Viral suppressors of RNA silencing hinder exogenous and endogenous small RNA pathways in Drosophila.
ABSTRACT In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs). While VSRs in Drosophila melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways remained unexplored.
Here we show that expression of various plant and insect VSRs in transgenic flies does not perturb the Drosophila microRNA (miRNA) pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed transposon silencing by endogenous siRNAs (endo-siRNAs).
Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections.
- SourceAvailable from: sciencedirect.comCurrent Opinion in Insect Science. 01/2015; 425.
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ABSTRACT: Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.Nucleic Acids Research 10/2014; · 8.81 Impact Factor
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ABSTRACT: The host RNA interference (RNAi) pathway of insects senses virus infection and induces an antiviral response to restrict virus replication. Dicer-2 detects viral double-stranded RNA, produced by RNA and DNA viruses, and generates viral small interfering RNAs (vsiRNAs). Recent small RNA profiling studies provided new insights into the viral RNA substrates that trigger vsiRNA biogenesis. The importance of the antiviral RNAi pathway is underscored by the observation that viruses have evolved sophisticated mechanisms to counteract this small RNA-based immune response. More recently, it was proposed that another small RNA silencing mechanism, the piRNA pathway, also processes viral RNAs in Drosophila and mosquitoes. Here, we review recent insights into the mechanism of antiviral RNAi, viral small RNA profiles, and viral counter-defense mechanisms in insects.Current opinion in virology. 04/2014; 7C:19-28.
Viral Suppressors of RNA Silencing Hinder Exogenous
and Endogenous Small RNA Pathways in Drosophila
Bassam Berry1, Safia Deddouche2, Doris Kirschner1, Jean-Luc Imler2, Christophe Antoniewski1*
1Institut Pasteur, Drosophila Genetics and Epigenetics, CNRS-URA2578, Paris, France, 2IBMC, CNRS-UPR9022, Strasbourg, France
Background: In plants and insects, RNA interference (RNAi) is the main responder against viruses and shapes the basis of
antiviral immunity. Viruses counter this defense by expressing viral suppressors of RNAi (VSRs). While VSRs in Drosophila
melanogaster were shown to inhibit RNAi through different modes of action, whether they act on other silencing pathways
Methodology/Principal Findings: Here we show that expression of various plant and insect VSRs in transgenic flies does
not perturb the Drosophila microRNA (miRNA) pathway; but in contrast, inhibits antiviral RNAi and the RNA silencing
response triggered by inverted repeat transcripts, and injection of dsRNA or siRNA. Strikingly, these VSRs also suppressed
transposon silencing by endogenous siRNAs (endo-siRNAs).
Conclusions/Significance: Our findings identify VSRs as tools to unravel small RNA pathways in insects and suggest a
cosuppression of antiviral RNAi and endo-siRNA silencing by viruses during fly infections.
Citation: Berry B, Deddouche S, Kirschner D, Imler J-L, Antoniewski C (2009) Viral Suppressors of RNA Silencing Hinder Exogenous and Endogenous Small RNA
Pathways in Drosophila. PLoS ONE 4(6): e5866. doi:10.1371/journal.pone.0005866
Editor: Thomas Preiss, Victor Chang Cardiac Research Institute (VCCRI), Australia
Received April 2, 2009; Accepted May 13, 2009; Published June 10, 2009
Copyright: ? 2009 Berry et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a fellowship from the Lebanese CNRS to B.B. and by grants from the CNRS, the NIH (PO1 AI070167) and the ANR (MIME) to
J.-L. I. and from the CNRS and the ANR (AKROSS) to C. A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
RNA silencing is a eukaryotic gene regulation mechanism by
which RNA expression is shut down in a sequence specific manner
through the intervention of homologous small non coding RNAs
. Three types of small RNAs, miRNAs, piRNAs and siRNAs,
accumulate in different tissues and developmental stages of
The ,22 nucleotides (nt) miRNAs derive from stem-loop
precursor transcripts through the action of the Drosha/Pasha and
Dicer-1/Loqs complexes. In D. melanogaster, they are mostly loaded
into the Argonaute-1 (Ago1) protein and guide translational
repression of mRNAs [2,3]. The ,25-nt piRNAs are produced
from the cleavage of selfish and repetitive genetic element
transcripts by the Piwi Argonautes. They are restricted to the
gonads where they silence Transposable Elements (TEs) . The
,21 nt siRNAs originate from the processing by Dicer-2 (Dcr2) of
long dsRNA precursors, such as those produced by inverted-repeat
(IR) transgenes. They load into Argonaute-2 (Ago2) and guide the
cleavage of target mRNAs with perfect complementary sequence
matches . siRNAs produced from endogenous precursors
(endo-siRNAs) were recently described in flies. Beside piRNAs,
they contribute to the silencing of TEs and some endogenes in
both gonads and somatic tissues [1,4,6].
