Antiviral RNA interference targets viral transcripts but not genomes of RNA viruses in Drosophila melanogaster

RNA interference (RNAi) mediated by the small interfering RNA (siRNA) pathway is a major antiviral mechanism in insects. This pathway is triggered when double-stranded RNA (dsRNA) produced during virus replication is recognized by Dicer-2, leading to the formation of virus-derived siRNA duplexes. These siRNAs are loaded onto the programmable nuclease Argonaute-2 (AGO2), with one strand serving as a guide to target and cleave fully complementary sequences of viral RNAs. While siRNAs are generated from viral dsRNA, the specific viral RNA species targeted for silencing during RNA virus replication remains unclear. In this study, we characterized the primary viral RNA targets of the Drosophila siRNA pathway during infections caused by negative and positive RNA viruses, namely Vesicular stomatitis virus (VSV) and Sindbis virus (SINV). Our findings reveal that polyadenylated transcripts of VSV and SINV are the major targets of silencing by the siRNA pathway during infection, likely when they are poised for translation. Consistent with earlier findings, we confirmed that AGO2 copurifies with ribosomes, and this is not affected by virus infection. Therefore, we propose that the inhibition of the replication of RNA viruses in Drosophila results from the silencing of incoming viral transcripts, facilitated by the association of AGO2 with ribosomes. Author SummaryThe small interfering RNA (siRNA) pathway mediates major antiviral immune response in insects, functioning to cleave viral RNA. While this pathway has been extensively studied in the fruit fly Drosophila melanogaster, the specific molecular targets of inhibition by the siRNA pathway have remained unclear. In this study, we aimed to elucidate these targets. Our findings demonstrate that polyadenylated transcripts produced during viral infection are the primary targets of the Drosophila siRNA pathway, in the case of both negative and positive single-stranded RNA viruses. The silencing of these transcripts accounts for the antiviral effect of the siRNA pathway, suggesting that direct targeting of viral RNA genomes is unlikely to occur. We confirmed that Argonaute-2 (AGO2), the core component of the silencing complex, co-purifies with ribosomes and also show that this association is not affected by viral infection. This suggests that AGO2 is in permanent association with ribosomes where it can efficiently scan viral transcripts before they undergo translation by the cellular machinery, thereby preventing viral replication. These results provide valuable insights into the mechanism of gene silencing the siRNA pathway.