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

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.