Viral Suppressors of RNA Silencing Hinder Exogenous and Endogenous Small RNA Pathways in Drosophila

Victor Chang Cardiac Research Institute (VCCRI), Australia
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(6):e5866. DOI: 10.1371/journal.pone.0005866
Source: PubMed


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.

Download full-text


Available from: Christophe Antoniewski,
  • Source
    • "The affinity of p19 drops off dramatically for any dsRNA longer or shorter than those generated by the dicer enzyme [6]. Because of this p19 is becoming an attractive tool in biotechnology [7], in studies of microRNAs [8e18], in diverse organisms such as human cells, C. elegans, and Drosophila [8] [19] [20], and protein overexpression [21e26]. Chemical biology approaches are increasingly being applied to the RNA silencing pathway, and small molecule screens have identified several modulators of the RNA silencing machinery [27] [28]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC50 = 0.43 μM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC50 = 430 μM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway. Copyright © 2015. Published by Elsevier Inc.
    Biochemical and Biophysical Research Communications 06/2015; 463(4). DOI:10.1016/j.bbrc.2015.06.071 · 2.30 Impact Factor
  • Source
    • "Recently, it was shown that Ago-2, Dcr-2 and R2D2 belong to the top 3% of fastest evolving genes, in sharp contrast to the miRNA pathway genes (Obbard et al., 2006). Moreover, viral suppressors of RNAi (VSRs) have been identified that inhibit efficiently the siRNA, but not the miRNA pathway (Berry et al., 2009; Li et al., 2002; van Rij et al., 2006; Wang et al., 2006). Drosophila siRNA pathway mutants are highly susceptible to viral infections that result in high viral titers compared with wild-type as well as in the detection of viral siRNAs (Kemp and Imler, 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Experiments of dsRNA-mediated gene silencing in lepidopteran insects in vivo are characterized by high variability although lepidopteran cell cultures have shown an efficient response to RNAi in transfection experiments. In order to identify the core RNAi factors that regulate the RNAi response of Lepidoptera, we employed the silkmoth ovary-derived Bm5 cells as a test system since this cell line is known to respond potently in silencing after dsRNA transfection. Two parallel approaches were used; involving knock-down of the core RNAi genes or over-expression of the main siRNA pathway factors, in order to study possible inhibition or stimulation of the RNAi silencing response, respectively. Components from all three main small RNA pathways (BmAgo-1 for miRNA, BmAgo-2/BmDcr-2 for siRNA, and BmAgo-3 for piRNA) were found to be involved in the RNAi response that is triggered by dsRNA. Since BmAgo-3, a factor in the piRNA pathway that functions independent of Dicer in Drosophila, was identified as a limiting factor in the RNAi response, sense and antisense ssRNA was also tested to induce gene silencing but proved to be ineffective, suggesting a dsRNA-dependent role for BmAgo-3 in Bombyx mori. After efficient over-expression of the main siRNA factors, immunofluorescence staining revealed a predominant cytoplasmic localization in Bm5 cells. This is the first study in Lepidoptera to provide evidence for possible overlapping of all three known small RNA pathways in the regulation of the dsRNA-mediated silencing response using transfected B. mori-derived Bm5 cells as experimental system.
    Insect biochemistry and molecular biology 05/2013; 43(8). DOI:10.1016/j.ibmb.2013.05.001 · 3.45 Impact Factor
  • Source
    • "More specifically, it was observed that the conserved PAZ and the adjacent ND domains were necessary for P0-mediated destabilization in all AGO proteins tested [136]. Although the PAZ domain is conserved in animal Argonaute homologs, the strong P0 from Curcubit aphid-borne yellows virus (CAbYV) is not able to suppress RNA silencing in adult Drosophila triggered by dsRNAs or sRNAs [139]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The F-box domain is a protein structural motif of about 50 amino acids that mediates protein-protein interactions. The F-box protein is one of the four components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes. Several discoveries have been made on the use of the ubiquitin-proteasome system by viruses of several families to complete their infection cycle. On the other hand, F-box proteins can be used in the defense response by the host. This review describes the role of F-box proteins and the use of the ubiquitin-proteasome system in virus-host interactions.
    International Journal of Molecular Sciences 02/2013; 14(2):4030-49. DOI:10.3390/ijms14024030 · 2.86 Impact Factor
Show more