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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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Viral polyadenylated RNA is a major target of slicing by Ago2.(A,B) Levels of genome RNA, antigenome RNA, and polyadenylated virus RNA from Dcr-2 mutants (hollow bars) and wildtype controls (solid bars) at different times post infection with VSV (A) and SINV (B). Asterisks indicate p<0.05 comparing RNA levels between mutant and wildtype samples. (C) Fold increase in polyadenylated viral RNA, genome RNA, and antigenome RNA in Dcr-2 mutants relative to wildtype at different times post infection with VSV or SINV. (D) VSV RNA levels in  Ago2414, Ago2V966M/Ago2414, and wildtype heterozygous animals at different days post infection. Asterisks indicate p<0.05 comparing RNA levels between mutant and matched wildtype samples; (@) indicates p<0.05 comparing RNA levels between Ago2414 and Ago2V966M/Ago2414 mutants.
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ppat-1003579-g006: Viral polyadenylated RNA is a major target of slicing by Ago2.(A,B) Levels of genome RNA, antigenome RNA, and polyadenylated virus RNA from Dcr-2 mutants (hollow bars) and wildtype controls (solid bars) at different times post infection with VSV (A) and SINV (B). Asterisks indicate p<0.05 comparing RNA levels between mutant and wildtype samples. (C) Fold increase in polyadenylated viral RNA, genome RNA, and antigenome RNA in Dcr-2 mutants relative to wildtype at different times post infection with VSV or SINV. (D) VSV RNA levels in Ago2414, Ago2V966M/Ago2414, and wildtype heterozygous animals at different days post infection. Asterisks indicate p<0.05 comparing RNA levels between mutant and matched wildtype samples; (@) indicates p<0.05 comparing RNA levels between Ago2414 and Ago2V966M/Ago2414 mutants.

Mentions: vsiRNAs originate from both strands along the entire length of the VSV and SINV genomes, and so they could potentially inhibit positive-stranded, negative-stranded, and transcript viral RNAs. During infection, production of each viral RNA species is dependent on the others; genomes make transcripts; transcripts make replication proteins, which make genomes and antigenomes. Thus, vsiRNAs that directly inhibit one class of RNAs would indirectly inhibit production of other viral RNAs. We hypothesized that loss of inhibition would lead to more pronounced changes in the levels of direct vsiRNA targets than downstream RNAs. To measure the abundance of negative- and positive-stranded viral RNAs, we employed strand-specific RT-qPCR. We confirmed that mispriming did not significantly affect our measurements by using no-primer control reactions [49] (data not shown). We measured the abundance of polyadenylated viral RNAs by oligo dT-directed RT-qPCR. We then compared the abundance of viral RNAs extracted from wildtype hosts versus Dcr-2 mutants. Levels of all SINV and VSV RNA species were derepressed in Dcr-2 mutants (Fig. 6A,B). We calculated the level of derepression for each species of viral RNA. Polyadenylated viral RNA was more strongly derepressed than either negative- or positive-stranded viral RNA; 2.3-fold for SINV (p = 0.004) and 3.9-fold for VSV (p = 0.001) (Fig. 6C). This greater sensitivity of polyadenylated RNA to inhibition suggests that it is the primary target of 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)

Viral polyadenylated RNA is a major target of slicing by Ago2.(A,B) Levels of genome RNA, antigenome RNA, and polyadenylated virus RNA from Dcr-2 mutants (hollow bars) and wildtype controls (solid bars) at different times post infection with VSV (A) and SINV (B). Asterisks indicate p<0.05 comparing RNA levels between mutant and wildtype samples. (C) Fold increase in polyadenylated viral RNA, genome RNA, and antigenome RNA in Dcr-2 mutants relative to wildtype at different times post infection with VSV or SINV. (D) VSV RNA levels in  Ago2414, Ago2V966M/Ago2414, and wildtype heterozygous animals at different days post infection. Asterisks indicate p<0.05 comparing RNA levels between mutant and matched wildtype samples; (@) indicates p<0.05 comparing RNA levels between Ago2414 and Ago2V966M/Ago2414 mutants.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757037&req=5

ppat-1003579-g006: Viral polyadenylated RNA is a major target of slicing by Ago2.(A,B) Levels of genome RNA, antigenome RNA, and polyadenylated virus RNA from Dcr-2 mutants (hollow bars) and wildtype controls (solid bars) at different times post infection with VSV (A) and SINV (B). Asterisks indicate p<0.05 comparing RNA levels between mutant and wildtype samples. (C) Fold increase in polyadenylated viral RNA, genome RNA, and antigenome RNA in Dcr-2 mutants relative to wildtype at different times post infection with VSV or SINV. (D) VSV RNA levels in Ago2414, Ago2V966M/Ago2414, and wildtype heterozygous animals at different days post infection. Asterisks indicate p<0.05 comparing RNA levels between mutant and matched wildtype samples; (@) indicates p<0.05 comparing RNA levels between Ago2414 and Ago2V966M/Ago2414 mutants.
Mentions: vsiRNAs originate from both strands along the entire length of the VSV and SINV genomes, and so they could potentially inhibit positive-stranded, negative-stranded, and transcript viral RNAs. During infection, production of each viral RNA species is dependent on the others; genomes make transcripts; transcripts make replication proteins, which make genomes and antigenomes. Thus, vsiRNAs that directly inhibit one class of RNAs would indirectly inhibit production of other viral RNAs. We hypothesized that loss of inhibition would lead to more pronounced changes in the levels of direct vsiRNA targets than downstream RNAs. To measure the abundance of negative- and positive-stranded viral RNAs, we employed strand-specific RT-qPCR. We confirmed that mispriming did not significantly affect our measurements by using no-primer control reactions [49] (data not shown). We measured the abundance of polyadenylated viral RNAs by oligo dT-directed RT-qPCR. We then compared the abundance of viral RNAs extracted from wildtype hosts versus Dcr-2 mutants. Levels of all SINV and VSV RNA species were derepressed in Dcr-2 mutants (Fig. 6A,B). We calculated the level of derepression for each species of viral RNA. Polyadenylated viral RNA was more strongly derepressed than either negative- or positive-stranded viral RNA; 2.3-fold for SINV (p = 0.004) and 3.9-fold for VSV (p = 0.001) (Fig. 6C). This greater sensitivity of polyadenylated RNA to inhibition suggests that it is the primary target of 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