2. Academic Validation
  3. NS2B is indispensable for NS3/NS5 interaction of Tembusu virus in living cells

NS2B is indispensable for NS3/NS5 interaction of Tembusu virus in living cells

  • Poult Sci. 2025 Dec 15;105(2):106287. doi: 10.1016/j.psj.2025.106287.
Yu He 1,  Yanfan Shi 2,  Zhen Wu 1,  Tao Wang 3,  Mingshu Wang 3,  Renyong Jia 3,  Dekang Zhu 3,  Mafeng Liu 3,  Xinxin Zhao 3,  Qiao Yang 3,  Ying Wu 3,  Shaqiu Zhang 3,  Juan Huang 3,  Xumin Ou 1,  Di Sun 3,  Bin Tian 4,  Anchun Cheng 5,  Shun Chen 6
Affiliations

Affiliations

  • 1 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology for Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 2 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 3 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology for Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 4 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
  • 5 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology for Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Institute of Veterinary Immunology and Green Drugs, Veterinary Department in College of Animal Science, State Key Laboratory of Green Pesticide, Guizhou University, Guiyang 550025, China. Electronic address: [email protected].
  • 6 Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Agricultural Animal Diseases and Veterinary Public Health Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology for Ministry of Education, Sichuan Agricultural University, Chengdu, Sichuan 611130, China. Electronic address: [email protected].
PMID: 41435701 DOI: 10.1016/j.psj.2025.106287
Abstract

Tembusu virus (TMUV) causes egg-drop syndrome and neurological signs in duck, goose and chicken, posing a great threat to the waterfowl industry. In the present study, we established an in vivo protein-protein interaction system for the NS2B-3 and NS5, together with ​co-immunoprecipitation (Co-IP)​, we demonstrated that NS2B3 (polyprotein) directly binds NS5 in live cells, while NS3 alone fails to interact with NS5-indicating NS2B is indispensable for NS3/NS5 binding. Mutations disrupting NS3's protease active site (to abrogate NS2B3 self-cleavage) or truncations of NS2B both abolished NS2B3/NS5 interactions, highlighting the structural integrity of NS2B-including its transmembrane domains (TMDs) is essential for stabilizing NS3's overall conformation, or cellular localization and facilitating NS3-NS5 binding. Furthermore, we confirmed that NS3-​helicase domain and NS5-RdRp domain mediated NS3/NS5 interaction, and the residues G567, N571, and T584​ were identified as key interaction sites with NS5, validated by reduced viral replication in replicon assays. Our findings reveal that NS2B stabilizes NS3's protease-helicase overall structure, positions NS3 at the ER membrane, finally enabling NS3 to DOCK NS5. This study elucidates the critical role of TMUV NS2B-NS3 complex in mediating NS3-NS5 interaction within the viral replication complex, advances understanding of Flavivirus replication mechanisms.

Keywords

NS2B-NS3; NS3-NS5 interaction; Protease cofactor; Replication complex; Tembusu virus.

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