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MODEL

Microbes and Infection xx (2014) 1e9

www.elsevier.com/locate/micinf

Viral RNA recognition by the Drosophila small interfering RNA pathway

Zamira Guerra Soares, Andr
e Nicolau Aquime Gonalves, Karla Pollyanna Vieira de Oliveira,
Jo~ao Trindade Marques*
Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Received 28 June 2014; accepted 1 September 2014

Abstract
Viral RNA is a common activator of antiviral responses. In this review, we dissect the mechanism of viral RNA recognition by the small
interfering RNA pathway in Drosophila melanogaster. This antiviral response in fruit flies can help understand general principles of nucleic acid
recognition.
2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: RNA interference; Small interfering RNA; dsRNA; Viral infection; Drosophila melanogaster

1. Introduction
Nucleic acid sensing is a common strategy that prokaryotic
and eukaryotic cells utilize to recognize invading viruses [1].
A variety of DNA and RNA recognition proteins have been
linked to activation of both cell-autonomous and systemic
immunity. These nucleic acid sensors must be able to
discriminate molecular patterns found in infected cells that are
mostly absent in healthy conditions. Nucleic acids sensing
systems must cope with the ubiquitous presence of RNA and
DNA. Indeed, erroneous sensing of nucleic acids is often
associated with autoimmunity [2]. The RNA interference
(RNAi) pathway is a highly efficient RNA recognition system
activated by diverse sources of nucleic acids [3]. In this review, we will focus on the recognition of viral RNA in the fruit
fly Drosophila melanogaster, which is mediated by a highly
specialized RNAi mechanism known as the small interfering
RNA (siRNA) pathway. The major trigger for the activation of

* Corresponding author. Universidade Federal de Minas Gerais, Departamento de Bioqumica e Imunologia-ICB, Av. Ant^onio Carlos, 6627, Pampulha,
Belo Horizonte, MG CEP 31270-901, Brazil. Tel.: 55 31 3409 2623;
fax: 55 31 3409 2614.
E-mail address: jtm@ufmg.br (J.T. Marques).

this pathway is double stranded RNA (dsRNA), which is

generated as a byproduct of viral replication but is also found
in uninfected cells. Thus, the Drosophila siRNA pathway
needs to deal with viral and cellular sources of dsRNA. Understanding this mechanism can help understand general aspects of nucleic acid sensing.
2. RNA interference pathways
RNAi was first described as dsRNA-mediated gene
silencing in the nematode worm Caenorhabiditis elegans [4].
Currently, the term RNAi is used to broadly describe pathways that utilize small non-coding RNAs (ncRNAs) in association with an Argonaute protein to regulate gene
expression and other biological processes ranging from DNA
replication to translation [5e7]. The active complex formed
by the small ncRNA associated with the Argonaute protein is
often referred to as the RNA-induced silencing complex
(RISC) [6]. RNAi pathways and Argonaute proteins are
present in most eukaryotes although their diversity and
function can vary significantly [5,6]. In animals, such as the
fruit fly, D. melanogaster, there are at least three major
classes of small ncRNAs, known as microRNAs (miRNAs),
siRNAs and piwi-interacting RNAs (piRNAs) [8]. These
distinct classes of small RNAs define separate RNAi

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pathways that differ in function, biogenesis and mechanism

