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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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Characterization of vsiRNAs.(A,B) Coverage of vsiRNAs along viral genomes in samples from wildtype (wt), Dcr-2, R2D2 and loqs mutant animals. Shown is read density in 20-nt bins for positive-stranded RNAs (blue) and negative-stranded RNAs (red) matching VSV (A) and SINV (B). Genome structures of the viruses are also shown oriented 5′ – 3′ for the positive strand. Protein-coding genes are highlighted. (C,D) Shown are the regions in the VSV (C) and SINV (D) genomes in which no vsiRNAs were detected by high-throughput sequencing. These gaps in vsiRNA coverage are scaled to the genome. Vertical lines in each plot mark the gene promoters within the VSV genome and the 5′ end of the subgenomic RNA in the SINV genome, respectively. The probability that each gap did not occur by chance is shown as the inverse expected value (E-value) on a log10 scale. The horizontal line in each plot represents a significance cutoff of p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Gaps are present in samples from wildtype (wt), R2D2 and loqs mutant infected animals. Since there were fewer sequence reads in wildtype samples, the number of gaps are greater and their significance is smaller.
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ppat-1003579-g003: Characterization of vsiRNAs.(A,B) Coverage of vsiRNAs along viral genomes in samples from wildtype (wt), Dcr-2, R2D2 and loqs mutant animals. Shown is read density in 20-nt bins for positive-stranded RNAs (blue) and negative-stranded RNAs (red) matching VSV (A) and SINV (B). Genome structures of the viruses are also shown oriented 5′ – 3′ for the positive strand. Protein-coding genes are highlighted. (C,D) Shown are the regions in the VSV (C) and SINV (D) genomes in which no vsiRNAs were detected by high-throughput sequencing. These gaps in vsiRNA coverage are scaled to the genome. Vertical lines in each plot mark the gene promoters within the VSV genome and the 5′ end of the subgenomic RNA in the SINV genome, respectively. The probability that each gap did not occur by chance is shown as the inverse expected value (E-value) on a log10 scale. The horizontal line in each plot represents a significance cutoff of p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Gaps are present in samples from wildtype (wt), R2D2 and loqs mutant infected animals. Since there were fewer sequence reads in wildtype samples, the number of gaps are greater and their significance is smaller.

Mentions: Processing of dsRNA by Dcr-2 is dependent on dsRNA substrate concentration in vitro [32], and thus substrate abundance is likely to affect the abundance of siRNAs in vivo. The ratio of siRNA product to dsRNA substrate is therefore an indirect measure of processing activity. Therefore, we normalized the levels of vsiRNAs to the levels of viral genomes (see Methods for details). We found that Dcr-2 mutants had virtually no VSV vsiRNAs when compared to wildtype (Fig. 2B). This result confirmed that the 21 nt RNAs can be considered canonical vsiRNAs. In R2D2 mutants, SINV vsiRNAs levels were similar to wildtype, and VSV vsiRNA abundance was slightly reduced (Fig. 2B,C). loqs mutants had little or no effect on the levels of VSV and SINV vsiRNAs. The distributions of vsiRNAs from R2D2 and loqs mutants were homogeneous along the length of the viral genomes, as was also observed for wildtype (Fig. 3A,B). In contrast, vsiRNAs in Dcr-2 mutants were strongly biased towards the 5′ ends of the VSV genome and antigenome (Fig. 3A). To summarize, R2D2 and Loqs-PD appear largely dispensable for Dcr-2-mediated biogenesis of VSV and SINV vsiRNAs.


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)

Characterization of vsiRNAs.(A,B) Coverage of vsiRNAs along viral genomes in samples from wildtype (wt), Dcr-2, R2D2 and loqs mutant animals. Shown is read density in 20-nt bins for positive-stranded RNAs (blue) and negative-stranded RNAs (red) matching VSV (A) and SINV (B). Genome structures of the viruses are also shown oriented 5′ – 3′ for the positive strand. Protein-coding genes are highlighted. (C,D) Shown are the regions in the VSV (C) and SINV (D) genomes in which no vsiRNAs were detected by high-throughput sequencing. These gaps in vsiRNA coverage are scaled to the genome. Vertical lines in each plot mark the gene promoters within the VSV genome and the 5′ end of the subgenomic RNA in the SINV genome, respectively. The probability that each gap did not occur by chance is shown as the inverse expected value (E-value) on a log10 scale. The horizontal line in each plot represents a significance cutoff of p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Gaps are present in samples from wildtype (wt), R2D2 and loqs mutant infected animals. Since there were fewer sequence reads in wildtype samples, the number of gaps are greater and their significance is smaller.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757037&req=5

ppat-1003579-g003: Characterization of vsiRNAs.(A,B) Coverage of vsiRNAs along viral genomes in samples from wildtype (wt), Dcr-2, R2D2 and loqs mutant animals. Shown is read density in 20-nt bins for positive-stranded RNAs (blue) and negative-stranded RNAs (red) matching VSV (A) and SINV (B). Genome structures of the viruses are also shown oriented 5′ – 3′ for the positive strand. Protein-coding genes are highlighted. (C,D) Shown are the regions in the VSV (C) and SINV (D) genomes in which no vsiRNAs were detected by high-throughput sequencing. These gaps in vsiRNA coverage are scaled to the genome. Vertical lines in each plot mark the gene promoters within the VSV genome and the 5′ end of the subgenomic RNA in the SINV genome, respectively. The probability that each gap did not occur by chance is shown as the inverse expected value (E-value) on a log10 scale. The horizontal line in each plot represents a significance cutoff of p = 0.05 that the gap occurred by chance. E-values above the line are even more significant. Gaps are present in samples from wildtype (wt), R2D2 and loqs mutant infected animals. Since there were fewer sequence reads in wildtype samples, the number of gaps are greater and their significance is smaller.
Mentions: Processing of dsRNA by Dcr-2 is dependent on dsRNA substrate concentration in vitro [32], and thus substrate abundance is likely to affect the abundance of siRNAs in vivo. The ratio of siRNA product to dsRNA substrate is therefore an indirect measure of processing activity. Therefore, we normalized the levels of vsiRNAs to the levels of viral genomes (see Methods for details). We found that Dcr-2 mutants had virtually no VSV vsiRNAs when compared to wildtype (Fig. 2B). This result confirmed that the 21 nt RNAs can be considered canonical vsiRNAs. In R2D2 mutants, SINV vsiRNAs levels were similar to wildtype, and VSV vsiRNA abundance was slightly reduced (Fig. 2B,C). loqs mutants had little or no effect on the levels of VSV and SINV vsiRNAs. The distributions of vsiRNAs from R2D2 and loqs mutants were homogeneous along the length of the viral genomes, as was also observed for wildtype (Fig. 3A,B). In contrast, vsiRNAs in Dcr-2 mutants were strongly biased towards the 5′ ends of the VSV genome and antigenome (Fig. 3A). To summarize, R2D2 and Loqs-PD appear largely dispensable for Dcr-2-mediated biogenesis of VSV and SINV vsiRNAs.

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