RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in
            <i>Drosophila</i>

Mueller, Stefanie; Gausson, Valérie; Vodovar, Nicolas; Deddouche, Safia; Troxler, Laurent; Perot, Jonathan; Pfeffer, Sébastien; Hoffmann, Jules A.; Saleh, María-Carla; Imler, Jean‐Luc

doi:10.1073/pnas.1014378107

Cited by 131 publications

(158 citation statements)

References 47 publications

(40 reference statements)

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“…In contrast to the sharp 21-nt peak in WT flies and AGO2 mutants, small RNAs in Dcr-2 mutants showed a broader size distribution, with only a minor enrichment at 21 nt. A similar shift toward small RNAs with sizes other than 21 nt was noted before in Dcr-2 mutant flies infected with vesicular stomatitis virus (14). Recombinant and immunoprecipitated Dicer-1 (Dcr-1) is able to process long dsRNA into siRNA in in vitro assays, albeit with low efficiency (39)(40)(41).…”

Section: Iiv-6 As a Model To Study Antiviral Immunity Against Dna Virmentioning

confidence: 80%

“…Indeed, early seminal work in mosquitoes and Drosophila cells directly detected viral small RNAs by Northern blot analysis and demonstrated that knockdown of core RNAi genes resulted in an increase in virus replication (8)(9)(10). In accordance, in the genetic model organism D. melanogaster, flies with defects in Dcr-2, R2D2, or AGO2 are unable to control RNA virus replication and, consequently, are hypersensitive to virus infection and succumb more rapidly than their wild-type (WT) controls (11)(12)(13)(14).…”

mentioning

confidence: 98%

“…Indeed, vsiRNAs mapped in similar proportions to (+) and (−) viral RNA strands in Aedes aegypti, Aedes albopictus, and Culex pipiens mosquitoes infected with a number of arthropodborne viruses, including Sindbis virus, Semliki Forest virus, West Nile virus, Dengue virus, and Chikungunya virus, as well as in Drosophila infected with (+) RNA viruses from different families (5,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). In addition, in infections of Drosophila with the negative-strand RNA virus vesicular stomatitis virus, similar numbers of (+) over (−) vsiRNAs were recovered (14). These results, together with the distribution of vsiRNAs all along the viral genome, imply that viral replication intermediates of RNA viruses are the main targets for Dcr-2.…”

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confidence: 99%

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The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

Bronkhorst

Cleef²,

Vodovar³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.insect immunity | Iridoviridae | siRNA | antiviral immunity | small RNA profiling | next-generation sequencing

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Section: Iiv-6 As a Model To Study Antiviral Immunity Against Dna Virmentioning

confidence: 80%

mentioning

confidence: 98%

mentioning

confidence: 99%

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The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

Bronkhorst

Cleef²,

Vodovar³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

132

155

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“…The monoclonal anti-dsRNA J2 antibody binds dsRNAs at least 40 bp long in a sequence-independent fashion (12)(13)(14). The J2 dsRNA monoclonal antibody was previously used to detect viral dsRNA by dsRNA immunoblotting, immunohistochemistry, ELISA, and immunoprecipitation in eukaryotic cells (13)(14)(15)(16)(17)(18)(19)(20). In this study, we used the J2 dsRNA antibody in an immunoprecipitation assay to enrich dsRNA from total RNA extracted from E. coli.…”