of action.
2.1. Drosophila RNA interference pathways
The piRNA pathway is mostly active in the animal germline although it is can also be present in somatic tissues [9].
Loss of piRNAs causes genomic instability and leads to sterility in animals. Drosophila piRNAs are 24e27 nt long small
RNAs that originate from single-stranded RNA (ssRNA) precursors arising mostly from transposable elements and other
specific clusters in the genome [10]. The mechanism of piRNA
biogenesis from ssRNA precursors is still not completely understood. Once primary piRNAs are generated they can also
enter an amplification loop, called the ping-pong mechanism,
to drive the production of secondary piRNAs [10,11]. Mature
piRNAs associate with an animal specific subfamily of animal
Argonaute proteins represented by three different members in
Drosophila: piwi, Aubergine (Aub) and Argonaute-3 (AGO3)
[6]. More detailed reviews about the piRNA pathway can be
found elsewhere [9,12].
The miRNA pathway is absolutely necessary for animal
development and cell differentiation [13]. miRNAs are noncoding genes transcribed by RNA polymerase II (RNA pol
II) as ssRNA transcripts known as primary miRNAs (primiRNA) that fold onto a hairpin structure with long arms
[14,15]. In Drosophila, pri-miRNAs are processed by the nuclear RNase III enzyme Drosha associated with a dsRNA
binding protein (dsRBP) called Pasha [16]. This first nuclear
processing event generates precursor miRNAs (pre-miRNA)
that are approximately 65-nt long ssRNA hairpins, which are
exported to the cytoplasm by exportin-5 (Exp5) [17]. In the
cytoplasm, pre-miRNAs are processed by another RNase III
called Dicer-1 (Dcr-1) associated with the isoform PB of
loquacious (loqs-PB), a small dsRBP [18,19]. This second
processing event generates 22-nt long small duplex RNAs
known as mature miRNAs that associate with Argonaute-1
(AGO1) [20]. The miRNA duplex is then dissociated to
release the passenger strand from AGO1 that remains associated with the guide strand to generate miRNA programmed
RISC (miRISC) [21,22]. miRISC will search for partially
complementary binding sites in the 30 untranslated region
(UTR) of messenger RNAs (mRNAs) where binding leads to
inhibition of translation by different mechanisms [23,24].
There are several hundreds of miRNAs in Drosophila that
potentially regulate the expression of half of all coding genes
in the fly genome [3,25]. An overview of the Drosophila
miRNA pathway is shown in Fig. 1A.
In Drosophila, long dsRNA (>30 bp) is recognized and
processed into siRNAs by Dicer-2 (Dcr-2), another cytoplasmic RNase III enzyme distinct from Dcr-1 [26,27]. siRNAs are 21-nt long duplex RNAs that are bound by a
heterodimer between Dcr-2 and r2d2, a dsRBP partner [28].
The Dcr-2/r2d2 complex transfers the duplex siRNA to
Argonaute-2 (AGO2) to form siRNA programmed RISC
(siRISC) [29,30]. Mature siRISC that carries only the guide
strand of the siRNA duplex is formed after AGO2 cleaves the