siRNAs are also produced by Dcr-2 from dsRNA viral
intermediates upon viral infection [7–9], playing an essential role
in the fly’s defense against viruses. To counterattack, viruses
evolved to encode proteins that suppress their host’s antiviral
silencing. Viral suppressors of RNAi (VSRs) have been reported in
all types of plant viruses . More recently, insect viral
suppressors, the Flock House virus (FHV) B2, the Cricket Paralysis
virus (CrPV) and the Drosophila C virus (DCV) 1A proteins where
shown to act as potent inhibitors of antiviral RNAi in Drosophila
[8,9,11]. VSRs are very diverse in sequence and structure across
viral kingdoms, but operate through a few evolutionarily
convergent strategies: they most commonly bind to long dsRNAs
to inhibit their processing by Dicer proteins [12–14]; sequester
siRNA duplexes to prevent their loading to Argonaute complexes
[15–17]; or may directly interact with Dicers or Argonautes to
impair their antiviral activities [18,19].
Alongside inhibiting antiviral RNAi, plant VSRs including
HcPro, P21, P19, P15 and P0 were shown to suppress miRNA- or
endo-siRNA-mediated silencing, therefore corrupting develop-
ment or homeostasis of their host and contributing to viral
pathogenicity [18,20–23]. In contrast, the effect of VSRs on endo-
siRNAs in animals and its potential contribution to host disorders
has not been explored in detail. To address this issue, we
established Drosophila transgenic lines expressing a panel of 9 VSRs
from plant and insect viruses (Table 1) and performed a
comparative analysis of their effects on RNAi induced by (i) viral
infection, (ii) inverted repeat transgenes, (iii) in vitro transcribed
dsRNA, (iv) synthetic siRNAs, (v) endogenous siRNAs and (vi)
Our study reveals that none of these VSRs perturb the miRNA
pathway. In contrast, the insect VSRs FHV B2 and DCV 1A, as
well as the plant VSRs P21, P19 and P15, inhibited anti-viral
PLoS ONE | www.plosone.org1 June 2009 | Volume 4 | Issue 6 | e5866
RNAi in flies and suppressed silencing induced by siRNAs either
exogenously supplied or endogenously produced, strongly sup-
porting the relevance of RNAi mechanisms throughout evolution
and kingdoms. In addition, B2, 1A and P19 suppressed the
silencing of TEs in somatic tissues and in gonads, suggesting that
effects of VSRs on endo-siRNA-mediated silencing may account
for viral pathogenicity in insects. Our results demonstrate the
effectiveness of using viral silencing suppressor proteins as a tool to
dissect small RNA pathways in animals, as previously established
VSRs do not inhibit the Drosophila miRNA pathway
Drosophila transgenic lines were established to express a panel of
9 Flag-tagged VSRs (Table 1) under the control of a GAL4
inducible UAS promoter . Two VSRs, B2 and 1A, derive from
the insect viruses FHV and DCV, respectively. The remaining
VSRs P0, HcPro, P25, P38, P21, P15 and P19 derive from plant
viruses. The VSR transgenic lines were crossed with a daughterless-
GAL4 driver line, which ubiquitously expresses GAL4 throughout
development. Western blot assay using a Flag antibody indicated
that all suppressors are expressed at a high level in the progenies
from these crosses (Fig. S1).
In plants, HcPro, P21, P19, P15 and P14 interfere with
miRNAs, causing developmental defects that resemble those
observed in mutant plants deficient in their miRNA pathway
[20–23]. In addition, it has been reported that the P19 VSR might
interfere with the silencing activity of miR-32 in human cells .
Thus, we assessed the effect of the overexpression of the 9 VSRs
on miRNA activity. Expression of the VSRs in the posterior
compartment of the wing imaginal discs did not suppress the
silencing of a GFP reporter targeted by the bantam miRNA (Fig.
