The role of RNA silencing in Drosophila antiviral immunity

Abstract

Innate immune mechanisms are essential components of virus-host interactions and play a critical role in determining the outcome of infection. Cell-intrinsic antiviral pathways have been identified in humans, but the mechanisms governing their function are not fully understood. Moreover, arboviruses, which are transmitted by insect vectors, represent an emerging worldwide threat to human health, but insect antiviral pathways are only beginning to be uncovered. It is likely that additional factors involved in mediating conserved or insect-specific antiviral responses remain to be characterized. Therefore, I sought to identify novel host factors involved in antiviral immunity using image-based high-throughput RNAi screening in Drosophila cells. I found that a previously uncharacterized host factor Ars2 plays an essential role in restricting RNA viruses. Depletion of Ars2 from cells and flies renders them hypersusceptible to viral infection. Specifically, Ars2 controls infection at the level of RNA replication. One known antiviral pathway in insects that responds to viral RNA is RNA interference (RNAi). Using a variety of molecular and biochemical approaches, I found that Ars2 is important for RNAi-mediated silencing at the level of siRNA biogenesis. Additionally, I found that miRNA biogenesis is impaired in the absence of Ars2 in both Drosophila and mammals. Thus, Ars2 is a conserved component of RNA silencing that is critical for RNAi-mediated virus restriction in Drosophila.^ The Drosophila antiviral RNAi response is initiated by Dicer-2, which generates virus-derived siRNAs (vsiRNAs) from viral RNA. Viral dsRNA is thought to be the target of Dicer-2, although the precise viral precursors of vsiRNAs have not been well characterized. To determine the identity of these precursors, I employed small RNA deep sequencing of Drosophila cells infected with a diverse panel of viruses, and mapped the cloned vsiRNAs onto the corresponding viral genomes. Each virus generated a signature pattern of vsiRNAs, and these patterns suggest that dsRNA replication intermediates, structured single-stranded RNAs, and unique hairpins are all Dicer-2 targets. Therefore, the RNAi pathway is capable of recognizing a variety of viral substrates to mount a successful antiviral response.