SFTSV NSs interacts with AGO2 to regulate the RNAi pathway for viral replication.

RNA interference (RNAi) is a posttranscriptional gene silencing mechanism acting as an antiviral defense in eukaryotes. During viral replication, intermediate double-stranded RNAs are processed into virus-derived small interfering RNAs (vsiRNAs) by the host enzyme, DICER. These vsiRNAs are incorporated into the RNA-induced silencing complex (RISC), where AGO2 cleaves viral genomic RNAs. However, viruses have evolved mechanisms to suppress this pathway. Here, we report that the nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome virus (SFTSV) interacts with RISC to suppress the RNAi pathway. NSs forms a ternary complex by interacting with both DICER and AGO2 of the RNAi pathway. The interaction between NSs and DICER, facilitated by the two RNase III domains of DICER, is disrupted in the absence of AGO2 or the DICER-interacting domain (PIWI) of AGO2, indicating a direct interaction between NSs and AGO2. Functional assays using shRNA- and siRNA-mediated silencing of GFP signal, along with co-localization studies, demonstrated that NSs competes with siRNA to interact with AGO2, thereby abolishing RNAi activity. Mutational analysis identified an NSs-A26 mutant that no longer interacts with AGO2 and is unable to suppress RNAi activity, suggesting that NSs acts as a viral suppressor of RNAi (VSR) for SFTSV. Viral infection led to the generation of vsiRNA and showed higher replication in