S2 and Movies S1 and S2). Additionally, the transgenic stocks
ubiquitously expressing each of the 9 VSRs progressed normally
from embryogenesis to adulthood, showing neither developmental
defects nor altered viability. These results indicate that the VSRs
here tested do not appreciably perturb the miRNA pathway in
Suppression of antiviral RNAi.
construct was previously engineered to autonomously replicate in
the presence of the viral RNA-dependent RNA polymerase
(RdRp) encoded by RNA1 . Non-sense mutations in the B2
ORF of RNA1 (Fig. 1A) strongly impair the replication of a
derivative RNA1DB2 transgenic construct (Fig. 1B, compare
A FHV RNA1 transgenic
RNA1 and RNA1DB2 controls), an effect that was attributed to
the inability of RNA1DB2 to limit the RNAi host response
through the activity of the B2 VSR . Accordingly, RNA1DB2
expression was restored in flies expressing the B2 transgene
(Fig. 1B). We therefore decided to monitor the ability of our panel
of VSRs to restore the replication level of RNA1DB2 (Fig. 1B).
RNA1DB2 replication was restored in DCV 1A transgenic flies
at levels similar to those observed in B2 transgenic flies. High levels
of RNA1DB2 replication were also measured in flies expressing
P15, P21 or P19, suggesting that these proteins, in a similar way to
B2 and 1A, suppress fly antiviral RNAi. In contrast, no significant
increase of RNA1DB2 replication was observed in flies expressing
P38, P25, HcPro or P0.
To further characterize the impact of the suppressors on
antiviral RNAi, we tested their effect on fly susceptibility to
infection by the Drosophila C virus (DCV), a common Drosophila
pathogen. Upon DCV inoculation of wild type flies, 50% of
animals died after 6 to 7 days (Fig. 2A). The survival was similar
for flies expressing P25, P38, HcPro or P0 under the control of the
ubiquitous Actin5C-GAL4 driver (data not shown), paralleling the
lack of effect of these proteins on the replication of the FHV
RNA1DB2. In contrast, flies expressing B2, 1A, P19, P21 and P15
under the control of the ubiquitous Actin5C.GAL4 driver
showed a more rapid course of disease and died faster after
inoculation with DCV (Fig. 2A and data not shown). Similar
results were obtained when these suppressors were expressed
under the control of the heat-shock.GAL4 driver after heat shock
induction (Fig. 2B). These data show that the B2 and 1A insect
VSRs, as well as the P19, P15 and P21 plant VSRs act as potent
inhibitors of antiviral RNAi in flies.
Because one of the modes of action of VSR is the
binding to long dsRNA species, sequestering them from Dicer
access, or to siRNAs hindering their loading into the RISC
complex, we next tested the ability of the suppressors to inhibit
RNAi induced by injection of dsRNA or siRNA in embryos. 500-
nt dsRNAs or 21-nt siRNAs matching the fushi tarazu gene (ftz)
induced a high frequency (.80%) of cuticle defects in wild type
survivors, but not in ago22/2survivors, indicating that the two
types of molecules elicited potent RNAi against the ftz gene (Fig. 3).
Injection of long dsRNAs or siRNAs in transgenic embryos P0,
HcPro, P25 or P38 also induced a strong ftz RNAi, with a ,80%
frequency of cuticule defects (Fig. 3B–C). In contrast, only 10–
30% of cuticule defects were observed in transgenic embryos P21,
P15, P19 or B2, indicating that maternal stocks and/or embryonic
Table 1. Viral Suppressors of RNAi used in this study.
HostVSRVirusGenus Mode of action
InsectsB2 Flock house virus Nodavirus dsRNA and siRNA binding [12,14,26,27]
1A Drosophila C virusCripaviruslong dsRNAs binding 
PlantsP0Cucurbit aphid born yellows virus Luteovirus ubiquitilation degradation of Argonautes proteins
P15 Peanut clump virus PecluvirussiRNA binding 
P19Tomato bushy stunt virus Tombusvirus siRNA binding [15,16]
P21Beet yellows virus Closterovirus siRNA binding 
P25Potato virus X Potexvirusunknown [21,42]
P38Turnip crinkle virusCarmovirus interferes with DCL4 
HcPro Zucchini yellow mosaic virus PotyvirussiRNA binding 
RNAi Suppressors in Flies
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expression of these proteins efficiently suppressed ftz RNAi. In
contrast to B2 transgenic embryos, 1A transgenic embryos were
able to mount an efficient ftz silencing when injected with short
siRNAs but not with long dsRNAs (Compare Fig. 3B and C). This
result is fully consistent with previous data indicating that DCV-
1A specifically binds long dsRNAs in vitro , while B2 bind to
both long dsRNA [14,26] and short siRNA [12,27].