passenger strand that is then released and further degraded

[31,32]. The guide strand is then 20 -O-methylated within
AGO2 by the Drosophila 20 -O-methytranferase DmHen1,
which stabilizes the silencing complex [33]. siRISC searches
for fully complementary sequences within mRNAs which results in direct target cleavage by AGO2 [34]. Silencing of
mRNAs by siRISC is highly efficient because AGO2 is a
multiple turnover enzyme that catalyzes cleavage of numerous
targets [34]. Polyadenylated RNAs, presumably mRNAs, seem
to be preferred targets of silencing by the siRNA pathway [35].
Interestingly, AGO2 is found in association with cellular ribosomes, which suggests it can monitor incoming mRNAs
before they can be translated [36,37]. An overview of the
Drosophila siRNA pathway is shown in Fig. 1B.
A close comparison of the three small RNA pathways in
Drosophila suggests there are important similarities and distinctions. The biogenesis of both miRNAs and siRNAs involve
processing of dsRNA precursors by RNase III enzymes while
piRNAs arise from ssRNA precursors [3,12]. Argonaute proteins associated with miRNAs and siRNAs, AGO1 and AGO2,
respectively, belong to the AGO clade and are significantly
different from piwi, Aub and AGO3 that belong to the PIWI
clade [6]. Both miRNAs and piRNAs originate mostly from
RNA precursors transcribed from the Drosophila genome. In
contrast, the siRNA pathway can be activated by long dsRNA
precursors that are artificially introduced into cells, produced
as a byproduct of viral replication during infection or originate
from the Drosophila genome [3]. The ability to recognize
different sources of dsRNA is an important characteristic of
the Drosophila siRNA pathway as represented in Fig. 2.
2.2. Endogenous and exogenous siRNA pathways in
Drosophila
The dsRNA trigger for the siRNA pathway can originate
from diverse sources, either endogenous or exogenous. Several
distinct sources of dsRNA are commonly labeled as exogenous
including transgenes, in vitro synthesized RNAs and viral
infection [38e42]. In contrast, the term endogenous usually
refers to RNA molecules synthesized by nuclear transcription
using the host genome as template. Endogenous dsRNA
commonly originates from structured loci, sense-antisense
pairs and bidirectional transcription of transposable elements
[43,44]. Although these diverse sources of dsRNA commonly
activate the Drosophila siRNA pathway, there are subtle differences in the mechanisms of siRNA biogenesis and action
that will be discussed in this review.
Historically, the mechanism of gene silencing by the siRNA
pathway was first characterized using Drosophila embryos or
cell
culture
and
in
vitro
synthesized
dsRNA
[27,29,41,42,45e47]. In these cases, dsRNA was often injected into Drosophila embryos or delivered to cultured cells by
soaking or transfection to induce gene silencing [45,48,49]. In
addition, mechanisms of gene silencing triggered by dsRNA
were also characterized by biochemistry utilizing protein extracts from Drosophila embryos, adult heads or cultured S2
cells and in vitro synthesized dsRNA [27,29,42,50]. Thus, the

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Fig. 1. Endogenous miRNA and siRNA pathways in Drosophila melanogaster. (A) The miRNA pathway: miRNA genes are generally transcribed by RNA pol II as
ssRNA precursors known as pri-miRNAs. The drosha/pasha complex recognizes a hairpin secondary structure within the pri-miRNA transcript that is processed in
the nucleus to generate the pre-miRNA. Pre-miRNAs are exported to the cytoplasm by exportin-5 where they are processed by Dcr-1 associated with loqs-PB to
generate the mature miRNA duplex. Mature duplex miRNAs are bound by AGO1 that releases the passenger strand (also known as miRNA*) and remains
associated with the guide strand to form miRISC. This complex will search for partially complementary sites in the 30 UTR of mRNAs leading to inhibition of
translation by several mechanisms. (B) The siRNA pathway: dsRNAs originating from nuclear transcription of structured loci, natural sense-antisense pairs of
transcripts (NAT) and transposable elements are exported to the cytoplasm where they are recognized and processed into siRNAs by the Dcr-2/loqs-PD complex.
Duplex siRNAs are recognized by the Dcr-2/r2d2 complex and loaded onto AGO2. The passenger strand of the siRNA duplex is cleaved by AGO2 that remains
associated with the guide strand. Within AGO2, the guide strand is 20 O-methylated by Hen1 to generate the mature siRISC. Recognition of fully complementary
sequences within mRNAs by siRISC will result in degradation of the target by AGO2-mediated cleavage.

dsRNA source for these initial experiments could all be classically considered exogenous. Genome-integrated transgenes
encoding inverted-repeat transcripts were also used to induce
efficient, heritable and durable gene silencing in Drosophila
adults [39]. In these cases, the dsRNA arises from nuclear
transcription similar to endogenous transcripts although
transgenes are technically exogenous. Screens in Drosophila
adults with transgenes and cell culture using in vitro synthesized dsRNA helped identify components of the siRNA
pathway [26,51]. These studies led to the characterization of
the mechanisms of gene silencing involving Dcr-2, r2d2 and
AGO2 in response to dsRNA classically defined as exogenous.
Endogenous dsRNA generated from long structured loci,
natural sense-antisense transcript (NAT) pairs and transposable

elements were later shown to activate the siRNA pathway.

