Viral suppressors of RNA silencing hinder exogenous and endogenous small RNA pathways in Drosophila.

Institut Pasteur, Drosophila Genetics and Epigenetics, CNRS-URA2578, Paris, France.
PLoS ONE (Impact Factor: 3.73). 02/2009; 4(6):e5866. DOI: 10.1371/journal.pone.0005866
Source: PubMed

ABSTRACT In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs). While VSRs in Drosophila melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways remained unexplored.
Here we show that expression of various plant and insect VSRs in transgenic flies does not perturb the Drosophila microRNA (miRNA) pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed transposon silencing by endogenous siRNAs (endo-siRNAs).
Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Nearly all RNA viruses produce double-stranded RNA (dsRNA) during their replication cycles-an important pathogen-associated molecular pattern recognized by the RNA interference (RNAi) pathway in invertebrates and plants. Nodamura virus (NoV) encodes a suppressor of RNA silencing termed B2, which binds to dsRNA and prevents the initiation of RNAi as well as the loading of silencing complexes. Using the published crystal structure of NoV-B2, we performed a series of molecular dynamics (MD) simulations to determine the relative electrostatic and van der Waals contributions of various residues in binding dsRNA, identifying four novel potential interactors: R56, E48, P68 and R69. Additionally, steered MD was used to simulate the binding affinity of NoV-B2 sequences bearing substitutions at positions F49, R56 or R59 to dsRNA, with F49S and R56L/R59L substitutions found to have a significant negative impact on the ability of NoV-B2 to bind dsRNA. NoV RNA1 variants were tested for self-directed replication in both vertebrate (RNAi(-)) and invertebrate (RNAi(+)) cultured cells. Consistent with a role in dsRNA binding, NoV replication in F49C and F49S variant constructs was affected negatively only in RNAi(+) cells. Thus, we used a combination of MD simulations and experimental mutagenesis to further characterize residues important for NoV-dsRNA interactions.
    Journal of Molecular Modeling 03/2014; 20(3):2092. · 1.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups - wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ∼21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.
    PLoS Genetics 12/2013; 9(12):e1003896. · 8.52 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The host RNA interference (RNAi) pathway of insects senses virus infection and induces an antiviral response to restrict virus replication. Dicer-2 detects viral double-stranded RNA, produced by RNA and DNA viruses, and generates viral small interfering RNAs (vsiRNAs). Recent small RNA profiling studies provided new insights into the viral RNA substrates that trigger vsiRNA biogenesis. The importance of the antiviral RNAi pathway is underscored by the observation that viruses have evolved sophisticated mechanisms to counteract this small RNA-based immune response. More recently, it was proposed that another small RNA silencing mechanism, the piRNA pathway, also processes viral RNAs in Drosophila and mosquitoes. Here, we review recent insights into the mechanism of antiviral RNAi, viral small RNA profiles, and viral counter-defense mechanisms in insects.
    Current opinion in virology. 04/2014; 7C:19-28.

Full-text (2 Sources)

Available from
May 16, 2014