To establish the role of VSRs on endo-siRNAs we
sought to use a GMR.IR[w] transgenic construct expressing an
RNA hairpin in eyes . This construct triggers the Dcr-2-
dependent production of siRNAs [29,30] and silences the white
gene (Fig. 4, control panels). When co-expressed under the control
of a GMR.GAL4 driver with the RNAi trigger GMR.IR[w]
transgene, B2, 1A and to a lesser extent, P19, P15 and P21,
suppressed this white silencing (Fig. 4). This result indicates that in
addition to antiviral and exogenously induced RNAi, these 5
VSRs can block RNAi induced by an transgenic hairpin locus. In
contrast, P38, P25, HcPro and P0 failed to inhibit the white
silencing, paralleling their lack of effect on antiviral or exogenously
A class of endogenous siRNAs is produced from sense and
antisense transcription of transposable elements. In somatic tissues,
these endo-siRNAs were shown to silence TEs from where they
originate in a negative feedback loop [4,6]. To further characterize
the activity of B2, 1A and P19 on the fly endo-siRNA pathway, we
analyzed the effects of their overexpression on two retrotranspo-
sons in female adult heads and ovaries (Fig. 5).
As expected, the steady-state levels of the LTR retrotransposons
297 and ZAM increased in heads from ago22/2mutant control
flies. The ago2 mutation also derepressed ZAM and to a lesser
extent, 297, in ovaries, agreeing with previous expression level
analyses in this tissue [31,32].
Expression of ZAM increased in heads and to a lesser extent, in
ovaries, from B2, 1A and P19 transgenic animals driven by the
ubiquitous da.GAL4 driver. Expression of 297 also increased in
heads but not in ovaries of all three transgenic lines. These data
indicate that B2, 1A and P19 impair transposon silencing by endo-
siRNAs in somatic tissues.The modest effect of these VSRs inovaries
suggests that they do not interfere with piRNA silencing in this tissue.
Here we analyzed the ability of 9 VSRs to interfere with small
RNA silencing pathways in adult flies. We found that 2 insect
VSRs - B2 and 1A - and 3 plant VSRs - P19, P21 and P15 - inhibit
in vivo antiviral RNAi as well as RNAi induced by both
exogenously and endogenously supplied dsRNAs.
None of the VSRs tested in this study significantly interfered
with miRNA activity. Biogenesis of miRNAs involves primary
miRNA processing by Drosha/Pasha in the nucleus, exportin-5
dependent export of pre-miRNAs in the cytoplasm, pre-miRNA
processing by Dicer-1 and miRNA loading into RISC complexes.
It is possible that VSRs cannot access the miRNA machinery in
insect cells during this coordinated sequence of events. Alterna-
tively, the imperfect duplex structure of miRNAs may preclude
their binding by VSRs. In any case, the absence of effects of VSRs
on miRNAs might represent an adaptation of insect hosts during
evolution to limit the perturbation of gene expression upon viral
infection. Of note, the VSR B2 was recently shown to suppress an
Figure 1. Flies expressing insect and plant VSRs accumulate high levels of FHV RNA1DB2 transcripts. (A) RNA1 and RNA1DB2
constructs. The GAL4 inducible UAS-RNA1 transgene (left hand side) expresses a RNA1 of (+) polarity. RNA1 encodes an RdRp protein, which directs
autonomous replication of RNA1 of (2) polarity, then of both RNA1 and RNA3 of (+) polarity. Two point mutations (triangles) interrupt the B2 ORF in
the RNA1DB2 (right hand side) without affecting the RdRp ORF . (B) Fold changes in RNA1+RNA3 levels. The RNA1 transgenic line was crossed
with the da.GAL4 line (control). The RNA1DB2 transgenic line was crossed with the da.GAL4 line (control) or with da.GAL4; UAS-VSR lines as
indicated. RNA1+RNA3 expression was measured by quantitative RT-PCR from the progenies of these crosses. Error bars indicate the standard
deviation from triplicate qPCR experiments.
