Characterizing the role of RSD-6 in the biogenesis of virus-derived small interfering RNAs and the modulation of viral pathogenesis

Small interfering RNAs (siRNAs) produced through the processing of viral double-stranded RNAs mediate potent antiviral RNA interference (RNAi) in eukaryotes. In <i>Caenorhabditis elegans</i>, such an antiviral defense is further amplified through the production of secondary siRNAs, yet the mechanisms by which secondary virus-derived siRNAs (vsiRNAs) confer protection remain poorly understood. Here, we characterize the role of <i>rsd-6</i>, which encodes a Tudor domain protein and plays an important role in antiviral RNAi, in vsiRNA biogenesis and the modulation of viral pathogenesis. Using CRISPR Cas9-generated <i>rsd-6</i> null mutants, we show that both primary and secondary vsiRNAs accumulate normally in the absence of RSD-6, indicating that it functions downstream of secondary vsiRNA biogenesis. We further showed that secondary vsiRNAs generated in an <i>rrf-1</i>-independent manner remained detected in the absence of RSD-6 and that viral replication is further enhanced in <i>rrf-1;rsd-6</i> double mutants compared to <i>rrf-1</i> single mutants, suggesting a role of <i>rsd-6</i> in mediating antiviral guided by all secondary vsiRNAs. Consistently, <i>rsd-6</i> mutants exhibited more severe pathogenesis upon Orsay virus infection compared to <i>rrf-1</i> mutants, underscoring its role as a major determinant of viral disease outcome. Domain characterization established that the N-terminal tandem domains of RSD-6 are required for antiviral activity, while the C-terminal Tudor domains are dispensable. Functional conservation was confirmed in <i>C. briggsae</i>, where silencing of the <i>rsd-6</i> homolog enhanced viral replication. Together, our findings identify RSD-6 as a key effector acting downstream of secondary vsiRNA production and highlight its conserved role in modulating viral replication and pathogenesis across <i>Caenorhabditis</i> species.IMPORTANCEIn <i>C. elegans</i>, the RNAi-mediated antiviral defense relies on the production of secondary virus-derived siRNAs (vsiRNAs) to achieve an amplified antiviral effect. However, the mechanism by which these secondary vsiRNAs confer protection remains poorly understood. This is primarily due to the limited number of identified key effector genes. To address this knowledge gap, we profiled vsiRNA biogenesis in loss-of-function mutants and discovered that <i>rsd-6</i> is dispensable for the production of secondary vsiRNAs, suggesting a role of <i>rsd-6</i> in mediating antiviral defense downstream of secondary vsiRNA biogenesis. Worm survival assay further confirmed that <i>rsd-6</i> is a critical modulator of viral pathogenesis and its antiviral function is conserved across Caenorhabditis species. The RSD-6 protein features three N-terminal tandem domains of unknown function and two tandem Tudor domains at its C-terminus. Our domain analyses demonstrated that the N-terminal tandem domains, but not the C-terminal Tudor domains, are essential for antiviral function. The identification of <i>rsd-6</i> as a key effector gene acting downstream of vsiRNA biogenesis provides a solid foundation for elucidating the mechanism of antiviral RNAi amplification.