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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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Requirement for Dcr-2 helicase activity and detection of vsiRNA phasing in SINV.(A) SINV genome RNA levels in Dcr-2A500V and wildtype animals at different times post infection. Asterisks indicate p<0.05. (B) Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from the SINV positive strand. Shown are all vsiRNAs mapping to the 5′-most 1000 nts of the positive strand. The sample was derived from infected R2D2 mutants. ACF values above the dotted line are statistically significant (p<0.05).
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ppat-1003579-g005: Requirement for Dcr-2 helicase activity and detection of vsiRNA phasing in SINV.(A) SINV genome RNA levels in Dcr-2A500V and wildtype animals at different times post infection. Asterisks indicate p<0.05. (B) Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from the SINV positive strand. Shown are all vsiRNAs mapping to the 5′-most 1000 nts of the positive strand. The sample was derived from infected R2D2 mutants. ACF values above the dotted line are statistically significant (p<0.05).

Mentions: Our data indicates that vsiRNA production by Dcr-2 is an active mechanism that requires efficient processing of viral dsRNA substrates of diverse origins. Dcr-2 has RNase III domains that cleave dsRNA, and it also has an ATP-dependent helicase domain that is required for efficient processing [32]. To determine if the Dcr-2 helicase is essential, we infected a Dcr-2 mutant that specifically disables the helicase domain (Dcr-2A500V) with SINV [44]. Similar to Dcr-2 mutants, Dcr-2A500V mutants showed increased levels of SINV replication (Fig. 5A). This result suggests that helicase activity is essential for the antiviral response. The helicase has been shown to enhance two features of dsRNA dicing. It is required for Dcr-2 to recognize dsRNA ends that are blunt or have 5′ overhangs [45]. It also allows multiple siRNAs to be produced along the length of a dsRNA without Dcr-2 dissociation [32]. One of the consequences of this Dcr-2 processivity is the production of siRNAs with defined spacing between the 5′ end of one siRNA and its nearest neighbors on the same strand [32]. This phasing has been detected in siRNAs generated from dsRNA substrates in vitro and in vivo as a discrete end-to-end distance peak [46], [47].


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)

Requirement for Dcr-2 helicase activity and detection of vsiRNA phasing in SINV.(A) SINV genome RNA levels in Dcr-2A500V and wildtype animals at different times post infection. Asterisks indicate p<0.05. (B) Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from the SINV positive strand. Shown are all vsiRNAs mapping to the 5′-most 1000 nts of the positive strand. The sample was derived from infected R2D2 mutants. ACF values above the dotted line are statistically significant (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003579-g005: Requirement for Dcr-2 helicase activity and detection of vsiRNA phasing in SINV.(A) SINV genome RNA levels in Dcr-2A500V and wildtype animals at different times post infection. Asterisks indicate p<0.05. (B) Autocorrelation functions (ACF) of the distance in nucleotides between 5′ ends of vsiRNAs from the SINV positive strand. Shown are all vsiRNAs mapping to the 5′-most 1000 nts of the positive strand. The sample was derived from infected R2D2 mutants. ACF values above the dotted line are statistically significant (p<0.05).
Mentions: Our data indicates that vsiRNA production by Dcr-2 is an active mechanism that requires efficient processing of viral dsRNA substrates of diverse origins. Dcr-2 has RNase III domains that cleave dsRNA, and it also has an ATP-dependent helicase domain that is required for efficient processing [32]. To determine if the Dcr-2 helicase is essential, we infected a Dcr-2 mutant that specifically disables the helicase domain (Dcr-2A500V) with SINV [44]. Similar to Dcr-2 mutants, Dcr-2A500V mutants showed increased levels of SINV replication (Fig. 5A). This result suggests that helicase activity is essential for the antiviral response. The helicase has been shown to enhance two features of dsRNA dicing. It is required for Dcr-2 to recognize dsRNA ends that are blunt or have 5′ overhangs [45]. It also allows multiple siRNAs to be produced along the length of a dsRNA without Dcr-2 dissociation [32]. One of the consequences of this Dcr-2 processivity is the production of siRNAs with defined spacing between the 5′ end of one siRNA and its nearest neighbors on the same strand [32]. This phasing has been detected in siRNAs generated from dsRNA substrates in vitro and in vivo as a discrete end-to-end distance peak [46], [47].

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