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Functional specialization of the small interfering RNA pathway in response to virus infection.

Marques JT, Wang JP, Wang X, de Oliveira KP, Gao C, Aguiar ER, Jafari N, Carthew RW - PLoS Pathog. (2013)

Bottom Line: R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression.Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD.We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America. jtm@ufmg.br

ABSTRACT
In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) 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 siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

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R2D2 but not Loqs-PD is required for defense against RNA viruses in Drosophila.(A,B) Viral genome RNA levels in Dcr-2, R2D2 and loqs mutant animals (open bars) compared to their wildtype controls, Dcr-2/+, R2D2/+ and loqs/+ (closed bars). Animals were infected with VSV (A) or SINV (B) for the indicated times. p values below 0.05 are shown for differences between mutants and wildtype. (C) Median survival of control, mock-, VSV- and SINV-infected animals of the indicated genotypes. (*) indicates p<0.05 comparing mock- to virus-infected animals of the same genotype, (#) indicates p<0.05 comparing an infected mutant to its matched wildtype control, and (@) indicates p<0.05 comparing the loqs R2D2 mutant to its matched loqs mutant. Statistical analysis of median survival is shown in Table S1 and the survival curves are shown in Fig. S1. (D) VSV RNA levels at various days post infection in loqsKO  mutant animals with rescue transgenes for Loqs-PB only or Loqs-PB+Loqs-PD. Also shown are loqs heterozygous mutants as wildtype controls and the loqs mutant genotype used in (A). (E) GFP RNA levels in wildtype control and mutant animals infected with recombinant VSV or SINV expressing GFP. (F) Fold increase of SINV RNA levels in wildtype and mutant embryos at 24 h post injection compared to 2 h post injection of purified RNA. p values are shown for significant differences in SINV RNA levels between wildtype and mutant.
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ppat-1003579-g001: R2D2 but not Loqs-PD is required for defense against RNA viruses in Drosophila.(A,B) Viral genome RNA levels in Dcr-2, R2D2 and loqs mutant animals (open bars) compared to their wildtype controls, Dcr-2/+, R2D2/+ and loqs/+ (closed bars). Animals were infected with VSV (A) or SINV (B) for the indicated times. p values below 0.05 are shown for differences between mutants and wildtype. (C) Median survival of control, mock-, VSV- and SINV-infected animals of the indicated genotypes. (*) indicates p<0.05 comparing mock- to virus-infected animals of the same genotype, (#) indicates p<0.05 comparing an infected mutant to its matched wildtype control, and (@) indicates p<0.05 comparing the loqs R2D2 mutant to its matched loqs mutant. Statistical analysis of median survival is shown in Table S1 and the survival curves are shown in Fig. S1. (D) VSV RNA levels at various days post infection in loqsKO mutant animals with rescue transgenes for Loqs-PB only or Loqs-PB+Loqs-PD. Also shown are loqs heterozygous mutants as wildtype controls and the loqs mutant genotype used in (A). (E) GFP RNA levels in wildtype control and mutant animals infected with recombinant VSV or SINV expressing GFP. (F) Fold increase of SINV RNA levels in wildtype and mutant embryos at 24 h post injection compared to 2 h post injection of purified RNA. p values are shown for significant differences in SINV RNA levels between wildtype and mutant.

Mentions: Although Loqs-PD and R2D2 execute different steps in the endo-/exo-siRNA pathway, their roles in the antiviral siRNA pathway are less clear. To explore this issue, we infected Drosophila adults by injecting either SINV or VSV into their hemocoelic cavities. We monitored viral RNA genome levels for three days post-infection (dpi), and observed significantly higher levels of SINV and VSV genomes in Dcr-2 and R2D2 mutants, compared to wildtype (Fig. 1A,B). In contrast, loqs mutants showed viral genome levels indistinguishable from wildtype. We also analyzed host survival after viral infection. When wildtype adults were injected with VSV or SINV, they showed a weak reduction in survival compared to mock-injected animals (Figs. 1C and S1, Table S1). Likewise, loqs mutants showed a comparably weak reduction in lifespan due to VSV or SINV injection when compared to mock-injected. In contrast, R2D2 mutants had a significantly reduced lifespan upon injection of either VSV or SINV (Figs. 1C and S1, Table S1).


Functional specialization of the small interfering RNA pathway in response to virus infection.

