viral rna recognition by the drosophila small interfering rna pathway

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immunity. These nucleic acid sensors must be able to

systems must cope with the ubiquitous presence of RNA and RNAi was first described as dsRNA-mediated gene

fruit fly, D. melanogaster, there are at least three majorclasses of small ncRNAs, known as microRNAs (miRNAs),siRNAs and piwi-interacting RNAs (piRNAs) [8]. Thesedistinct classes of small RNAs define separate RNAi

mento de Bioqumica e Imunologia-ICB, Av. Anto^nio Carlos, 6627, Pampulha,Belo Horizonte, MG CEP 31270-901, Brazil. Tel.: 55 31 3409 2623;fax: 55 31 3409 2614.

E-mail address: [email protected] (J.T. Marques).

Microbes and Infect

+ MODELDNA. Indeed, erroneous sensing of nucleic acids is oftenassociated with autoimmunity [2]. The RNA interference(RNAi) pathway is a highly efficient RNA recognition systemactivated by diverse sources of nucleic acids [3]. In this re-view, we will focus on the recognition of viral RNA in the fruitfly Drosophila melanogaster, which is mediated by a highlyspecialized RNAi mechanism known as the small interferingRNA (siRNA) pathway. The major trigger for the activation of

silencing in the nematode worm Caenorhabiditis elegans [4].Currently, the term RNAi is used to broadly describe path-ways that utilize small non-coding RNAs (ncRNAs) in as-sociation with an Argonaute protein to regulate geneexpression and other biological processes ranging from DNAreplication to translation [5e7]. The active complex formedby the small ncRNA associated with the Argonaute protein isoften referred to as the RNA-induced silencing complex(RISC) [6]. RNAi pathways and Argonaute proteins arepresent in most eukaryotes although their diversity andfunction can vary significantly [5,6]. In animals, such as the

* Corresponding author. Universidade Federal de Minas Gerais, Departa-discriminate molecular patterns found in infected cells that aremostly absent in healthy conditions. Nucleic acids sensing2. RNA interference pathwaysKeywords: RNA interference; Small interfering RNA; dsRNA; Viral infection; Drosophila melanogaster

1. Introduction

Nucleic acid sensing is a common strategy that prokaryoticand eukaryotic cells utilize to recognize invading viruses [1].A variety of DNA and RNA recognition proteins have beenlinked to activation of both cell-autonomous and systemic

this pathway is double stranded RNA (dsRNA), which isgenerated as a byproduct of viral replication but is also foundin uninfected cells. Thus, the Drosophila siRNA pathwayneeds to deal with viral and cellular sources of dsRNA. Un-derstanding this mechanism can help understand general as-pects of nucleic acid sensing.Viral RNA recognition by the Droso

Zamira Guerra Soares, Andre Nicolau AquimeJo~ao Trinda

Department of Biochemistry and Immunology, Univer

Received 28 June 2014;

Abstract

Viral RNA is a common activator of antiviral responses. In this reinterfering RNA pathway in Drosophila melanogaster. This antiviral rerecognition. 2014 Institut Pasteur. Published by Elsevier Masson SAS. All righPlease cite this article in press as: Soares ZG, et al., Viral RNA recognition by the

http://dx.doi.org/10.1016/j.micinf.2014.09.001

http://dx.doi.org/10.1016/j.micinf.2014.09.001

1286-4579/ 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rightsila small interfering RNA pathway

onalves, Karla Pollyanna Vieira de Oliveira,Marques*

de Federal de Minas Gerais, Belo Horizonte, Brazil

pted 1 September 2014

w, we dissect the mechanism of viral RNA recognition by the smallnse in fruit flies can help understand general principles of nucleic acid

served.

ion xx (2014) 1e9www.elsevier.com/locate/micinfDrosophila small interfering RNA pathway, Microbes and Infection (2014),

reserved.

plasmic RNase III enzyme distinct from Dcr-1 [26,27]. siR-y a

heterodimer between Dcr-2 and r2d2, a dsRBP partner [28].

different from piwi, Aub and AGO3 that belong to the PIWIfrome. In

contrast, the siRNA pathway can be activated by long dsRNA

s anThe Dcr-2/r2d2 complex transfers the duplex siRNA toArgonaute-2 (AGO2) to form siRNA programmed RISCNAs are 21-nt long duplex RNAs that are bound bpathways that differ in function, biogenesis and mechanismof action.

