-
ph
Gde
sida
acce
viespo
ts re
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
Please cite this article in press as: Soares ZG, et al., Viral
RNA recognition by the
http://dx.doi.org/10.1016/j.micinf.2014.09.0013d 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.
Drosophila small interfering RNA pathway, Microbes and Infection
(2014),
-
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
Please cite this article in press as: Soares ZG, et al., Viral
RNA recognition by the
http://dx.doi.org/10.1016/j.micinf.2014.09.001d 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.
Drosophila small interfering RNA pathway, Microbes and Infection
(2014),
-
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.
Please cite this article in press as: Soares ZG, et al., Viral
RNA recognition by the
http://dx.doi.org/10.1016/j.micinf.2014.09.001activated 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
Drosophila small interfering RNA pathway, Microbes and Infection
(2014),
-
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
Please cite this article in press as: Soares ZG, et al., Viral
RNA recognition by the
http://dx.doi.org/10.1016/j.micinf.2014.09.001siRNAs 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.
Drosophila small interfering RNA pathway, Microbes and Infection
(2014),
-
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.
References
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during viral
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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