Interestingly, this work also showed that processing of
endogenous siRNAs by Dcr-2 required the isoform PD of loqs
(loqs-PD) [43,44]. As mentioned before, the loqs gene encodes
isoforms with distinct functions including the role of loqs-PB
in the biogenesis of miRNAs but its role in the siRNA pathway
had not been noted before [18,19]. Some of these initial
studies also suggested that r2d2 was dispensable for the
loading of endogenous siRNAs onto AGO2 although these
studies were mostly based on cultured S2 cells [43,52,53].
Later, it was observed that in Drosophila embryos and adult
flies, the siRNA pathway activated not only by endogenous but
also exogenous dsRNA required loqs-PD for substrate processing by Dcr-2 and, subsequently, also in both cases, needed

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Fig. 2. Discrimination of different sources of dsRNA by the Drosophila siRNA pathway. dsRNA can originate from different sources that include endogenous
transcripts, exogenous synthetic molecules or viral replication. Endogenous and exogenous dsRNAs are recognized and processed into siRNA by the loqs-PD/Dcr2 complex while processing of viral dsRNA by Dcr-2 is carried out largely independent of loqs-PD. Exogenous, endogenous or virus-derived siRNAs are bound by
the Dcr-2/r2d2 complex and loaded onto AGO2 to form siRISC. The core siRISC component AGO2 is normally associated with cellular ribosomes where it can
scan incoming cellular or viral mRNAs containing fully complementary sequences that will be directly cleaved by siRISC to prevent translation of target mRNA.

r2d2 for siRNA loading onto AGO2 [28e30,54]. These results

have suggested a common unified siRNA pathway where loqsPD and r2d2 act sequentially in response to either endogenous
or exogenous sources of dsRNA (Fig 2) [29,30,54,55].
2.3. The siRNA pathway activated by viral infection in
Drosophila
The antiviral role of RNAi was shown very early in plants
and, later, in animals including insects, worms and mammals
[40,56e59]. The Drosophila siRNA pathway is activated by
viral infection in cultured S2 cells and adult animals as indicated by the production of virus-derived siRNAs
[35,40,60e62]. AGO2, r2d2 and Dcr-2 deficient flies are more
susceptible to a range of different viruses reinforcing the idea
that 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 of
blocking different steps of the siRNA pathway to favor virus
replication [66]. A good example is the B2 protein encoded by
Flock House virus (FHV), which is able to block both the
biogenesis as well as loading of siRNAs onto AGO2 [40]. The
Drosophila siRNA pathway is mostly a cell-autonomous

antiviral mechanism but viral dsRNA released from infected

cells can mediate some systemic silencing [67].
Exogenous dsRNA is often utilized as a surrogate for viral
infection because virus replication leads to the accumulation
of dsRNA [68]. Thus it would be reasonable to assume that a
similar siRNA pathway is activated in response to exogenous
dsRNA and viruses. Indeed, Dcr-2, AGO2 and r2d2 are all
required for the response against viruses and exogenous
dsRNA [35]. In contrast, loqs-PD that is required for the
biogenesis of exogenous siRNAs, is completely dispensable
for the biogenesis of virus-derived siRNAs and inhibition of
RNA viruses in vivo [35]. Genes involved in hostepathogen
interactions tend to be under strong pressure due to natural
selection. AGO2, r2d2 and Dcr-2 are among the 3% fastest
evolving genes in the Drosophila genome suggesting the
components of the antiviral siRNA pathway are under positive
selection [69]. In contrast, loqs is not among the fastest
evolving genes which reinforces the idea that loqs is not
required for the antiviral defense. Thus, a separate loqsindependent siRNA pathway seems to be dedicated to the
antiviral response while Dcr-2, AGO2 and r2d2 are required
for silencing triggered by both exogenous dsRNA and viruses
(Fig. 2).

