Host OTUB2 and viral PLpro stabilize NSP8 to promote SARS-CoV-2 replication

Wenying Gao; Hongfei Wang; Jingguo Xin; Chenjia Gu; Lu Qiu; Xue Zhang; Chunlei Wang; Qingxiang Zhang; Shuai Li; Guangquan Li; Wenyan Zhang

doi:10.1016/j.virs.2026.07.008

August 2026

Citation: Wenying Gao, Hongfei Wang, Jingguo Xin, Chenjia Gu, Lu Qiu, Xue Zhang, Chunlei Wang, Qingxiang Zhang, Shuai Li, Guangquan Li, Wenyan Zhang. Host OTUB2 and viral PLpro stabilize NSP8 to promote SARS-CoV-2 replication .VIROLOGICA SINICA, 2026, 41(4) : 806-819. http://dx.doi.org/10.1016/j.virs.2026.07.008

Host OTUB2 and viral PLpro stabilize NSP8 to promote SARS-CoV-2 replication

Wenying Gao ^a ,
Hongfei Wang ^b ,
Jingguo Xin ^a ,
Chenjia Gu ^a ,
Lu Qiu ^c ,
Xue Zhang ^b ,
Chunlei Wang ^a ,
Qingxiang Zhang ^a ,
Shuai Li ^{c
,,} ,
Guangquan Li ^{b
,,} ,
Wenyan Zhang ^{a
,,}

a. Institute of Virology and AIDS Research, Centre of Infectious Diseases and Pathogen Biology, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, 130000, China;
b. Jilin Provincial Key Laboratory on Molecular and Chemical Genetics, The Second Hospital of Jilin University, Changchun, 130000, China;
c. Changchun Institute of Biological Products Co., Ltd, Changchun, 130000, China

Corresponding author: Shuai Li, lishuai3@sinopharm.com
Guangquan Li, liguangquan@jlu.edu.cn
Wenyan Zhang, zhangwenyan@jlu.edu.cn
Received Date: 24 March 2026
Accepted Date: 17 July 2026
Available online: 27 July 2026

Abstract

The ubiquitin-proteasome system (UPS) plays a central role in antiviral defense but is also frequently hijacked by viruses to facilitate their replication. Here, we demonstrate that the host deubiquitinase OTUB2 stabilizes the viral replication factor NSP8 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through a dual-track mechanism. OTUB2 directly removes polyubiquitin chains from NSP8 to prevent its degradation. In parallel, OTUB2 stabilizes the viral papain-like protease (PLpro), which further promotes NSP8 stability through deubiquitination. Together, these effects preserve the functional integrity of the viral replication-transcription complex. Mechanistically, OTUB2-mediated stabilization of NSP8 potentiates NSP8-dependent suppression of type I interferon signaling, thereby promoting viral replication and immune evasion. Importantly, inhibition of OTUB2 disrupts OTUB2-mediated stabilization of NSP8 and PLpro, resulting in a marked reduction in viral replication and disease severity in cell culture systems and a hamster infection model. Collectively, our findings reveal a previously unrecognized mechanism by which SARS-CoV-2 utilizes the host deubiquitination system to stabilize its replication machinery and identify OTUB2 as a potential target for host-directed antiviral intervention.
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
- , NSP8
- , PLpro
- , OTUB2
- , Deubiquitination

