Identification of new proviral and antiviral factors through the study of the Dicer-2 interactomein vivoduring viral infection inDrosophila melanogaster

Abstract

ABSTRACTRNA interference, which has a major role in the control of viral infection in insects, is initialized by the sensing of double stranded RNA (dsRNA) by the RNAse III enzyme Dicer-2. Although manyin vitrostudies have helped understand how Dicer-2 is able to discriminate between different dsRNA substrate termini, much less is known about how this translates to thein vivorecognition of viral dsRNA. Indeed, although Dicer-2 associates with several dsRNA-binding proteins (dsRBPs) that can modify its specificity for a substrate, it remains unknown how Dicer-2 is able to recognize the protected termini of viral dsRNAs. In order to study how the ribonucleoprotein network of Dicer-2 impacts antiviral immunity, we used an IP-MS approach to identifyin vivointeractants of different versions of GFP::Dicer-2 in transgenic lines. We provide a global overview of the partners of Dicer-2in vivo, and reveal how this interactome is modulated by different factors such as the viral infection and/or different point mutations inactivating the helicase or RNase III domains of GFP::Dicer-2. Our analysis uncovers several previously unknown Dicer-2 interactants associated with RNA granules (i.e. Me31B, Rump, eIF4E1 & Syp). Functional characterization of the candidates reveals pro- and antiviral factors in the context of the infection by the picorna-like DCV virus. In particular, the protein Rasputin has been identified as a novel antiviral candidate. The resources provided by this work can be used to gain a better understanding of the molecular complexes assembled around Dicer-2 in the context of antiviral RNAi and beyond.