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How AGO2, r2d2 and Dcr-2 participate in two separate

siRNA pathways remains unclear. The different pathways
seem to differ mostly in the requirement for loqs-PD, a
dsRBPs, required to help Dcr-2 in the processing step. Interestingly, small dsRBPs are not necessary for dsRNA processing mediated by Dicers in other organisms such as yeast
[55,70]. Recombinant Drosophila Dcr-2 is able to carry out
dsRNA processing in vitro without any accessory proteins
although loqs-PD seems to increase the affinity of Dcr-2 for
the substrate [55]. It remains unclear whether Dcr-2 requires a
dsRBP or any other protein partner for processing viral dsRNA
in vivo. Dicer accessory proteins could help diversify types of
dsRNA activators recognized by the siRNA pathways.
These results indicate that viruses cannot be viewed solely
as exogenous sources of dsRNA but it is unclear how the
siRNA pathway can discriminate viral infection. One could
speculate that the delivery of RNA mediated by infectious
virus particles could trigger a specific recognition mechanism.
However, at least in the case of a positive strand RNA virus
whose genome is infectious, activation of the antiviral siRNA
pathway does not require the intact virus particle [35].
Therefore discrimination is likely an intrinsic feature of the
viral RNA such as secondary structures or modifications but
also its subcellular localization [1]. Indeed, viral nucleic acids
are often concentrated in specific compartments within
infected cells where viral genome replication and transcription
can occur in relative isolation from cellular proteins [71]. Dcr2 could be recruited to dsRNA present at sites of viral replication within infected cells independently of loqs-PD.
Accordingly, Dcr-2 seems to recognize viral dsRNA generated during replication but not incoming viral genomic RNA
[35,60,62]. In contrast, dsRNA arising from the nucleus or
from exogenous origins will certainly be more accessible to
cytoplasmic proteins than viral RNA. Thus, we propose that
the siRNA pathway could be defined in terms of access to the
dsRNA substrate. A default siRNA pathway would recognize
readily available dsRNA as opposed to less accessible viral
RNA, which would presumably require a specialized antiviral
pathway. From this model, crosstalk can occur between the
two separate siRNA pathways depending on the accessibility
of the dsRNA.
3. A parallel between the Drosophila siRNA and other
antiviral pathways
Discrimination between viral and exogenous dsRNA seems
to be a common feature of different antiviral systems [1]. In
addition to RNAi in animals and plants, another viral RNA
recognition mechanism is mediated by RNA helicases such as
Retinoic-acid inducible gene I (RIG-I) in vertebrates [1,72].
The Drosophila siRNA pathway is similar in many ways to
two the antiviral defense mediated by RNAi in C. elegans and
RNA helicases in mammals. Different nucleic acid sensing
mechanisms likely had to overcome comparable intricacies to
detect viral infection. Comparative analysis of these antiviral
pathways can help define conserved features of viral RNA
recognition.