RNAi Suppressors in Flies
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inducible antiviral response in Drosophila, in addition to its role in
suppressing RNAi . It will be interesting to test if other VSRs
can also block the upregulation of the antiviral gene Vago in
The B2 and 1A proteins are encoded by FHV and DCV
respectively, which belong to distinct families of (+) single stranded
RNA viruses naturally infecting Drosophila. Both proteins inhibit
the silencing of FHVDB2 and sensitize flies to infection with DCV.
Figure 2. Hypersensitivity to DCV infection. (A) An Act5C.GAL4 driver line was crossed to UAS.VSR transgenic lines (&) or to w1118 control
strain (%). 20 females from the progeny of these crosses were challenged by an intrathoracic injection of a control Tris solution (---) or a DCV
suspension corresponding to 102LD50 (–) and survival was monitored daily. (B) An hsp.GAL4 driver line was crossed to UAS.VSR transgenic lines.
20 females from the progeny of these crosses were challenged by an injection of the control Tris solution (---) or a the DCV suspension (–) under heat
shock (&) or non heat shock (%) conditions. Plotted values represent the mean6SEM (standard error to the mean) of three independent
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Synergistic viral diseases in plants, namely the co-infection of a
host plant with two unrelated viruses which elicits disease
symptoms that are more severe than the sum of those induced
in either single infection, have been shown to be dependent in
some instances on VSRs . Our data suggest that such viral
synergism may also exist in insects and that VSRs could act as
‘‘synergism genes’’ becoming permissive agents for the develop-
ment of infection.
The plant VSRs P19, P15 and P21 inhibit the antiviral silencing
machinery of their host by binding to 21 nt siRNAs . P19 also
inhibits RNAi in mammalian cultured cells  and in vitro studies
demonstrated that P19 and P21 proteins block siRNA-directed
target RNA cleavage in Drosophila embryonic extracts by sequester-
ing siRNA duplexes . Here, we show that in vivo P19, P21 and
P15 promote the accumulation of the FHVDB2 replicon, sensitize
flies to DCV infection and inhibit ftz silencing triggered either by
long dsRNA or siRNA. Altogether, these data indicate that, in flies,
these three VSRs mimic their function in plants by sequestering
siRNAs. It is worthy to note that in a number of cases, plant viruses
are transmitted by insect vectors  and may then have to face the
antiviral RNAi response in distant organisms. Sequestering siRNAs,
whose structure is conserved throughout evolution, may indeed
represent an optimal strategy to adapt to otherwise evolutionarily
divergent antiviral responses.
Although P0, P25, P38 and HcPro were characterized as potent
VSRs in plants , they did not show activity in flies in any of
our assays. We cannot exclude that this lack of activity results from
imperfect biosynthesis of the viral suppressors in flies. Neverthe-
less, it is unlikely due to a deleterious effect of the Flag epitope as
non-Flagged versions of these suppressors gave identical results
(data not shown). P0 is an F-box like protein that induces the
ubiquitinylation and the degradation of plant Argonautes .
Figure 3. Suppression of silencing induced by injection of long dsRNAs or siRNAs in embryos. RNAi of the fushi tarazu gene (ftz) in wild
type syncytial embryos results in loss of denticle belts in the cuticle of prehatching larvae (A). Early embryos homozygous for the ago2414mutant
allele or expressing the indicated VSR under the control of the da.GAL4 driver were injected with long dsRNAs (B) or siRNAs (C) targeted to the ftz
gene and scored 48 h after injection for the ftz mutant phenotype. Confidence intervals (alpha risk 0.05) for the observed ratio are indicated.
