Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

Mierlo, Joël T. van; Bronkhorst, Alfred W; Overheul, Gijs J.; Sadanandan, Sajna Anand; Ekström, Jens‐Ola; Heestermans, Marco; Hultmark, Dan; Antoniewski, Christophe; Rij, Ronald P. van

doi:10.1371/journal.ppat.1002872

Cited by 95 publications

(141 citation statements)

References 55 publications

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“…We thus analyzed 21-nt viral small RNAs for the presence of this Dcr-2 signature, as previously described (37). In contrast to our observations in infections with Nora virus, a (+) RNA virus (37), we did not observe enrichment for a 19-nt overlap among viral small RNAs mapping to opposite strands of the IIV-6 genome (Fig.…”

Section: Iiv-6 As a Model To Study Antiviral Immunity Against Dna Virmentioning

confidence: 49%

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

Bronkhorst

Cleef²,

Vodovar³

et al. 2012

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

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134

155

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RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6-infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2-dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.insect immunity | Iridoviridae | siRNA | antiviral immunity | small RNA profiling | next-generation sequencing

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Section: Iiv-6 As a Model To Study Antiviral Immunity Against Dna Virmentioning

confidence: 49%

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

Bronkhorst

Cleef²,

Vodovar³

et al. 2012

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

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134

155

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“…The MoNV B2 ORF was PCR amplified using the primers 5=-ACGTGGTACCCAAA ATGACAGAAAATCAGCAGCAACTTC-3= and 5=-ACGTGCGGCCGC CACTTCGCTTCGACGATGGGGGGC-3=. PCR products were cloned into the Acc65I and NotI restriction sites of pAc5.1-V5-His-Ntag (47). To detect expression of V5-tagged MoNV protein, Western blotting was performed using a monoclonal mouse anti-V5 antibody (Invitrogen) and IRDye 680 goat anti-mouse IgG (Li-Cor Biosciences) for detection on the Odyssey infrared imager (Li-Cor Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Antiviral RNAi is activated by viral doublestranded RNA (dsRNA), which is processed into viral small interfering RNAs (vsiRNAs) of 21 nucleotides (nt) by the RNase Dicer-2 (Dcr-2) (28,29,(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). The current model proposes that these vsiRNAs are loaded onto Argonaute-2 (AGO2) in the RNAinduced silencing complex (RISC) to guide the recognition and cleavage of viral target RNA by AGO2, thereby restricting viral replication (29,33,37,38,(46)(47)(48). Indeed, fruit flies deficient in Dicer-2, its cofactor R2D2, and AGO2 are hypersensitive to virus infection and support higher levels of viral replication than wildtype flies (32, 35-38, 48, 49).…”

mentioning

confidence: 99%

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A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

Schuster

Zirkel

Kurth

et al. 2014

J Virol

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Insects are a reservoir for many known and novel viruses. We discovered an unknown virus, tentatively named mosinovirus (MoNV), in mosquitoes from a tropical rainforest region in Côte d'Ivoire. The MoNV genome consists of two segments of positive-sense RNA of 2,972 nucleotides (nt) (RNA 1) and 1,801 nt (RNA 2). Its putative RNA-dependent RNA polymerase shares 43% amino acid identity with its closest relative, that of the Pariacoto virus (family Nodaviridae). Unexpectedly, for the putative capsid protein, maximal pairwise identity of 16% to Lake Sinai virus 2, an unclassified virus with a nonsegmented RNA genome, was found. Moreover, MoNV virions are nonenveloped and about 50 nm in diameter, larger than any of the known nodaviruses. Mature MoNV virions contain capsid proteins of ϳ56 kDa, which do not seem to be cleaved from a longer precursor. Northern blot analyses revealed that MoNV expresses two subgenomic RNAs of 580 nt (RNA 3) and 292 nt (RNA 4). RNA 4 encodes a viral suppressor of RNA interference (RNAi) that shares its mechanism with the B2 RNAi suppressor protein of other nodaviruses despite lacking recognizable similarity to these proteins. MoNV B2 binds long double-stranded RNA (dsRNA) and, accordingly, inhibits Dicer-2-mediated processing of dsRNA into small interfering RNAs (siRNAs). Phylogenetic analyses indicate that MoNV is a novel member of the family Nodaviridae that acquired its capsid gene via reassortment from an unknown, distantly related virus beyond the family level. IMPORTANCEThe identification of novel viruses provides important information about virus evolution and diversity. Here, we describe an unknown unique nodavirus in mosquitoes, named mosinovirus (MoNV). MoNV was classified as a nodavirus based on its genome organization and on phylogenetic analyses of the RNA-dependent RNA polymerase. Notably, its capsid gene was acquired from an unknown virus with a distant relationship to nodaviruses. Another remarkable feature of MoNV is that, unlike other nodaviruses, it expresses two subgenomic RNAs (sgRNAs). One of the sgRNAs expresses a protein that counteracts antiviral defense of its mosquito host, whereas the function of the other sgRNA remains unknown. Our results show that complete genome segments can be exchanged beyond the species level and suggest that insects harbor a large repertoire of exceptional viruses.