Electronic Supplementary Material

10.1016j.virs.2026.07.008-ESM.docx
References
1. Bai, C., Zhong, Q., Gao, G.F., 2022. Overview of SARS-CoV-2 genome-encoded proteins. Sci China Life Sci 65, 280-294.
2. Chen, B., Farzan, M., Choe, H., 2025. SARS-CoV-2 spike protein: structure, viral entry and variants. Nat Rev Microbiol 23, 455-468.
3. Fan, L., Zhou, Y., Wei, X., Feng, W., Guo, H., Li, Y., Gao, X., Zhou, J., Wen, Y., Wu, Y., Shen, X., Liu, L., Xu, G., Zhang, Z., 2024. The E3 ligase TRIM22 restricts SARS-CoV-2 replication by promoting proteasomal degradation of NSP8. mBio 15, e0232023.
4. Gao, W., Wang, L., Ju, X., Zhao, S., Li, Z., Su, M., Xu, J., Wang, P., Ding, Q., Lv, G., Zhang, W., 2022. The Deubiquitinase USP29 Promotes SARS-CoV-2 Virulence by Preventing Proteasome Degradation of ORF9b. mBio 13, e0130022.
5. Gao, Y., Yan, L., Huang, Y., Liu, F., Zhao, Y., Cao, L., Wang, T., Sun, Q., Ming, Z., Zhang, L., Ge, J., Zheng, L., Zhang, Y., Wang, H., Zhu, Y., Zhu, C., Hu, T., Hua, T., Zhang, B., Yang, X., Li, J., Yang, H., Liu, Z., Xu, W., Guddat, L.W., Wang, Q., Lou, Z., Rao, Z., 2020. Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science 368, 779-782.
6. Guo, G., Gao, M., Gao, X., Zhu, B., Huang, J., Luo, K., Zhang, Y., Sun, J., Deng, M., Lou, Z., 2021. SARS-CoV-2 non-structural protein 13 (nsp13) hijacks host deubiquitinase USP13 and counteracts host antiviral immune response. Signal Transduct Target Ther 6, 119.
7. Holmes, E.C., Goldstein, S.A., Rasmussen, A.L., Robertson, D.L., Crits-Christoph, A., Wertheim, J.O., Anthony, S.J., Barclay, W.S., Boni, M.F., Doherty, P.C., Farrar, J., Geoghegan, J.L., Jiang, X., Leibowitz, J.L., Neil, S.J.D., Skern, T., Weiss, S.R., Worobey, M., Andersen, K.G., Garry, R.F., Rambaut, A., 2021. The origins of SARS-CoV-2: A critical review. Cell 184, 4848-4856.
8. Hou, Y., Shi, H., Wang, H., Tian, L., Huan, C., Liu, Y., Wang, H., Zhang, W., 2025. HERC5-mediated ISGylation of SARS-CoV-2 nsp8 facilitates its degradation and inhibits viral replication. Int J Biol Macromol 315, 144546.
9. Hsieh, H.C., Ling, L.L., Wang, Y.C., 2026. Post-translational modifications of immune checkpoints: molecular mechanisms, tumor microenvironment remodeling, and therapeutic implications. J Biomed Sci 33, 3.
10. Isaacson, M.K., Ploegh, H.L., 2009. Ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection. Cell Host Microbe 5, 559-570.
11. Kato, K., Nakajima, K., Ui, A., Muto-Terao, Y., Ogiwara, H., Nakada, S., 2014. Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice. Mol Cell 53, 617-630.
12. Martin-Vicente, M., Resino, S., Martinez, I., 2022. Early innate immune response triggered by the human respiratory syncytial virus and its regulation by ubiquitination/deubiquitination processes. J Biomed Sci 29, 11.
13. Mei, F., Deng, D., Cao, Z., Lou, L., Chen, K., Hu, M., Zhu, Z., Shen, J., Zhang, J., Liang, J., Huang, J., Bao, M., Waisman, A., Wang, X., 2025. Deubiquitination of RIPK3 by OTUB2 potentiates neuronal necroptosis after ischemic stroke. EMBO Mol Med 17, 679-695.