2.1. Drosophila RNA interference pathways

The piRNA pathway is mostly active in the animal germ-line although it is can also be present in somatic tissues [9].Loss of piRNAs causes genomic instability and leads to ste-rility in animals. Drosophila piRNAs are 24e27 nt long smallRNAs that originate from single-stranded RNA (ssRNA) pre-cursors arising mostly from transposable elements and otherspecific clusters in the genome [10]. The mechanism of piRNAbiogenesis from ssRNA precursors is still not completely un-derstood. Once primary piRNAs are generated they can alsoenter an amplification loop, called the ping-pong mechanism,to drive the production of secondary piRNAs [10,11]. MaturepiRNAs associate with an animal specific subfamily of animalArgonaute proteins represented by three different members inDrosophila: piwi, Aubergine (Aub) and Argonaute-3 (AGO3)[6]. More detailed reviews about the piRNA pathway can befound elsewhere [9,12].

The miRNA pathway is absolutely necessary for animaldevelopment and cell differentiation [13]. miRNAs are non-coding genes transcribed by RNA polymerase II (RNA polII) as ssRNA transcripts known as primary miRNAs (pri-miRNA) that fold onto a hairpin structure with long arms[14,15]. In Drosophila, pri-miRNAs are processed by the nu-clear RNase III enzyme Drosha associated with a dsRNAbinding protein (dsRBP) called Pasha [16]. This first nuclearprocessing event generates precursor miRNAs (pre-miRNA)that are approximately 65-nt long ssRNA hairpins, which areexported to the cytoplasm by exportin-5 (Exp5) [17]. In thecytoplasm, pre-miRNAs are processed by another RNase IIIcalled Dicer-1 (Dcr-1) associated with the isoform PB ofloquacious (loqs-PB), a small dsRBP [18,19]. This secondprocessing event generates 22-nt long small duplex RNAsknown as mature miRNAs that associate with Argonaute-1(AGO1) [20]. The miRNA duplex is then dissociated torelease the passenger strand from AGO1 that remains associ-ated with the guide strand to generate miRNA programmedRISC (miRISC) [21,22]. miRISC will search for partiallycomplementary binding sites in the 30 untranslated region(UTR) of messenger RNAs (mRNAs) where binding leads toinhibition of translation by different mechanisms [23,24].There are several hundreds of miRNAs in Drosophila thatpotentially regulate the expression of half of all coding genesin the fly genome [3,25]. An overview of the DrosophilamiRNA pathway is shown in Fig. 1A.

In Drosophila, long dsRNA (>30 bp) is recognized andprocessed into siRNAs by Dicer-2 (Dcr-2), another cyto-

2 Z.G. Soares et al. / Microbe(siRISC) [29,30]. Mature siRISC that carries only the guidestrand of the siRNA duplex is formed after AGO2 cleaves the

Please cite this article in press as: Soares ZG, et al., Viral RNA recognition by the

http://dx.doi.org/10.1016/j.micinf.2014.09.001precursors that are artificially introduced into cells, producedas a byproduct of viral replication during infection or originatefrom the Drosophila genome [3]. The ability to recognizedifferent sources of dsRNA is an important characteristic ofthe Drosophila siRNA pathway as represented in Fig. 2.

2.2. Endogenous and exogenous siRNA pathways inDrosophila

The dsRNA trigger for the siRNA pathway can originatefrom diverse sources, either endogenous or exogenous. Severaldistinct sources of dsRNA are commonly labeled as exogenousincluding transgenes, in vitro synthesized RNAs and viralinfection [38e42]. In contrast, the term endogenous usuallyrefers to RNA molecules synthesized by nuclear transcriptionusing the host genome as template. Endogenous dsRNAcommonly originates from structured loci, sense-antisensepairs and bidirectional transcription of transposable elements[43,44]. Although these diverse sources of dsRNA commonlyactivate the Drosophila siRNA pathway, there are subtle dif-ferences in the mechanisms of siRNA biogenesis and actionthat will be discussed in this review.