3.1. dsRNA recognition by the C. elegans antiviral RNAi

pathway
The separation between siRNA pathways that recognize
different sources of dsRNA came initially from work in the
nematode worm C. elegans [73]. Worms have a large variety
of functionally distinct RNAi pathways. There are 27 genes for
Argonaute proteins in the C. elegans genome but only one
Dicer gene, DCR-1, that is responsible for the biogenesis of
different classes of small ncRNAs, such as miRNAs and
siRNAs [74]. When the siRNA pathway is activated in C.
elegans, primary siRNAs are generated from direct processing
of the original dsRNA trigger by DCR-1 [73]. Primary siRNAs
associate with the worm Argonaute protein RDE-1 to form the
complex that recognizes target RNAs and directs the synthesis
of secondary siRNAs by RNA-dependent RNA polymerases
(RdRPs) [75]. These secondary siRNAs then associate with
different worm Argonautes that direct silencing of the target
[74]. This two-step mechanism allows for rapid, specific and
efficient amplification of the initial signal given by the dsRNA
trigger. Different sources of dsRNA require distinct processing
complexes that seem to compete for the same pool of DCR-1
[73]. Thus, accessory proteins present in the processing
complexes play a crucial role in the recognition, loading and
processing of different sources of dsRNA by DCR-1.
Recognition and processing of viral RNA by the C. elegans
RNAi pathway depends on a complex containing Dicer related
helicase-1 (DRH-1) in addition to DCR-1 [76,77]. The DCR1/DRH-1 complex seems to exist even in the absence of
infection, likely allowing cells to respond more rapidly to
viruses [76]. DRH-1 contains a conserved C-terminus motif
that is also present in mammalian RIG-I where it mediates the
recognition of 50 triphosphorylated RNA ends [76,78]. Thus,
DRH-1 might mediate recruitment of DCR-1 to viral dsRNA
by interacting with 50 triphosphorylated ends of viral genomes
[76]. In contrast to its requirement for the recognition of viral
RNA, DRH-1 is largely dispensable for processing of exogenous dsRNA [76]. Notably, the DRH-1 dependent RNAi
pathway is also triggered when viral RNA is expressed from a
genome-integrated replicon suggesting recognition of intrinsic
characteristics of the RNA itself [77]. These results are in
accordance with the model where recognition of viral or
exogenous dsRNA are carried out by separate DCR-1 complexes defined by their dependence on DRH-1 [73,76]. This is
similar to the Drosophila siRNA pathway where separate Dcr2 complexes recognize and process viral or exogenous dsRNA.
3.2. dsRNA recognition by mammalian antiviral
pathways
In mammals, the synthetic dsRNA analog, polyinosinicpolycytidylic acid (poly(I:C)), has long been utilized as a
surrogate for virus infection [1]. There are several dsRNAactivated proteins that mediate mammalian antiviral defense
including the dsRNA-activated protein kinase (PKR), oligodenylate synthetases (OAS), toll-like receptor-3 (TLR-3) and
RNA helicases such as RIG-I [79]. These proteins induce a

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complex antiviral response in mammalian cells that include

translation inhibition and apoptosis of infected cells in addition 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 to
as RIG-like helicases (RLHs) [72]. Recognition of viruses by
RIG-I and MDA-5 triggers signaling pathways that result in
the production of type I IFNs. These two RNA helicases are
very similar in sequence and structure but perform nonredundant roles in the recognition of mammalian viruses
[72]. Influenza virus, Vesicular Stomatitis virus, and Japanese
Encephalitis virus (JEV) and several paramyxoviruses are
exclusively recognized by RIG-I while MDA-5 is required for
the recognition of picornaviruses such as Encephalomyocarditis virus, Mengo virus, and Theiler's virus [80]. Other viruses, such as Dengue virus, seem to require both RIG-I and
MDA-5 [81]. Despite this specificity in the recognition of
viruses, both RIG-I and MDA-5 are similarly capable of
recognizing exogenous dsRNA [82].
In addition to these classical antiviral pathways, the
mammalian siRNA pathway is also capable of recognizing
different sources of dsRNA. Dicer-dependent siRNAs are
generated from endogenous sources such as transposable elements and sense-antisense pairs of transcripts as well as
exogenous or viral sources of dsRNA [57,58,86,87]. Notably,
viral dsRNA sensing by the siRNA pathway seems to be
inhibited in differentiated cells where classical antiviral
pathways, such as the RLHs, are functional [88]. Processing
of endogenous dsRNA by the siRNA pathway seems to prevent erroneous activation of RLHs, which could be detrimental to the cell [89]. In addition, RIG-I is inhibited by the
presence of 30 overhangs at the end of dsRNA which are
naturally found in Dicer products [84]. This suggests that, in
differentiated mammalian cells, there is functional specialization between the siRNA pathway and RLHs to recognize
endogenous and viral dsRNA, respectively. This competition
seems restricted to differentiated cells since the siRNA
pathway in stem cells recognizes all types of dsRNA
[58,86,87]. Expression of accessory proteins required for the
recognition of viral dsRNA by the siRNA pathway could
explain the difference between stem cells and differentiated
cells.
The recognition of viruses by RLHs and the siRNA pathway
in mammals also illustrate complexities of viral RNA recognition. Therefore, the paradigm of exogenous dsRNA as a
surrogate for viral infection is a dangerous oversimplification.
4. Concluding remarks
The analysis of the Drosophila siRNA pathway suggests
that viral RNA sensing and recognition of exogenous dsRNA
are mediated by distinct mechanisms. To help understand the
discrimination between exogenous and viral dsRNA, it is
worth considering the separation between miRNA and siRNA
pathways. The biogenesis and function of siRNAs shares
important similar features with miRNAs (Fig. 1) [3]. Both

