature Immunology 21, no. 2 (2020): 158–167.

[57] S. Venkatasubramanian, C. R. Plumlee, K. A. Dill-McFarland, et al., “TOLLIP Inhibits Lipid Accumulation and the Integrated Stress Response in Alveolar Macrophages to Control Mycobacterium Tuberculosis Infection,” Nature Microbiology 9, no. 4 (2024): 949–963.

[58] J. A. Shah, R. Emery, B. Lee, et al., “TOLLIP Deficiency is Associated With Increased Resistance to Legionella Pneumophila Pneumonia,” Mucosal Immunology 12, no. 6 (2019): 1382–1390.

[59] Z. Zhang, N. Nomura, Y. Muramoto, et al., “Structure of SARS-CoV-2 Membrane Protein Essential for Virus Assembly,” Nature Communications 13, no. 1 (2022): 4399.

[60] L. Sui, Y. Zhao, W. Wang, et al., “SARS-CoV-2 Membrane Protein Inhibits Type I Interferon Production Through Ubiquitin-Mediated Degradation of TBK1,” Frontiers in Immunology 12 (2021): 662989.

[61] Y. Ren, A. Wang, Y. Fang, et al., “SARS-CoV-2 Membrane Glycoprotein M Triggers Apoptosis with the Assistance of Nucleocapsid Protein N in Cells,” Frontiers in Cellular and Infection Microbiology 11 (2021): 706252.

[62] Y. Li, C. Li, C. Zhao, et al., “Coronavirus M Protein Promotes Mitophagy Over Virophagy by Recruiting PDPK1 to Phosphorylate SQSTM1 at T138,” Nature Communications 15, no. 1 (2024): 8927.

[63] R. Cannalire, C. Cerchia, A. R. Beccari, F. S. Di Leva, and V. Summa, “Targeting SARS-CoV-2 Proteases and Polymerase for COVID-19 Treatment: State of the Art and Future Opportunities,” Journal of Medicinal Chemistry 65, no. 4 (2022): 2716–2746.

[64] E. Van Damme, P. Abeywickrema, Y. Yin, et al., “A Small-Molecule SARS-CoV-2 Inhibitor Targeting the Membrane Protein,” Nature 640, no. 8058 (2025): 506–513.

[65] M. Laporte, D. Jochmans, D. Bardiot, et al., “A Coronavirus Assembly Inhibitor That Targets the Viral Membrane Protein,” Nature 640, no. 8058 (2025): 514–523.

[66] D. Shi, C. Ma, S. Xu, and P. Zhan, “Targeting the Coronavirus Membrane Protein: A Promising Novel Therapeutic Strategy,” Acta Pharmaceutica Sinica B 15, no. 8 (2025): 4309–4312.

[67] A. D. Proal, S. Aleman, M. Bomsel, et al., “Targeting the SARS-CoV-2 Reservoir in Long COVID,” The Lancet Infectious Diseases 25, no. 5 (2025): e294–e306.

Rights & permissions

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.