Historically, the mechanism of gene silencing by the siRNApathway was first characterized using Drosophila embryos orcell culture and in vitro synthesized dsRNA[27,29,41,42,45e47]. In these cases, dsRNA was often injec-ted into Drosophila embryos or delivered to cultured cells bysoaking or transfection to induce gene silencing [45,48,49]. Inaddition, mechanisms of gene silencing triggered by dsRNAwere also characterized by biochemistry utilizing protein ex-clade [6]. Both miRNAs and piRNAs originate mostlyRNA precursors transcribed from the Drosophila genompassenger strand that is then released and further degraded[31,32]. The guide strand is then 20-O-methylated withinAGO2 by the Drosophila 20-O-methytranferase DmHen1,which stabilizes the silencing complex [33]. siRISC searchesfor fully complementary sequences within mRNAs which re-sults in direct target cleavage by AGO2 [34]. Silencing ofmRNAs by siRISC is highly efficient because AGO2 is amultiple turnover enzyme that catalyzes cleavage of numeroustargets [34]. Polyadenylated RNAs, presumably mRNAs, seemto be preferred targets of silencing by the siRNA pathway [35].Interestingly, AGO2 is found in association with cellular ri-bosomes, which suggests it can monitor incoming mRNAsbefore they can be translated [36,37]. An overview of theDrosophila siRNA pathway is shown in Fig. 1B.

A close comparison of the three small RNA pathways inDrosophila suggests there are important similarities and dis-tinctions. The biogenesis of both miRNAs and siRNAs involveprocessing of dsRNA precursors by RNase III enzymes whilepiRNAs arise from ssRNA precursors [3,12]. Argonaute pro-teins associated with miRNAs and siRNAs, AGO1 and AGO2,respectively, belong to the AGO clade and are significantly

d Infection xx (2014) 1e9tracts from Drosophila embryos, adult heads or cultured S2cells and in vitro synthesized dsRNA [27,29,42,50]. Thus, the

Drosophila small interfering RNA pathway, Microbes and Infection (2014),

s anZ.G. Soares et al. / MicrobedsRNA source for these initial experiments could all be clas-sically considered exogenous. Genome-integrated transgenesencoding inverted-repeat transcripts were also used to induceefficient, heritable and durable gene silencing in Drosophilaadults [39]. In these cases, the dsRNA arises from nucleartranscription similar to endogenous transcripts althoughtransgenes are technically exogenous. Screens in Drosophilaadults with transgenes and cell culture using in vitro synthe-sized dsRNA helped identify components of the siRNApathway [26,51]. These studies led to the characterization ofthe mechanisms of gene silencing involving Dcr-2, r2d2 andAGO2 in response to dsRNA classically defined as exogenous.

Endogenous dsRNA generated from long structured loci,natural sense-antisense transcript (NAT) pairs and transposable

Fig. 1. Endogenous miRNA and siRNA pathways in Drosophila melanogaster. (A) T

ssRNA precursors known as pri-miRNAs. The drosha/pasha complex recognizes a h

the nucleus to generate the pre-miRNA. Pre-miRNAs are exported to the cytoplasm

generate the mature miRNA duplex. Mature duplex miRNAs are bound by AGO

associated with the guide strand to form miRISC. This complex will search for pa

translation by several mechanisms. (B) The siRNA pathway: dsRNAs originating

transcripts (NAT) and transposable elements are exported to the cytoplasm where t

Duplex siRNAs are recognized by the Dcr-2/r2d2 complex and loaded onto AGO2

associated with the guide strand. Within AGO2, the guide strand is 20O-methylatedsequences within mRNAs by siRISC will result in degradation of the target by AG


http://dx.doi.org/10.1016/j.micinf.2014.09.0013d Infection xx (2014) 1e9elements were later shown to activate the siRNA pathway.Interestingly, this work also showed that processing ofendogenous siRNAs by Dcr-2 required the isoform PD of loqs(loqs-PD) [43,44]. As mentioned before, the loqs gene encodesisoforms with distinct functions including the role of loqs-PBin the biogenesis of miRNAs but its role in the siRNA pathwayhad not been noted before [18,19]. Some of these initialstudies also suggested that r2d2 was dispensable for theloading of endogenous siRNAs onto AGO2 although thesestudies were mostly based on cultured S2 cells [43,52,53].Later, it was observed that in Drosophila embryos and adultflies, the siRNA pathway activated not only by endogenous butalso exogenous dsRNA required loqs-PD for substrate pro-cessing by Dcr-2 and, subsequently, also in both cases, needed

he miRNA pathway: miRNA genes are generally transcribed by RNA pol II as

airpin secondary structure within the pri-miRNA transcript that is processed in

by exportin-5 where they are processed by Dcr-1 associated with loqs-PB to

1 that releases the passenger strand (also known as miRNA*) and remains

rtially complementary sites in the 30 UTR of mRNAs leading to inhibition offrom nuclear transcription of structured loci, natural sense-antisense pairs of

hey are recognized and processed into siRNAs by the Dcr-2/loqs-PD complex.

