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.