RNAi Suppressors in Flies
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Although the mechanisms by which P25 and P38 inhibit RNA
silencing remain unclear, these two proteins do not appear to act
by sequestering siRNAs . It is therefore likely that the lack of
activity of P0, P25 and P38 in flies reflects interactions of these
VSR with host plant proteins whose structure and/or function are
not conserved in flies. The HcPro protein from Tobacco etch virus
(TEV) was shown to inhibit dsRNA-induced silencing in Drosophila
S2 cells  and sequester siRNA from embryonic extracts,
although less efficiently than P19 . The lack of activity of
HcPro from the ZYMV that we observed in our study may be due
a divergence of sequence between HcPro proteins from TEV and
B2, 1A, P19, P15 and P21 VSRs that inhibit silencing induced
by exogenously supplied dsRNAs were also capable of inhibiting
the silencing mediated by siRNAs produced from an inverse
repeat transgene. In addition, the insect VSRs B2 and 1A, and the
plant VSR P19 inhibited the silencing of the Drosophila 297 and
ZAM retrotransposons in heads and ovaries of adult flies,
indicating that these suppressors interfere with the endo-siRNA
pathway in flies [4,6]. Their modest effect in gonads also suggests
Figure 4. VSRs inhibit silencing induced a white inverse-repeat transgene. Silencing of the white gene induced by an IR[w] inverted-repeat
transgene in flies expressing insect and plant VSRs in eyes. (A) In all heterozygous combinations GMR.GAL4; UAS.GFP or GMR.GAL4; UAS.VSR,
the mini-white markers of the two transgenes produce equivalent strong red eye pigmentation. (B) Silencing of the mini-white markers by one copy
of the GMR.IR[w] transgene (note that the GMR.IR[w] construct has no mini-white marker ). The effect of the GMR.IR[w] transgene on the two
mini-white markers in GMR.GAL4; UAS.VSR genetic combinations depends on the efficiency of the suppressor to inhibit white silencing. Eye
pigment levels were measured in 3 separate experiments from 2 days-old adult eyes and are expressed as a percentage of pigment levels measured
in the GMR.GAL4; UAS.GFP combination. Error bars correspond to standard deviations.
RNAi Suppressors in Flies
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that they do not interfere with the piRNA-mediated silencing of
TEs in this tissue.
The DCV-1A protein specifically binds long dsRNAs and does
not interfere with RNAi when directly triggered by siRNAs .
Immunoprecipitation experiments showed that P19 and to a lesser
extent B2 bind to TE-derived endo-siRNA in S2 cultured cell;
additionally, B2 was specifically able to bind longer RNA species
(Fagegaltier et al, in preparation). Together, these data suggest that
the effects of VSRs on TE silencing is achieved through
sequestration of TE-derived endo-siRNAs by P19, of long
double-stranded TE RNA precursors by 1A and of both endo-
siRNAs and their double-stranded precursors by B2.
It is remarkable that plants as well as insect VSRs can, besides
their well-known roles in viral counterdefense mechanisms,
consistently interfere with endo-siRNA pathway. Whether viruses
can manipulate this pathway to improve their interaction with the
host during the course of a natural infection and what the
physiological implications would be are open questions.
Materials and Methods
Suppressor transgenic constructs
VSR cDNAs were first PCR amplified using the following
templates : pBWDi-FHV-B2 kindly provided by C. Vaury (B2,
Swiss-Prot P68831); pAC-DCV1A (1A, ; pBIN-P15, -P19, -P21, -
P25 and -P38 constructs kindly provided by O. Voinnet (P15, P19,
P21, P25 and P38, ; Zucchini yellows mosaic virus HcPro cDNA
kindly provided by C. Desbiez (HcPro, Swiss-Prot P18479);
pGBKT7-P0CAB(P0, . PCR primers were as follows: B2-F, 59-
GTGG-39; 1A-F, 59-CACCATGGAATCTGATAAAAGTAT-39;
1A-R, 59-CTTGTCATCGTCATCCTTGTAAT-39; P19-F, 59-
CACCATGGAACGAGC-39; P19-R, 59-CTCGCTTTCTTTT-
TCG-39; P15-F, 59-CACCATGCCTAAGTCG-39; P15-R, 59-
CAGTTTAGAACGAAG-39; P21-F 59-CACCATGAAGTTTTT-
CTTTAATGA-39; P21-R 59-TACAGCTATACCGAGGATTT-
TGGCCCTGCGCGGAC-39; P38-F, 59-CACCATGGAAAAT-
GATC-39; P38-R, 59-AATTCTGAGTGCTTGC-39; HcPro-F, 59-
AGC-39; P0-R, 59-GCGTTGTAGCTCCTTTTG-39
VSR cDNAs were cloned into the pENTR-D-TOPO vector
(Invitrogen) and were subsequently recombined into pUASp
vectors  adapted for the Gateway system (Drosophila Gate-
wayH Vector collection, Carnegie Institution) to give C-terminal
Flag tagged pPWF-VSR and non tagged pPW-VSR transgenesis
vectors. VSR sequences in these constructs were verified by
Figure 5. VSRs inhibit retrotransposon silencing by endo-siRNAs. 297 and ZAM retrotransposon silencing is impaired by B2, 1A and P19 VSRs.