. The passenger strand of the siRNA duplex is cleaved by AGO2 that remains

by Hen1 to generate the mature siRISC. Recognition of fully complementary

O2-mediated cleavage.


s an4 Z.G. Soares et al. / Microber2d2 for siRNA loading onto AGO2 [28e30,54]. These resultshave suggested a common unified siRNA pathway where loqs-PD and r2d2 act sequentially in response to either endogenousor exogenous sources of dsRNA (Fig 2) [29,30,54,55].

2.3. The siRNA pathway activated by viral infection inDrosophila

The antiviral role of RNAi was shown very early in plantsand, later, in animals including insects, worms and mammals[40,56e59]. The Drosophila siRNA pathway is activated byviral infection in cultured S2 cells and adult animals as indi-cated by the production of virus-derived siRNAs[35,40,60e62]. AGO2, r2d2 and Dcr-2 deficient flies are moresusceptible to a range of different viruses reinforcing the ideathat the siRNA pathway mediates a powerful antiviral defense[35,61,63e65]. In addition, viral suppressors of RNAi (VSRs)are commonly found in insect viruses and are capable ofblocking different steps of the siRNA pathway to favor virusreplication [66]. A good example is the B2 protein encoded byFlock House virus (FHV), which is able to block both thebiogenesis as well as loading of siRNAs onto AGO2 [40]. TheDrosophila siRNA pathway is mostly a cell-autonomous

Fig. 2. Discrimination of different sources of dsRNA by the Drosophila siRNA p

transcripts, exogenous synthetic molecules or viral replication. Endogenous and exo

2 complex while processing of viral dsRNA by Dcr-2 is carried out largely independ

the Dcr-2/r2d2 complex and loaded onto AGO2 to form siRISC. The core siRISC

scan incoming cellular or viral mRNAs containing fully complementary sequences


http://dx.doi.org/10.1016/j.micinf.2014.09.001d Infection xx (2014) 1e9antiviral mechanism but viral dsRNA released from infectedcells can mediate some systemic silencing [67].

Exogenous dsRNA is often utilized as a surrogate for viralinfection because virus replication leads to the accumulationof dsRNA [68]. Thus it would be reasonable to assume that asimilar siRNA pathway is activated in response to exogenousdsRNA and viruses. Indeed, Dcr-2, AGO2 and r2d2 are allrequired for the response against viruses and exogenousdsRNA [35]. In contrast, loqs-PD that is required for thebiogenesis of exogenous siRNAs, is completely dispensablefor the biogenesis of virus-derived siRNAs and inhibition ofRNA viruses in vivo [35]. Genes involved in hostepathogeninteractions tend to be under strong pressure due to naturalselection. AGO2, r2d2 and Dcr-2 are among the 3% fastestevolving genes in the Drosophila genome suggesting thecomponents of the antiviral siRNA pathway are under positiveselection [69]. In contrast, loqs is not among the fastestevolving genes which reinforces the idea that loqs is notrequired for the antiviral defense. Thus, a separate loqs-independent siRNA pathway seems to be dedicated to theantiviral response while Dcr-2, AGO2 and r2d2 are requiredfor silencing triggered by both exogenous dsRNA and viruses(Fig. 2).

athway. dsRNA can originate from different sources that include endogenous

genous dsRNAs are recognized and processed into siRNA by the loqs-PD/Dcr-

ent of loqs-PD. Exogenous, endogenous or virus-derived siRNAs are bound by

component AGO2 is normally associated with cellular ribosomes where it can

that will be directly cleaved by siRISC to prevent translation of target mRNA.


