Project description:We observed several hundreds of thousand reads matching the IIV6 genome in IIV6 infected S2 and wild-type flies. The large majority of these reads have a size of 21 nt. This peak was absent from the library prepared from S2 non-infected and infected Dcr-2-/- mutant flies. The virus-derived siRNAs were not uniformally distributed along the viral genome, but rather several hotspots were observed, revealing that specific regions of the viral genome generate the siRNAs. Small RNA profiles of IIV6 infected S2 cells and adult flies were generated by deep sequencing using Illumina 2G Analyzer.
Project description:In Drosophila, the siRNA pathway is initiated when exogenous or endogenous double stranded RNA (dsRNA) is processed into siRNAs by Dicer-2 (Dcr-2) and a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs). The siRNAs are then loaded onto Argonaute-2 (Ago2) protein by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to the siRNA. Dcr-2, R2D2, and Ago2 have also been shown to be required for innate antiviral defense in Drosophila. However, the biogenesis of virus-derived siRNAs (vsiRNAs) and their targets in virus-infected cells remain unclear. Here, we analyzed the antiviral response in Drosophila by monitoring the replication of different RNA viruses and deep sequencing of small RNAs in infected animals. We show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and transcription. These vsiRNAs then directly target viral transcripts but not genomes, to inhibit viral replication. The biogenesis of vsiRNAs was virtually independent of Loqs and R2D2. R2D2, however, was essential for sorting and loading of vsiRNAs onto Ago2 and effective silencing of viral RNA expression. Loqs was completely dispensable for silencing of viruses in contrast to its role in silencing of endogenous targets. Our results suggest the existence of a specific siRNA pathway triggered by viral infection independent of conserved dsRBP cofactors and separate from the endogenous pathway. Inhibition of virus replication resulting from direct injection of viral RNA into Drosophila embryos was also not dependent on Loqs, suggesting the distinction of the two pathways is not related to the mode of entry but recognition of intrinsic features of viral RNA or its mode of replication. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response We analyzed the small RNA reponse to viral infection by deep sequencing of small RNA libraries from wild type and RNAi mutant adult flies infected with Sindbis birus and Vesicular Stomatatis virus.
Project description:Bacterial and fungal infections induce a potent immune response in Drosophila melanogaster, but it is unclear whether viral infections induce a similar immune response. Using microarrays, we examined the changes in gene expression in Drosophila that occur in response to infection with the sigma virus, a negative-stranded RNA virus (Rhabdoviridae) that occurs in wild populations of D. melanogaster. We detected many changes in gene expression in infected flies, but found no evidence for the activation of the Toll, IMD or Jak-STAT pathways, which control immune responses against other pathogens. We identified a number of functional categories of genes, including serine proteases, ribosomal proteins and chorion proteins that were overrepresented among the differentially expressed genes. We also found that the sigma virus alters the expression of many more genes in males than in females. In contrast to previous results, our data suggest that either Drosophila do not mount an immune response against the sigma virus, or that the immune response is not controlled by known immune pathways. The genes which we identified as differentially expressed after infection are promising candidates for controlling the host’s response to the sigma virus.
2009-09-11 | GSE17377 | GEO
Project description:Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV6) Small RNAs deep sequencing
Project description:The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms of innate immunity, and has led to some important discoveries on the sensing and signalling of microbial infections. The response of drosophila to virus infections remains poorly characterized, and appears to involve two facets. On one hand RNA interference (RNAi) involves the recognition and processing of dsRNA into small interfering (si) RNAs by the host ribonuclease Dicer-2 (Dcr-2), whereas on the other hand an inducible response controlled by the evolutionarily conserved JAK/STAT pathway contributes to the antiviral host defence. In order to clarify the contribution of the siRNA and JAK/STAT pathways to the control of viral infections, we have compared the resistance of flies wild-type or mutant for Dcr-2 or the JAK kinase Hopscotch (Hop) to infections by seven RNA or DNA viruses belonging to different families. Our results reveal a unique susceptibility of hop mutant flies to infection by DCV and CrPV, two members of the Dicistroviridae family. Genome-wide microarray analysis confirmed that different sets of genes were induced following infection by DCV (GSE2828) or two unrelated RNA viruses, FHV and SINV. Overall, our data reveal that RNAi is an efficient antiviral mechanism, operating against a large range of viruses, including a DNA virus. By contrast, the antiviral contribution of the JAK/STAT pathway appears to be virus-specific. For each experimental challenge (FHV, 48 or 72 hours after infection; SINV, 4 or 8 days after infection), three biologically independent samples composed of 45 male Oregon R flies were used. Infection has been performed by injecting viral stocks prepared in Tris solution. Injection of the same volume of Tris has been used as control. Infected flies were then incubated for 48 or 72 hours in the case of FHV and 4 days or 8 days in the case of SINV.
Project description:We report the application of ribosomal profiling based RNA sequencing technology for high-throughput profiling of the Gr5a cells of male Drosophila melanogaster adults. By expressing the UAS-Rpl3-3XFLAG transgene using the Gr5a-GAL4 driver on Pclc429 mutant flies.
Project description:High throughput seqeuncing of small RNAs (PAGE isolated from total RNA or Argonaute immunoprecipitates) from Drosophila melanogaster using the Illumina platform. Adapter ligation requires 5' monophosphate and 3' OH. Full analysis of all libraries in this set is published (Czech B. et al. 2008), leading to the description of endogenous siRNAs in flies. Keywords: Solexa sequences