RNA levels of 297 and ZAM were measured in heads and ovaries of 1 day-old female flies heterozygous for the da.GAL4 driver and the indicated
UAS.VSR transgene. Fold changes in RNA levels were calculated relative to the 297 and ZAM RNA levels measured in heterozygous da.GAL4 control
flies. Fold changes in homozygous ago2414mutants were calculated relative to the 297 and ZAM RNA levels measured in heterozygous ago2414flies.
Therefore, the weakereffect oftheago2 mutationon297expression observed in adultheadsmayresultfrom variations incopy numberof297between
the ago2 mutant stock and the VSR transgenic stocks. Error bars indicate the standard deviation from triplicate qPCR experiments.
RNAi Suppressors in Flies
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sequencing before establishing UAS.VSR transgenic stocks by
standard injection procedure. For each VSR construct, three
independent transgenic lines were tested in western blot assay as
well as in GMR.IR[w] desilencing test. One highly expressed
transgene insertion for each suppressor (Fig. S1) was chosen for
Transgenic and mutant stocks
The FHV RNA1 and RNA1DB2 flies are described in . We
obtained the GMR.IR[w] transgenic line from R. Carthew ,
the Ago2414mutant stock from H. Siomi , the engrailed.GAL4,
Tubulin.GFP and Tubulin.GFP-ban transgenic stocks from S.
Cohen , the GMR.GAL4, da.GAL4, hsp.GAL4 and
UAS.GFP lines from the Bloomington Stock Center and the
Act5C.GAL4 17a driver line (nuU192) from the Fly stocks of
National Institute for Genetics from Japan (http://www.shigen.
To study the effect of the VSRs on the replication of FHV
RNA1DB2, we genetically recombined the da.GAL4 driver with
the UAS.VSR transgenic insertions. The resulting UAS.VSR;
da.GAL4 stocks were then crossed to the UAS.RNA1DB2
transgenic line. For control, the UAS.RNA1DB2 line was crossed
to the da.GAL4 driver line.
To monitor fly sensitivity to infection by DCV, the UAS.VSRs
transgenic lines were crossed to the Act5C.GAL4 or hsp.GAL4
To analyze the effect of the suppressors on white silencing, we
constructed an homozygous w1118, GMR.IR[w]; GMR.GAL4
stock by genetic recombination and crossed it to the homozygous
UAS.GFP and UAS.VSR stocks. For controls, the UAS.GFP
line, as well as all the UAS.VSR transgenic stocks were crossed to
the homozygous w1118; GMR.GAL4 stock.
To analyze the effect of suppressors of bantam silencing, we
recombined the Tubulin.GFP-ban sensor transgene with the
engrailed.GAL4 driver on the third chromosome and crossed the
resulting homozygous stock to the UAS.VSR lines.
Quantitative RT PCR
To measure the steady state level of the FHV RNA1DB2, ten 6-
days old female flies were collected from the progeny of each
UASp.VSR; da.GAL4 stock crossed to homozygous UAS.
RNA1DB2 flies. Same aged UAS.RNA1; da.GAL4 or UAS.
RNA1DB2; da.GAL4 heterozygous flies were collected as
controls. Total RNA extracts were prepared with Trizol Reagent
(Invitrogen) according to the manufacturer’s instructions, treated
with DNAse I then reverse transcribed with iScript cDNA
synthesis kit (Biorad). FHV RNA1 levels were then analyzed by
real-time PCR on an Opticon2 Instrument (Biorad) using the
Faststart Sybr-Green Mix (Roche) and the 59ACCTCGATGG-
CAGGGTTT 39 and 59CTTGAACCATGGCCTTTTG 39
primers. Template concentrations were normalized to endogenous
reference RpL23 and to the heterozygous UAS.RNA1DB2;
da.GAL4 control calibrator using the DDCTmethod.
297 and ZAM transposon expression levels were measured in
double heterozygous UASp.VSR; da.GAL4 and control hetero-
zygous da.GAL4 2-days old females. Fifty heads and ovaries were
separated manually and processed for total RNA extraction and
reverse transcription as described above. FHV RNA1 levels were
TAAAATGGTTCG-39 , ZAM-F 59-ACTTGACCTGGATA-
CACTCACAAC-39 and ZAM-R 59-GAGTATTACGGCGAC-
TAGGGATAC-39  primers. Template concentrations were
normalized to endogenous referenceRpL23 and to the heterozygous
da.GAL4 control calibrator using the DDCTmethod.
