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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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vsiRNA abundance is dependent on Dcr-2 but not Loqs-PD.(A) Levels of the genome RNA strand, antigenome RNA strand, and total virus RNA from VSV and SINV infected animals 48 hours post infection. The polarity of SINV and VSV genomes are indicated. (B,C) Normalized levels of sequenced small RNAs of different size that match the VSV (B) and SINV (C) genomes. Shown are levels after infection of wildtype (wt), Dcr-2, R2D2 and loqs mutants. Bars above the midline denote positive-stranded small RNAs, and bars below the midline denote negative-stranded small RNAs.
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ppat-1003579-g002: vsiRNA abundance is dependent on Dcr-2 but not Loqs-PD.(A) Levels of the genome RNA strand, antigenome RNA strand, and total virus RNA from VSV and SINV infected animals 48 hours post infection. The polarity of SINV and VSV genomes are indicated. (B,C) Normalized levels of sequenced small RNAs of different size that match the VSV (B) and SINV (C) genomes. Shown are levels after infection of wildtype (wt), Dcr-2, R2D2 and loqs mutants. Bars above the midline denote positive-stranded small RNAs, and bars below the midline denote negative-stranded small RNAs.

Mentions: SINV and VSV have single-stranded RNA genomes, and they synthesize an antigenome RNA of opposite polarity in order to synthesize more genomes [29], [30], [31]. The antigenome is typically less abundant than the genome since one antigenome template can be copied several times. In wildtype hosts, SINV and VSV produced a 6.3- and 5.5-fold excess of genomes over antigenomes, respectively (Fig. 2A). As expected for canonical siRNAs, the majority of VSV and SINV vsiRNAs were 21 nt in length (Fig. 2B,C). These mapped in roughly equal numbers to both genome and antigenome strands of SINV and VSV. Thus, the ratio of vsiRNAs derived from genome and antigenome strands was clearly different from the relative abundance of genomes and antigenomes (Fig. 2A–C). An equal distribution of vsiRNAs to both strands indicates that the preferred substrate for their biogenesis is sense-antisense viral dsRNA.


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)

vsiRNA abundance is dependent on Dcr-2 but not Loqs-PD.(A) Levels of the genome RNA strand, antigenome RNA strand, and total virus RNA from VSV and SINV infected animals 48 hours post infection. The polarity of SINV and VSV genomes are indicated. (B,C) Normalized levels of sequenced small RNAs of different size that match the VSV (B) and SINV (C) genomes. Shown are levels after infection of wildtype (wt), Dcr-2, R2D2 and loqs mutants. Bars above the midline denote positive-stranded small RNAs, and bars below the midline denote negative-stranded small RNAs.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757037&req=5

ppat-1003579-g002: vsiRNA abundance is dependent on Dcr-2 but not Loqs-PD.(A) Levels of the genome RNA strand, antigenome RNA strand, and total virus RNA from VSV and SINV infected animals 48 hours post infection. The polarity of SINV and VSV genomes are indicated. (B,C) Normalized levels of sequenced small RNAs of different size that match the VSV (B) and SINV (C) genomes. Shown are levels after infection of wildtype (wt), Dcr-2, R2D2 and loqs mutants. Bars above the midline denote positive-stranded small RNAs, and bars below the midline denote negative-stranded small RNAs.
Mentions: SINV and VSV have single-stranded RNA genomes, and they synthesize an antigenome RNA of opposite polarity in order to synthesize more genomes [29], [30], [31]. The antigenome is typically less abundant than the genome since one antigenome template can be copied several times. In wildtype hosts, SINV and VSV produced a 6.3- and 5.5-fold excess of genomes over antigenomes, respectively (Fig. 2A). As expected for canonical siRNAs, the majority of VSV and SINV vsiRNAs were 21 nt in length (Fig. 2B,C). These mapped in roughly equal numbers to both genome and antigenome strands of SINV and VSV. Thus, the ratio of vsiRNAs derived from genome and antigenome strands was clearly different from the relative abundance of genomes and antigenomes (Fig. 2A–C). An equal distribution of vsiRNAs to both strands indicates that the preferred substrate for their biogenesis is sense-antisense viral dsRNA.

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