Molecular mechanism of Tembusu virus nonstructural protein 5 antagonising RNA interference

Tembusu virus (TMUV), a member of the genus Orthoflavivirus within the family Flaviviridae , is an emerging pathogen that severely impacts waterfowl, causing decreased egg production in laying ducks and neurological symptoms in ducklings and growing birds. RNA interference (RNAi) is a conserved post-transcriptional regulatory mechanism mediated by small non-coding RNAs in eukaryotic cells, while many viruses can antagonise host RNAi-mediated antiviral immunity by encoding viral suppressors of RNAi (VSRs). In this study, we demonstrated that TMUV antagonises the RNAi pathway. Using enhanced green fluorescent protein (EGFP)-based RNAi reporter systems in both HEK293T and Drosophila S2 cells, we identified the TMUV nonstructural protein 5 (NS5) as a potent VSR. Furthermore, we found that NS5 not only interacts with DEAD-box helicase 3 X-linked (DDX3X), a key protein in the RNAi pathway, but also binds directly to double-stranded RNA (dsRNA). In summary, our findings indicated that TMUV NS5 can act as a VSR in vitro, thereby providing a theoretical foundation for the development of antiviral therapeutics.