Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes

Donaire, Livia; Wang, Yu; González-Ibeas, Daniel; Mayer, Klaus; Aranda, Miguel A.; Llave, César

doi:10.1016/j.virol.2009.07.005

Cited by 263 publications

(244 citation statements)

References 58 publications

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“…In addition to the DI-RNA-derived siRNA peaks in ANV RNA2, the distribution of viral siRNAs was not uniform along the remaining viral genomic RNAs (Fig. S4), as noted previously (16)(17)(18)(19)(20). However, our analyses indicated that the high siRNA density regions of the viral RNA genomes were associated with neither unusual adenine+uridine (AU) content nor strong secondary structures.…”

Section: Discovery Of Four Rna Viruses From a Drosophila Schneider 2 supporting

confidence: 64%

“…The Dicer proteins involved in the production of siRNAs targeting both +RNA viruses and DNA viruses have been identified in Arabidopsis thaliana (2,3), and plants encode AGOs in the AGO subfamily but none from the PIWI subfamily (15). Cloning and sequencing of plant viral siRNAs suggest that they may be processed either from vRI-dsRNAs or hairpin regions of single-stranded RNA precursors (16)(17)(18)(19)(20). Production of viral siRNAs has also been demonstrated in fungi, silkworms, mosquitoes, and nematodes in response to infection with +RNA viruses, and viral small silencing RNAs produced in fungi and mosquitoes have recently been cloned and sequenced (21)(22)(23)(24)(25).…”

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confidence: 99%

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

Luo

et al. 2010

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

433

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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Section: Discovery Of Four Rna Viruses From a Drosophila Schneider 2 supporting

confidence: 64%

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.

433

411

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“…In plants, where studies have focused on positive ssRNA viruses, there is some evidence for the first scenario. For example, infection by the Tombusvirus CymRSV generates ∼80% to 90% of viral small RNAs of sense polarity, which may reflect the excess of genome versus antigenome RNA present in infected cells (24,27,30). However, the profiles of vsiRNAs produced in response to several other plant viruses are consistent with processing of a dsRNA precursor: these profiles are characterized by an approximately equal number of vsiRNAs matching the positive or the negative strand of the viral RNA, and covering the whole length of the viral genome (24,26,29).…”

Section: Discussionmentioning

confidence: 99%

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Mueller

Gausson

Vodovar

et al. 2010

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

135

134

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Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5′ ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.deep sequencing | arbovirus | Dicer-2 | AGO2 | small RNA profiling

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“…(vsiRNAs) is the most remarkable feature of virus-induced gene silencing [4,[8][9][10][11][12][13]. Current research indicates that vsiRNAs are derived either from the dsRNAs formed during the virus replication stage or from single-stranded viral RNAs (ssRNAs) with a hairpin-like fold-back structure [8,10,[14][15][16].…”

Section: Special Topicmentioning

confidence: 99%

“…Current research indicates that vsiRNAs are derived either from the dsRNAs formed during the virus replication stage or from single-stranded viral RNAs (ssRNAs) with a hairpin-like fold-back structure [8,10,[14][15][16]. Both of these forms of viral RNA can be processed into vsiRNAs of a specific size by distinct DCLs proteins.…”

Section: Special Topicmentioning

confidence: 99%

The role of virus-derived small interfering RNAs in RNA silencing in plants

Zhu

Guo

2012

Sci. China Life Sci.

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Antiviral defense is one of the important roles of RNA silencing in plants. Virus-derived small interfering RNAs (vsiRNAs) are found in the infected host cells, indicating that the host RNA silencing machinery can target viral RNAs for destruction. With the development of high-throughput sequencing of vsiRNAs, recent genetic studies have shed light on the origin and composition of vsiRNAs and their potential functions in the regulation of gene expression. Here, we briefly describe the origin and biogenesis of vsiRNAs, and review the recent discoveries regarding vsiRNA-mediated RNA silencing of viral genomes and host transcripts. This will better our understanding of virus pathogenicity and RNA silencing-related host-pathogen interactions in plants.

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Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes

Cited by 263 publications

References 58 publications

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

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

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

The role of virus-derived small interfering RNAs in RNA silencing in plants

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