nematode worm C. elegans [73]. Worms have a large varietys forone

Dicer gene, DCR-1, that is responsible for the biogenesis of

surrogate for virus infection [1]. There are several dsRNA-fenseligo-

s anHow AGO2, r2d2 and Dcr-2 participate in two separatesiRNA pathways remains unclear. The different pathwaysseem to differ mostly in the requirement for loqs-PD, adsRBPs, required to help Dcr-2 in the processing step. Inter-estingly, small dsRBPs are not necessary for dsRNA pro-cessing mediated by Dicers in other organisms such as yeast[55,70]. Recombinant Drosophila Dcr-2 is able to carry outdsRNA processing in vitro without any accessory proteinsalthough loqs-PD seems to increase the affinity of Dcr-2 forthe substrate [55]. It remains unclear whether Dcr-2 requires adsRBP or any other protein partner for processing viral dsRNAin vivo. Dicer accessory proteins could help diversify types ofdsRNA activators recognized by the siRNA pathways.

These results indicate that viruses cannot be viewed solelyas exogenous sources of dsRNA but it is unclear how thesiRNA pathway can discriminate viral infection. One couldspeculate that the delivery of RNA mediated by infectiousvirus particles could trigger a specific recognition mechanism.However, at least in the case of a positive strand RNA viruswhose genome is infectious, activation of the antiviral siRNApathway does not require the intact virus particle [35].Therefore discrimination is likely an intrinsic feature of theviral RNA such as secondary structures or modifications butalso its subcellular localization [1]. Indeed, viral nucleic acidsare often concentrated in specific compartments withininfected cells where viral genome replication and transcriptioncan occur in relative isolation from cellular proteins [71]. Dcr-2 could be recruited to dsRNA present at sites of viral repli-cation within infected cells independently of loqs-PD.Accordingly, Dcr-2 seems to recognize viral dsRNA gener-ated during replication but not incoming viral genomic RNA[35,60,62]. In contrast, dsRNA arising from the nucleus orfrom exogenous origins will certainly be more accessible tocytoplasmic proteins than viral RNA. Thus, we propose thatthe siRNA pathway could be defined in terms of access to thedsRNA substrate. A default siRNA pathway would recognizereadily available dsRNA as opposed to less accessible viralRNA, which would presumably require a specialized antiviralpathway. From this model, crosstalk can occur between thetwo separate siRNA pathways depending on the accessibilityof the dsRNA.

3. A parallel between the Drosophila siRNA and otherantiviral pathways

Discrimination between viral and exogenous dsRNA seemsto be a common feature of different antiviral systems [1]. Inaddition to RNAi in animals and plants, another viral RNArecognition mechanism is mediated by RNA helicases such asRetinoic-acid inducible gene I (RIG-I) in vertebrates [1,72].The Drosophila siRNA pathway is similar in many ways totwo the antiviral defense mediated by RNAi in C. elegans andRNA helicases in mammals. Different nucleic acid sensingmechanisms likely had to overcome comparable intricacies todetect viral infection. Comparative analysis of these antiviral

Z.G. Soares et al. / Microbepathways can help define conserved features of viral RNArecognition.


http://dx.doi.org/10.1016/j.micinf.2014.09.001activated proteins that mediate mammalian antiviral deincluding the dsRNA-activated protein kinase (PKR), odifferent classes of small ncRNAs, such as miRNAs andsiRNAs [74]. When the siRNA pathway is activated in C.elegans, primary siRNAs are generated from direct processingof the original dsRNA trigger by DCR-1 [73]. Primary siRNAsassociate with the worm Argonaute protein RDE-1 to form thecomplex that recognizes target RNAs and directs the synthesisof secondary siRNAs by RNA-dependent RNA polymerases(RdRPs) [75]. These secondary siRNAs then associate withdifferent worm Argonautes that direct silencing of the target[74]. This two-step mechanism allows for rapid, specific andefficient amplification of the initial signal given by the dsRNAtrigger. Different sources of dsRNA require distinct processingcomplexes that seem to compete for the same pool of DCR-1[73]. Thus, accessory proteins present in the processingcomplexes play a crucial role in the recognition, loading andprocessing of different sources of dsRNA by DCR-1.

