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
  • 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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