RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster

Zambon, Robert A.; Vakharia, Vikram N.; Wu, Louisa P.

doi:10.1111/j.1462-5822.2006.00688.x

Cited by 275 publications

(266 citation statements)

References 50 publications

(72 reference statements)

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“…Previous studies showed that components of the canonical RNAi pathway mediate defense against various single-stranded RNA and double-stranded RNA viruses (1)(2)(3)(4). We tested whether RNAi activity was responsible for maintaining viral latency.…”

Section: Persistent Infection Of Multiple Drosophila S2 Lines By Flocmentioning

confidence: 99%

“…Argonaute2 ͉ Dicer-2 ͉ RNA interference ͉ virus G enetic analyses showed that core components of the RNA interference (RNAi) pathway generate viral small interfering RNAs (siRNAs) and restrict virus accumulation in flies (1)(2)(3)(4)(5), worms (6)(7)(8), and plants (9,10). Such studies indicate that the cellular defense to viral infection begins when double-stranded RNA (dsRNA) viral genomes or replication intermediates are cleaved by Dicer-class RNase III enzymes into small interfering RNAs (siRNAs).…”

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confidence: 99%

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Dicing of viral replication intermediates during silencing of latent Drosophila viruses

Flynt

Liu

Martı́n

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

107

103

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Previous studies revealed roles for RNA interference (RNAi) in the immediate cellular response to viral infection in plants, nematodes and flies. However, little is known about how RNAi combats viruses during persistent or latent infections. Our analysis of small RNAs cloned from Drosophila cells latently infected with Flock House Virus (FHV) failed to reveal signatures of bulk degradation of the viral genome. Instead, this ؉ strand virus specifically generated Dicer-2-dependent, 21-nucleotide siRNAs that derived in equal proportion from ؉ and ؊ strands. Curiously, luciferase reporters that are fully complementary to abundant viral siRNAs were poorly repressed. Moreover, although the viral siRNAs that were incorporated into an effector complex associated with Argonaute2, bulk FHV siRNAs in latently infected cells were not loaded into any Argonaute protein. Together, these data suggest that direct dicing of viral replication intermediates plays an important role in maintaining the latent viral state. In addition, the denial of bulk viral siRNAs from effector complexes suggests that criteria beyond the structural competency of RNA duplexes influence the assembly of functional silencing complexes.Argonaute2 ͉ Dicer-2 ͉ RNA interference ͉ virus G enetic analyses showed that core components of the RNA interference (RNAi) pathway generate viral small interfering RNAs (siRNAs) and restrict virus accumulation in flies (1-5), worms (6-8), and plants (9, 10). Such studies indicate that the cellular defense to viral infection begins when double-stranded RNA (dsRNA) viral genomes or replication intermediates are cleaved by Dicer-class RNase III enzymes into small interfering RNAs (siRNAs). The viral siRNAs are incorporated into Argonaute complexes that subsequently cleave and degrade viral coding RNAs, preventing completion of the viral lifecycle. To counteract the RNAi defense, many viruses have evolved proteins that inhibit various components of the RNAi pathway, thus permitting their successful replication and/or infection (11).In Drosophila, the RNAi and microRNA (miRNA) pathways are genetically distinct, but have substantial cross-talk (12). The canonical RNAi pathway uses Dicer-2 to process dsRNA into siRNAs, which are mostly loaded into Argonaute2 (AGO2) complex. AGO2-resident RNAs are then methylated at their 3Ј ends by the Hen1 methyltransferase (13,14). The miRNA pathway uses Dcr-1 to process premiRNA hairpins into mature miRNAs, which are mostly loaded into Argonaute1 (AGO1) complex; they remain unmethylated. Recent studies demonstrated that the sorting of diced small RNAs is influenced by the structural features of their duplex precursors. Small RNAs from perfectly double stranded duplexes are favored to enter AGO2, whereas central bulges favor entry into AGO1 (15, 16). However, these rules do not entirely explain the types of RNAs that are resident in AGO1 and AGO2 (17, 18); therefore, there are presumably additional determinants that affect small RNA loading.Because the studies to date focused on the role of R...

