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 AGO2(-/-) cells compared to wild-type (WT) cells, confirming the antiviral role of the RNAi pathway against SFTSV infection. These data suggest that the NSs-AGO2 interaction suppresses RNAi, counteracting the antiviral RNAi pathway, thereby facilitating SFTSV infection and pathogenesis.IMPORTANCERNA interference (RNAi) is the main antiviral defense pathway in plants and insects but is not predominant in mammals. While RNAi's role in countering severe fever with thrombocytopenia syndrome virus (SFTSV) infection has been studied in ticks, its role in humans is unknown. The SFTSV nonstructural protein (NSs) forms inclusion bodies (IBs) in the host, sequestering multiple antiviral proteins and facilitating pathogenesis, contributing to SFTSV's high mortality rate. Our study found that SFTSV NSs directly interacts with AGO2, a key RNAi protein, hindering its function. A novel NSs mutant failed to interact with AGO2 and lost its RNAi suppression ability, highlighting NSs as a viral suppressor of RNAi (VSR). Infection studies confirmed the RNAi pathway's critical role in combating SFTSV infection. This study demonstrates NSs's role in viral infection and suggests potential therapeutic approaches.