14. Moustaqil, M., Ollivier, E., Chiu, H.P., Van Tol, S., Rudolffi-Soto, P., Stevens, C., Bhumkar, A., Hunter, D.J.B., Freiberg, A.N., Jacques, D., Lee, B., Sierecki, E., Gambin, Y., 2021. SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species. Emerg Microbes Infect 10, 178-195.
15. Nanao, M.H., Tcherniuk, S.O., Chroboczek, J., Dideberg, O., Dessen, A., Balakirev, M.Y., 2004. Crystal structure of human otubain 2. EMBO Rep 5, 783-788.
16. Ren, W., Xu, Z., Chang, Y., Ju, F., Wu, H., Liang, Z., Zhao, M., Wang, N., Lin, Y., Xu, C., Chen, S., Rao, Y., Lin, C., Yang, J., Liu, P., Zhang, J., Huang, C., Xia, N., 2024. Pharmaceutical targeting of OTUB2 sensitizes tumors to cytotoxic T cells via degradation of PD-L1. Nat Commun 15, 9.
17. Shin, D., Mukherjee, R., Grewe, D., Bojkova, D., Baek, K., Bhattacharya, A., Schulz, L., Widera, M., Mehdipour, A.R., Tascher, G., Geurink, P.P., Wilhelm, A., van der Heden van Noort, G.J., Ovaa, H., Muller, S., Knobeloch, K.P., Rajalingam, K., Schulman, B.A., Cinatl, J., Hummer, G., Ciesek, S., Dikic, I., 2020. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587, 657-662.
18. Sivakumar, D., Stein, M., 2021. Binding of SARS-CoV Covalent Non-Covalent Inhibitors to the SARS-CoV-2 Papain-Like Protease and Ovarian Tumor Domain Deubiquitinases. Biomolecules 11.
19. Ton, A.T., Pandey, M., Smith, J.R., Ban, F., Fernandez, M., Cherkasov, A., 2022. Targeting SARS-CoV-2 papain-like protease in the postvaccine era. Trends Pharmacol Sci 43, 906-919.
20. Wu, S., Lei, X., Zhu, Z., Liu, Z., Gao, Y., Wei, J., Qin, Q., 2024a. Grouper OTUB1 and OTUB2 promote red-spotted grouper nervous necrosis virus (RGNNV) replication by inhibiting the host innate immune response. Fish Shellfish Immunol 151, 109715.
21. Wu, X., Go, M., Nguyen, J.V., Kuchel, N.W., Lu, B.G.C., Zeglinski, K., Lowes, K.N., Calleja, D.J., Mitchell, J.P., Lessene, G., Komander, D., Call, M.E., Call, M.J., 2024b. Mutational profiling of SARS-CoV-2 papain-like protease reveals requirements for function, structure, and drug escape. Nat Commun 15, 6219.
22. Wydorski, P.M., Osipiuk, J., Lanham, B.T., Tesar, C., Endres, M., Engle, E., Jedrzejczak, R., Mullapudi, V., Michalska, K., Fidelis, K., Fushman, D., Joachimiak, A., Joachimiak, L.A., 2023. Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin. Nat Commun 14, 2366.
23. Xiao, J., Han, Y., Liu, K., Wang, X., Li, S., Yi, J., Liu, X., 2025. Post-translational modifications: Bridging viral infections and inflammatory bowel disease. Mol Aspects Med 106, 101417.
24. Yang, H., Rao, Z., 2021. Structural biology of SARS-CoV-2 and implications for therapeutic development. Nat Rev Microbiol 19, 685-700.
25. Zhang, X., Yang, Z., Pan, T., Sun, Q., Chen, Q., Wang, P.H., Li, X., Kuang, E., 2023. SARS-CoV-2 Nsp8 suppresses MDA5 antiviral immune responses by impairing TRIM4-mediated K63-linked polyubiquitination. PLoS Pathog 19, e1011792.
26. Zhu, Q., Fu, Y., Li, L., Liu, C.H., Zhang, L., 2021. The functions and regulation of Otubains in protein homeostasis and diseases. Ageing Res Rev 67, 101303.
27. Zong, S., Wu, Y., Li, W., You, Q., Peng, Q., Wang, C., Wan, P., Bai, T., Ma, Y., Sun, B., Qiao, J., 2023. SARS-CoV-2 Nsp8 induces mitophagy by damaging mitochondria. Virol Sin 38, 520-530.
Proportional views