Article

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: 3.53). 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.

Download full-text

Full-text

Available from: Christophe Antoniewski, Aug 04, 2015
2 Followers
 · 
163 Views
  • 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 · 3.42 Impact Factor
  • Source
    • "Interestingly, p19 also suppresses RNA silencing by another mechanism unrelated to sequestration of sRNAs; in plants p19 expression induces the expression of a host miRNA, miR168, which reduces levels of AGO1, thereby inhibiting anti-viral RISC formation [59]. p19 has been broadly used as an effective suppresser of RNA silencing through expression in heterologous systems, including non-endogenous host plants [24], Drosophila [38], Drosophila embryo extracts [19], as well as human cells [24]. Furthermore, it's ability to sequester small RNAs in a variety of systems make it an ideal probe for examining the role these molecules play in diverse biological processes, not limited to host-pathogen interactions . "
    [Show abstract] [Hide abstract]
    ABSTRACT: The origins of the RNA silencing pathway are in defense against invading viruses and in response, viruses have evolved counter-measures to interfere with the host pathway. The p19 protein is expressed by tombusviruses as a suppressor of RNA silencing and functions to sequester small RNA duplexes, thereby preventing induction of the pathway. p19 exhibits size-specific and sequence-independent binding of its small RNA ligands, binding with high affinity to duplexes 20-22 nucleotides long. p19's binding specificity and its ability to sequester small RNAs has made it a unique protein-based tool for probing the molecular mechanisms of the highly complex RNA silencing pathway in a variety of systems. Furthermore, protein engineering of this 'molecular caliper' promises novel applications in biotechnology and medicine where small RNA molecules are of remarkable interest given their potent gene regulatory abilities.
    FEBS letters 01/2013; 587(8). DOI:10.1016/j.febslet.2013.01.036 · 3.34 Impact Factor
  • Source
    • "Author's personal copy becomes pathogenic (FHV B2, Cirimotich et al., 2009, DCV1A and CrPV1A, Nayak et al., 2010). Importantly, neither of these suppressors provokes such deleterious effects when over-expressed in Drosophila (Berry et al., 2009; Chou et al., 2007; Nayak et al., 2010). Altogether these data strongly suggest that: (i) VSRs are bona fide virulence factors as they reduce viral pathogenicity when mutated or absent (Galiana-Arnoux et al., 2006) but are sufficient to promote pathogenicity in an otherwise innocuous virus; (ii) RNAi reduces viral replication to below a threshold under which the virus is not pathogenic; this threshold is in turn reduced by VSRs and (iii) non-pathogenic viruses such as SINV have an intrinsic pathogenic potential that is revealed when the effects of RNAi are lessened. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract In the past decade, small RNA pathways have been identified as a major mechanism of gene regulation. From an immunity standpoint, these pathways play a central role either by regulating immune reactions or by acting as immune effectors. In insects, several studies have unravelled the role of RNA interference (RNAi) as an antiviral response and have uncovered a complex relationship between insects and viruses that co-evolve in an ongoing race for supremacy. In this review, we comment on the role of small RNA pathways in insect defence and the exploitation of these same pathways by pathogens. We illustrate the host–pathogen relationship under RNAi constraints using several examples and we discuss future directions in using RNAi as a tool to control insect immunity.
    Small RNAs Their Diversity, Roles and Practical uses, Edited by Elizabeth L. Jockusch, 01/2012: pages 1 - 36; Academic Press.
Show more