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Section: Persistent Infection Of Multiple Drosophila S2 Lines By Flocmentioning

confidence: 99%

mentioning

confidence: 99%

Dicing of viral replication intermediates during silencing of latent Drosophila viruses

Flynt

Liu

Martı́n

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

107

103

View full text Add to dashboard Cite

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“…The function of ARGONAUTE (AGO) proteins is clearly essential since genetic inactivation of a single AGO without altering dicing enhances virus susceptibility in fungal, insect, nematode, and plant hosts (Li et al, 2002;Morel et al, 2002;Lu et al, 2005Lu et al, , 2009Schott et al, 2005;Wilkins et al, 2005;van Rij et al, 2006;Zambon et al, 2006;Qu et al, 2008;Sun et al, 2009). In Arabidopsis, hypomorphic ago1 and null ago7 mutants accumulate higher levels of viral RNA, and AGO1, AGO2, and AGO5 bind to viral siRNAs in the infected cells, suggesting an antiviral role for these AGOs (Morel et al, 2002;Zhang et al, 2006;Qu et al, 2008;Takeda et al, 2008;Azevedo et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes inArabidopsis thaliana

et al. 2011

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Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-D2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-D2b requires SUP-PRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-D2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-D2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21-and 22-nucleotide secondary siRNAs are discussed.

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“…Cloning and sequencing of small RNAs from FHV-infected Drosophila cells further indicate that the viral dsRNA replicative intermediates (vRI-dsRNAs) are the substrate of DCR2 and the precursor of viral siRNAs (11,12). Drosophila susceptibility to Drosophila X virus (DXV), which contains a dsRNA genome, is influenced by components from both the siRNA (e.g., AGO2, R2D2) and piRNA (e.g., AUB, PIWI) pathways (13). However, detection of small RNAs derived from any dsRNA virus has not been reported yet (1,13).…”

mentioning

confidence: 99%

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

Luo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

424

411

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In response to infection, invertebrates process replicating viral RNA genomes into siRNAs of discrete sizes to guide virus clearance by RNA interference. Here, we show that viral siRNAs sequenced from fruit fly, mosquito, and nematode cells were all overlapping in sequence, suggesting a possibility of using siRNAs for viral genome assembly and virus discovery. To test this idea, we examined contigs assembled from published small RNA libraries and discovered five previously undescribed viruses from cultured Drosophila cells and adult mosquitoes, including three with a positive-strand RNA genome and two with a dsRNA genome. Notably, four of the identified viruses exhibited only low sequence similarities to known viruses, such that none could be assigned into an existing virus genus. We also report detection of virus-derived PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster that have not been previously described in any other host species and demonstrate viral genome assembly from viral piRNAs in the absence of viral siRNAs. Thus, this study provides a powerful culture-independent approach for virus discovery in invertebrates by assembling viral genomes directly from host immune response products without prior virus enrichment or amplification. We propose that invertebrate viruses discovered by this approach may include previously undescribed human and vertebrate viral pathogens that are transmitted by arthropod vectors.arboviruses | piRNAs | siRNAs | viral immunity | massively parallel sequencing T he Dicer family of host immune receptors mediates antiviral immunity in fungi, plants, and invertebrate animals by RNA interference (RNAi) or RNA silencing (1-3). In this immunity, a viral dsRNA is recognized by Dicer and diced into siRNAs. These virus-derived siRNAs are then loaded into an RNA silencing complex to act as specificity determinants and to guide slicing of the target viral RNAs by an Argonaute protein (AGO) present in the complex. Dicer proteins typically contain an RNA helicase domain, a PAZ domain shared with AGOs, and two tandem type III endoribonuclease (RNase III) domains. Dicer cleaves dsRNA with a simple preference toward a terminus of dsRNA, producing duplex small RNA fragments of discrete sizes progressively from the terminus (4).In addition to siRNAs, micro-RNAs (miRNAs) and PIWIinteracting RNAs (piRNAs) guide RNA silencing in similar complexes but with distinct AGOs (4-6). In Drosophila melanogaster, miRNAs and siRNAs are predominantly 22 and 21 nucleotides in length, dependent on Dicer-1 (DCR1) and DCR2 for their biogenesis, and act in silencing complexes containing AGO1 and AGO2 in the AGO subfamily, respectively (4-6). In contrast, ∼24-30-nt piRNAs are Dicer-independent and require AGO3, Aubergine (AUB), and PIWI in the PIWI subfamily for their biogenesis (4-6). Genetic analyses (7-10) have clearly demonstrated a role for D. melanogaster DCR2 in the immunity and biogenesis of viral siRNAs targeting diverse positive-strand (+) RNA viruses, including Flock house virus (FHV), cricket par...

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RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster

Cited by 275 publications

References 50 publications

Dicing of viral replication intermediates during silencing of latent Drosophila viruses

Dicing of viral replication intermediates during silencing of latent Drosophila viruses

The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes inArabidopsis thaliana

Virus discovery by deep sequencing and assembly of virus-derived small silencing RNAs

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