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“…In contrast, detection of viral double-stranded RNA (dsRNA) by Dicer endonucleases is associated with the production of small interfering RNAs (siRNAs), which subsequently direct sequencespecific antiviral immunity through RNA interference (RNAi) (2)(3)(4)(5). Antiviral RNAi is a major antiviral defense mechanism in fungi, plants, insects, and nematodes, so that suppression of the defense by a virus-encoded suppressor of RNAi is essential to establish infection (6)(7)(8). Although the RNAi machinery is highly conserved in mammals, conclusive evidence for a natural antiviral role of RNAi in mammals is not available (9).…”

mentioning

confidence: 99%

Homologous RIG-I–like helicase proteins direct RNAi-mediated antiviral immunity in C. elegans by distinct mechanisms

Guo

Zhang

Wang

et al. 2013

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

128

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RNAi-mediated antiviral immunity in Caenorhabditis elegans requires Dicer-related helicase 1 (DRH-1), which encodes the helicase and C-terminal domains homologous to the mammalian retinoic acid inducible gene I (RIG-I)-like helicase (RLH) family of cytosolic immune receptors. Here we show that the antiviral function of DRH-1 requires the RIG-I homologous domains as well as its wormspecific N-terminal domain. We also demonstrate that the helicase and C-terminal domains encoded by either worm DRH-2 or human RIG-I can functionally replace the corresponding domains of DRH-1 to mediate antiviral RNAi in C. elegans. Notably, substitutions in a three-residue motif of the C-terminal regulatory domain of RIG-I that physically interacts with viral double-stranded RNA abolish the antiviral activity of C-terminal regulatory domains of both RIG-I and DRH-1 in C. elegans. Genetic analysis revealed an essential role for both DRH-1 and DRH-3 in C. elegans antiviral RNAi targeting a natural viral pathogen. However, Northern blot and small RNA deep sequencing analyses indicate that DRH-1 acts to enhance production of viral primary siRNAs, whereas DRH-3 regulates antiviral RNAi by participating in the biogenesis of secondary siRNAs after Dicer-dependent production of primary siRNAs. We propose that DRH-1 facilitates the acquisition of viral double-stranded RNA by the worm dicing complex for the subsequent processing into primary siRNAs. The strong parallel for the antiviral function of RLHs in worms and mammals suggests that detection of viral doublestranded RNA may activate completely unrelated effector mechanisms or, alternatively, that the mammalian RLHs have a conserved activity to stimulate production of viral siRNAs for antiviral immunity by an RNAi effector mechanism.I nnate immunity refers to ancient host defense systems that act immediately after pathogen attack and are under the control of germline-encoded immune receptors. Pathogen sensing by several classes of immune receptors such as retinoic acid inducible gene I (RIG-I)-like cytosolic sensors in mammals leads to the transcriptional activation of effector genes in the nucleus (1). In contrast, detection of viral double-stranded RNA (dsRNA) by Dicer endonucleases is associated with the production of small interfering RNAs (siRNAs), which subsequently direct sequencespecific antiviral immunity through RNA interference (RNAi) (2-5). Antiviral RNAi is a major antiviral defense mechanism in fungi, plants, insects, and nematodes, so that suppression of the defense by a virus-encoded suppressor of RNAi is essential to establish infection (6-8). Although the RNAi machinery is highly conserved in mammals, conclusive evidence for a natural antiviral role of RNAi in mammals is not available (9).The nematode Caenorhabditis elegans has recently emerged as a small-animal model for innate immunity studies (10, 11). Genetic studies indicate that antiviral RNAi in C. elegans requires the genes essential for RNAi induced by exogenous dsRNA (12-17). These include Dicer-1 and dsRNA-bind...

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Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

Cited by 95 publications

References 55 publications

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

Homologous RIG-I–like helicase proteins direct RNAi-mediated antiviral immunity in C. elegans by distinct mechanisms

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