siRNAs and miRNAs are generated by Dicer cleavage of

dsRNA precursors and associate with somewhat similar
Argonaute proteins. The miRNA pathway is absolutely
required for cell differentiation and development in
Drosophila and other animals [13]. Due to the functional and
mechanistic similarities between miRNAs and siRNAs, interference between the two pathways would have a great impact
on animal cells. Notably, miRNAs likely appeared later in
evolution since several unicellular eukaryotes do not have
miRNAs [5]. In contrast, the siRNA pathway as an antiviral
mechanism appeared early in the evolution of eukaryotes.
Thus, once miRNAs appeared and acquired important developmental functions, sharing of components and mechanistic
similarities with the siRNA pathway became problematic.
Therefore, it was essential to find the right balance between
miRNA and antiviral siRNA pathways. Insects developed
separate miRNA and siRNA pathways with protein components that have little or no functional overlap [3]. Notably, loqs
is the only gene still shared between miRNA and siRNA
pathways although it is clearly not required for the antiviral
siRNA pathway [35]. This complete separation likely allowed
independent evolution for the antiviral siRNA pathway
without any restrictions imposed by the miRNA pathway. A
highly specialized antiviral siRNA pathway could acquire
unique features that make it more efficient against viruses. In
support of this hypothesis, Dcr-2 that is responsible for the
recognition of viral dsRNA seems to have acquired the ability
to also trigger the transcription of antiviral genes independent
of its function in the biogenesis of siRNAs [90]. Hence,
activation of the antiviral siRNA pathway likely has broader
effects and is more tightly regulated than recognition of
exogenous dsRNA.
The existence of separate siRNA pathways implies that
viruses can be discriminated from other sources of dsRNA.
Indeed, the specific discrimination of viral dsRNA seems to
depend on a variety of intrinsic characteristics. For example,
C. elegans RDE-4 and mammalian MDA-5 recognize dsRNA
by multimerizing along the molecule in a length dependent
manner [83,91]. Similarly, in Drosophila, the ability to carry
out efficient processing of long dsRNA molecules by Dcr-2
seems to be essential for its antiviral function [35]. The
discrimination of viral dsRNA by worm DRH-1 and
mammalian RIG-I, but likely not Drosophila Dcr-2, depends
on the presence of a 50 triphosphate [76,78,85]. Notably, fly
Dcr-2, worm DRH-1 and mammalian RIG-I, all share a
common ancestor suggesting the capacity to recognize 50
triphosphate was present in the ancestor of these proteins but
was likely lost in fly Dcr-2 [90,92]. In addition, other characteristics are likely to contribute to the discrimination of viral
RNA. For example, exogenous and viral dsRNA have very
distinct subcellular localizations [68].
In summary, we propose a model where a default siRNA
pathway is activated by any type of dsRNA while the
engagement of the antiviral pathway would require additional
intrinsic characteristics of the viral RNA. This mechanism
could limit the activation of the antiviral pathway to avoid side
effects and waste of energy.

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Conflict 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 for
any work that might not have been cited in this review due to
limitation on the number of references.
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