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.