Recognition and processing of viral RNA by the C. elegansRNAi pathway depends on a complex containing Dicer relatedhelicase-1 (DRH-1) in addition to DCR-1 [76,77]. The DCR-1/DRH-1 complex seems to exist even in the absence ofinfection, likely allowing cells to respond more rapidly toviruses [76]. DRH-1 contains a conserved C-terminus motifthat is also present in mammalian RIG-I where it mediates therecognition of 50 triphosphorylated RNA ends [76,78]. Thus,DRH-1 might mediate recruitment of DCR-1 to viral dsRNAby interacting with 50 triphosphorylated ends of viral genomes[76]. In contrast to its requirement for the recognition of viralRNA, DRH-1 is largely dispensable for processing of exoge-nous dsRNA [76]. Notably, the DRH-1 dependent RNAipathway is also triggered when viral RNA is expressed from agenome-integrated replicon suggesting recognition of intrinsiccharacteristics of the RNA itself [77]. These results are inaccordance with the model where recognition of viral orexogenous dsRNA are carried out by separate DCR-1 com-plexes defined by their dependence on DRH-1 [73,76]. This issimilar to the Drosophila siRNA pathway where separate Dcr-2 complexes recognize and process viral or exogenous dsRNA.

3.2. dsRNA recognition by mammalian antiviralpathways

In mammals, the synthetic dsRNA analog, polyinosinic-polycytidylic acid (poly(I:C)), has long been utilized as aof functionally distinct RNAi pathways. There are 27 geneArgonaute proteins in the C. elegans genome but only3.1. dsRNA recognition by the C. elegans antiviral RNAipathway

The separation between siRNA pathways that recognizedifferent sources of dsRNA came initially from work in the

5d Infection xx (2014) 1e9denylate synthetases (OAS), toll-like receptor-3 (TLR-3) andRNA helicases such as RIG-I [79]. These proteins induce a


s ancomplex antiviral response in mammalian cells that includetranslation inhibition and apoptosis of infected cells in addi-tion to a transcriptional response of type I Interferon (IFN)genes.

RIG-I and melanoma differentiation-associated gene 5(MDA-5), belong to a family of vertebrate proteins referred toas RIG-like helicases (RLHs) [72]. Recognition of viruses byRIG-I and MDA-5 triggers signaling pathways that result inthe production of type I IFNs. These two RNA helicases arevery similar in sequence and structure but perform non-redundant roles in the recognition of mammalian viruses[72]. Influenza virus, Vesicular Stomatitis virus, and JapaneseEncephalitis virus (JEV) and several paramyxoviruses areexclusively recognized by RIG-I while MDA-5 is required forthe recognition of picornaviruses such as Encephalomyocar-ditis virus, Mengo virus, and Theiler's virus [80]. Other vi-ruses, such as Dengue virus, seem to require both RIG-I andMDA-5 [81]. Despite this specificity in the recognition ofviruses, both RIG-I and MDA-5 are similarly capable ofrecognizing exogenous dsRNA [82].

In addition to these classical antiviral pathways, themammalian siRNA pathway is also capable of recognizingdifferent sources of dsRNA. Dicer-dependent siRNAs aregenerated from endogenous sources such as transposable el-ements and sense-antisense pairs of transcripts as well asexogenous or viral sources of dsRNA [57,58,86,87]. Notably,viral dsRNA sensing by the siRNA pathway seems to beinhibited in differentiated cells where classical antiviralpathways, such as the RLHs, are functional [88]. Processingof endogenous dsRNA by the siRNA pathway seems to pre-vent erroneous activation of RLHs, which could be detri-mental to the cell [89]. In addition, RIG-I is inhibited by thepresence of 30 overhangs at the end of dsRNA which arenaturally found in Dicer products [84]. This suggests that, indifferentiated mammalian cells, there is functional speciali-zation between the siRNA pathway and RLHs to recognizeendogenous and viral dsRNA, respectively. This competitionseems restricted to differentiated cells since the siRNApathway in stem cells recognizes all types of dsRNA[58,86,87]. Expression of accessory proteins required for therecognition of viral dsRNA by the siRNA pathway couldexplain the difference between stem cells and differentiatedcells.

The recognition of viruses by RLHs and the siRNA pathwayin mammals also illustrate complexities of viral RNA recog-nition. Therefore, the paradigm of exogenous dsRNA as asurrogate for viral infection is a dangerous oversimplification.