Marques JT, Wang JP, Wang X, de Oliveira KP, Gao C, Aguiar ER, Jafari N, Carthew RW - PLoS Pathog. (2013)

R2D2 but not Loqs-PD is required for defense against RNA viruses in Drosophila.(A,B) Viral genome RNA levels in Dcr-2, R2D2 and loqs mutant animals (open bars) compared to their wildtype controls, Dcr-2/+, R2D2/+ and loqs/+ (closed bars). Animals were infected with VSV (A) or SINV (B) for the indicated times. p values below 0.05 are shown for differences between mutants and wildtype. (C) Median survival of control, mock-, VSV- and SINV-infected animals of the indicated genotypes. (*) indicates p<0.05 comparing mock- to virus-infected animals of the same genotype, (#) indicates p<0.05 comparing an infected mutant to its matched wildtype control, and (@) indicates p<0.05 comparing the loqs R2D2 mutant to its matched loqs mutant. Statistical analysis of median survival is shown in Table S1 and the survival curves are shown in Fig. S1. (D) VSV RNA levels at various days post infection in loqsKO  mutant animals with rescue transgenes for Loqs-PB only or Loqs-PB+Loqs-PD. Also shown are loqs heterozygous mutants as wildtype controls and the loqs mutant genotype used in (A). (E) GFP RNA levels in wildtype control and mutant animals infected with recombinant VSV or SINV expressing GFP. (F) Fold increase of SINV RNA levels in wildtype and mutant embryos at 24 h post injection compared to 2 h post injection of purified RNA. p values are shown for significant differences in SINV RNA levels between wildtype and mutant.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757037&req=5

ppat-1003579-g001: R2D2 but not Loqs-PD is required for defense against RNA viruses in Drosophila.(A,B) Viral genome RNA levels in Dcr-2, R2D2 and loqs mutant animals (open bars) compared to their wildtype controls, Dcr-2/+, R2D2/+ and loqs/+ (closed bars). Animals were infected with VSV (A) or SINV (B) for the indicated times. p values below 0.05 are shown for differences between mutants and wildtype. (C) Median survival of control, mock-, VSV- and SINV-infected animals of the indicated genotypes. (*) indicates p<0.05 comparing mock- to virus-infected animals of the same genotype, (#) indicates p<0.05 comparing an infected mutant to its matched wildtype control, and (@) indicates p<0.05 comparing the loqs R2D2 mutant to its matched loqs mutant. Statistical analysis of median survival is shown in Table S1 and the survival curves are shown in Fig. S1. (D) VSV RNA levels at various days post infection in loqsKO mutant animals with rescue transgenes for Loqs-PB only or Loqs-PB+Loqs-PD. Also shown are loqs heterozygous mutants as wildtype controls and the loqs mutant genotype used in (A). (E) GFP RNA levels in wildtype control and mutant animals infected with recombinant VSV or SINV expressing GFP. (F) Fold increase of SINV RNA levels in wildtype and mutant embryos at 24 h post injection compared to 2 h post injection of purified RNA. p values are shown for significant differences in SINV RNA levels between wildtype and mutant.
Mentions: Although Loqs-PD and R2D2 execute different steps in the endo-/exo-siRNA pathway, their roles in the antiviral siRNA pathway are less clear. To explore this issue, we infected Drosophila adults by injecting either SINV or VSV into their hemocoelic cavities. We monitored viral RNA genome levels for three days post-infection (dpi), and observed significantly higher levels of SINV and VSV genomes in Dcr-2 and R2D2 mutants, compared to wildtype (Fig. 1A,B). In contrast, loqs mutants showed viral genome levels indistinguishable from wildtype. We also analyzed host survival after viral infection. When wildtype adults were injected with VSV or SINV, they showed a weak reduction in survival compared to mock-injected animals (Figs. 1C and S1, Table S1). Likewise, loqs mutants showed a comparably weak reduction in lifespan due to VSV or SINV injection when compared to mock-injected. In contrast, R2D2 mutants had a significantly reduced lifespan upon injection of either VSV or SINV (Figs. 1C and S1, Table S1).

Bottom Line: R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression.Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD.We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, United States of America. jtm@ufmg.br

ABSTRACT
In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) 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 siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response.

Show MeSH
Related in: MedlinePlus