Section: Resultsmentioning

confidence: 99%

The double-stranded transcriptome of Escherichia coli

Lybecker

Zimmermann

Bilusic

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

146

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Advances in high-throughput transcriptome analyses have revealed hundreds of antisense RNAs (asRNAs) for many bacteria, although few have been characterized, and the number of functional asRNAs remains unknown. We have developed a genomewide high-throughput method to identify functional asRNAs in vivo. Most mechanisms of gene regulation via asRNAs require an RNA-RNA interaction with its target RNA, and we hypothesized that a functional asRNA would be found in a double strand (dsRNA), duplexed with its cognate RNA in a single cell. We developed a method of isolating dsRNAs from total RNA by immunoprecipitation with a ds-RNA specific antibody. Total RNA and immunoprecipitated dsRNA from Escherichia coli RNase III WT and mutant strains were deep-sequenced. A statistical model was applied to filter for biologically relevant dsRNA regions, which were subsequently categorized by location relative to annotated genes. A total of 316 potentially functional asRNAs were identified in the RNase III mutant strain and are encoded primarily opposite to the 5′ ends of transcripts, but are also found opposite ncRNAs, gene junctions, and the 3′ ends. A total of 21 sense/antisense RNA pairs identified in dsRNAs were confirmed by Northern blot analyses. Most of the RNA steady-state levels were higher or detectable only in the RNase III mutant strain. Taken together, our data indicate that a significant amount of dsRNA is formed in the cell, that RNase III degrades or processes these dsRNAs, and that dsRNA plays a major role in gene regulation in E. coli.T he advent and development of high-throughput sequencing technologies has uncovered the presence of widespread antisense transcription in many bacteria, with the number of annotated genes associated with antisense RNA (asRNA) differing greatly among bacterial species (1, 2). asRNAs are encoded on the DNA strand opposite an annotated gene and overlap a portion of a gene or the entire gene, or span multiple genes with perfect complementarity. asRNAs range in size from tens to thousands of nucleotides. Although numerous chromosomally encoded asRNAs have been identified, few have been confirmed by traditional methods or functionally characterized. Raghavan et al. (3) reported that few asRNAs are conserved between Escherichia coli and Salmonella enterica, and that the predicted promoter sequences of the asRNAs are not conserved between these species, suggesting that most asRNA transcripts are products of spurious transcription and are not biologically functional RNAs.The majority of functionally characterized asRNAs are found on plasmids, phages, and transposons (4, 5). The mode of regulation by asRNAs can be classified according to molecular mechanism as transcription interference, transcription attenuation, alteration of transcript stability, and translation inhibition (1, 2, 6). With the exception of transcription interference, a physical RNA-RNA interaction between the sense RNAs and asRNAs is necessary for all of these mechanisms, requiring that both RNAs be expressed in the same...

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“…A classical mechanism of this category is accomplished by IFN, a cytokine that is induced by dsRNA and creates an antiviral state in host cells (6)(7)(8). The third mechanism involves RNA interference (RNAi), by which the viral genome or transcripts are destroyed (9)(10)(11)(12). A newly discovered mechanism adopts autophagy for the elimination of intracellular virus (13)(14)(15).…”

mentioning

confidence: 99%

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis

Nainu

Tanaka

Shiratsuchi

et al. 2015

The Journal of Immunology

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We investigated whether phagocytosis participates in the protection of insects from viral infection using the natural host–virus interaction between Drosophila melanogaster and Drosophila C virus (DCV). Drosophila S2 cells were induced to undergo apoptotic cell death upon DCV infection. However, UV-inactivated virus was unable to cause apoptosis, indicating the need for productive infection for apoptosis induction. S2 cells became susceptible to phagocytosis by hemocyte-derived l(2)mbn cells after viral infection, and the presence of phagocytes in S2 cell cultures reduced viral proliferation. Phagocytosis depended, in part, on caspase activity in S2 cells, as well as the engulfment receptors Draper and integrin βν in phagocytes. To validate the in vivo situation, adult flies were abdominally infected with DCV, followed by the analysis of fly death and viral growth. DCV infection killed flies in a dose-responding manner, and the activation of effector caspases was evident, as revealed by the cleavage of a target protein ectopically expressed in flies. Furthermore, hemocytes isolated from infected flies contained DCV-infected cells, and preinjection of latex beads to inhibit the phagocytic activity of hemocytes accelerated fly death after viral infection. Likewise, viral virulence was exaggerated in flies lacking the engulfment receptors, and was accompanied by the augmented proliferation of virus. Finally, phagocytosis of DCV-infected cells in vitro was inhibited by phosphatidylserine-containing liposome, and virus-infected flies died early when a phosphatidylserine-binding protein was ectopically expressed. Collectively, our study demonstrates that the apoptosis-dependent, phosphatidylserine-mediated phagocytosis of virus-infected cells plays an important role in innate immune responses against viral infection in Drosophila.

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RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Cited by 131 publications

References 47 publications

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

The double-stranded transcriptome of Escherichia coli

Protection of Insects against Viral Infection by Apoptosis-Dependent Phagocytosis

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