4. Concluding remarks

The analysis of the Drosophila siRNA pathway suggeststhat viral RNA sensing and recognition of exogenous dsRNAare mediated by distinct mechanisms. To help understand thediscrimination between exogenous and viral dsRNA, it isworth considering the separation between miRNA and siRNA

6 Z.G. Soares et al. / Microbepathways. The biogenesis and function of siRNAs sharesimportant similar features with miRNAs (Fig. 1) [3]. Both


http://dx.doi.org/10.1016/j.micinf.2014.09.001siRNAs and miRNAs are generated by Dicer cleavage ofdsRNA precursors and associate with somewhat similarArgonaute proteins. The miRNA pathway is absolutelyrequired for cell differentiation and development inDrosophila and other animals [13]. Due to the functional andmechanistic similarities between miRNAs and siRNAs, inter-ference between the two pathways would have a great impacton animal cells. Notably, miRNAs likely appeared later inevolution since several unicellular eukaryotes do not havemiRNAs [5]. In contrast, the siRNA pathway as an antiviralmechanism appeared early in the evolution of eukaryotes.Thus, once miRNAs appeared and acquired important devel-opmental functions, sharing of components and mechanisticsimilarities with the siRNA pathway became problematic.Therefore, it was essential to find the right balance betweenmiRNA and antiviral siRNA pathways. Insects developedseparate miRNA and siRNA pathways with protein compo-nents that have little or no functional overlap [3]. Notably, loqsis the only gene still shared between miRNA and siRNApathways although it is clearly not required for the antiviralsiRNA pathway [35]. This complete separation likely allowedindependent evolution for the antiviral siRNA pathwaywithout any restrictions imposed by the miRNA pathway. Ahighly specialized antiviral siRNA pathway could acquireunique features that make it more efficient against viruses. Insupport of this hypothesis, Dcr-2 that is responsible for therecognition of viral dsRNA seems to have acquired the abilityto also trigger the transcription of antiviral genes independentof its function in the biogenesis of siRNAs [90]. Hence,activation of the antiviral siRNA pathway likely has broadereffects and is more tightly regulated than recognition ofexogenous dsRNA.

The existence of separate siRNA pathways implies thatviruses can be discriminated from other sources of dsRNA.Indeed, the specific discrimination of viral dsRNA seems todepend on a variety of intrinsic characteristics. For example,C. elegans RDE-4 and mammalian MDA-5 recognize dsRNAby multimerizing along the molecule in a length dependentmanner [83,91]. Similarly, in Drosophila, the ability to carryout efficient processing of long dsRNA molecules by Dcr-2seems to be essential for its antiviral function [35]. Thediscrimination of viral dsRNA by worm DRH-1 andmammalian RIG-I, but likely not Drosophila Dcr-2, dependson the presence of a 50 triphosphate [76,78,85]. Notably, flyDcr-2, worm DRH-1 and mammalian RIG-I, all share acommon ancestor suggesting the capacity to recognize 50

triphosphate was present in the ancestor of these proteins butwas likely lost in fly Dcr-2 [90,92]. In addition, other char-acteristics are likely to contribute to the discrimination of viralRNA. For example, exogenous and viral dsRNA have verydistinct subcellular localizations [68].

In summary, we propose a model where a default siRNApathway is activated by any type of dsRNA while theengagement of the antiviral pathway would require additionalintrinsic characteristics of the viral RNA. This mechanism

d Infection xx (2014) 1e9could limit the activation of the antiviral pathway to avoid sideeffects and waste of energy.


7s and Infection xx (2014) 1e9Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank the International Society for Infectious Diseases,CNPq, CAPES and FAPEMIG for funding. We apologize forany work that might not have been cited in this review due tolimitation on the number of references.

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9Z.G. Soares et al. / Microbes and Infection xx (2014) 1e9Please cite this article in press as: Soares ZG, et al., Viral RNA recognition by the

http://dx.doi.org/10.1016/j.micinf.2014.09.001Drosophila small interfering RNA pathway, Microbes and Infection (2014),

Viral RNA recognition by the Drosophila small interfering RNA pathway1 Introduction2 RNA interference pathways2.1 Drosophila RNA interference pathways2.2 Endogenous and exogenous siRNA pathways in Drosophila2.3 The siRNA pathway activated by viral infection in Drosophila

3 A parallel between the Drosophila siRNA and other antiviral pathways3.1 dsRNA recognition by the C. elegans antiviral RNAi pathway3.2 dsRNA recognition by mammalian antiviral pathways

4 Concluding remarksConflict of interestAcknowledgmentsReferences

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