In both experiments, RpL23 levels were measured using the
RpL-F, 59-CGGATCGATATGCTAAGCTGT-39 and RpL-R,
59-GCGCTTGTTCGATCCGTA-39 primer pair.
4–6 days old double heterozygous Act5C.GAL4; UAS.VSR
or heat-shocked hsp.GAL4; UAS.VSR and control heterozy-
gous Act5C.GAL4 or hsp.GAL4 flies were used in infection
experiments. Viral stocks were prepared in 10 mM Tris-HCl,
pH 7.5. Infections were done as described in  by injection
(Nanoject II apparatus; Drummond Scientific) of 4.6 nL of a viral
suspension (DCV, 26105LD50 (50% lethal dose)/mL) into the
thorax of flies. Injection of the same volume of 10 mM Tris-HCl,
pH 7.5, was used as a control. For heat-shock induction of
transgene expression, flies were incubated for 20 min at 37uC,
followed by 30 min at 18uC and another 20 min at 37uC. After
the treatment, flies were allowed to recover for 6 h at 25uC before
infection. All survival experiments were performed at 22uC.
ftz dsRNA and siRNA injection in embryos
A region of the fushi tarazu (ftz) gene was amplified with the T7-
CATGTATCACCCCCA-39 and T7-ftz-R 59-GAATTGTAA-
39 primers using a w1118fly genomic DNA template. The PCR
product was then used for dsRNA synthesis, using MEGAScriptH
RNAi Kit (AmbionH) according to the manufacturer’s instructions.
59 phosphorylated siRNA (21 bp) with dTdT 39overhangs
corresponding to the ftz gene (siFTZ 59-UGCCUACUAUCA-
GAACACC-39) was purchased from Eurogentec.
Embryos were collected from UAS.VSR; da.GAL4 stocks
over a 30 min period at 25uC, hand dechorionated, and attached
to a coverslip coated with double-stick tape. Embryos were then
desiccated at room temperature, covered in 700 halocarbon oil
and injected with a 1 mg/ml solution of dsRNA or siRNA using
Eppendorf’s sterile FemtotipsH needles. After 36 hr recovery, the
surviving embryos were scored for the number of ventral denticle
belts. Embryos with four, five, or six denticle belts were scored
Eye pigment dosage
Assays were performed on 3 days old virgin females. Five heads
per genotype were manually collected and homogenized in 50 ml
of a freshly prepared solution of acidified methanol (0.1% HCl).
Pigment was extracted by rocking tubes for 36 hr at 4uC.
Homogenates were then incubated at 50uC for 5 min, clarified
by centrifugation and optical density of each sample was read at
480 nm. Three independent extractions were performed for each
genotype and the mean values of the absorption per extraction
For protein analysis, equal amounts of proteins were extracted
from heterozygous UAS.VSR; da.GAL4 L3 larvae, boiled in
Laemmli buffer and loaded on 15% SDS-PAGE. After transfer
onto nitrocellulose membrane, equal loading was verified by
Ponceau staining. Membranes were blocked in 5% milk, 16PBS,
0.1% Tween, and incubated overnight with mouse HRP coupled
anti-Flag antibody (1:10000, Sigma). Detection was performed
using Chemiluminescent Substrate (Pierce).
RNAi Suppressors in Flies
PLoS ONE | www.plosone.org8 June 2009 | Volume 4 | Issue 6 | e5866
larval instar protein extract were prepared from transgenic larvae
double heterozygous for the da.GAL4 driver and the indicated
UAS.VSR transgene and analyzed by western blot using an anti-
Flag antibody. Ponceau staining (bottom) shows equal protein
loading between lanes.
Found at: doi:10.1371/journal.pone.0005866.s001 (0.98 MB EPS)
Expression of VSRs in transgenic animals. Third
miRNA. Confocal microscopy of wing imaginal discs expressing
the engrailed-GAL4 driver in their posterior half compartment (P)
and either a control Tubulin.GFP or a Tubulin.GFP-ban
sensor with bantam miRNA target sites in 39 UTR. The quadrant
pattern of GFP silencing by bantam in the wing pouch is not
affected by expression of the indicated UAS.VSR transgenes in
imaginal disc posterior compartment, indicating no obvious
interference with the miRNA pathway by either VSR.
Found at: doi:10.1371/journal.pone.0005866.s002 (4.43 MB TIF)
VSRs do not suppress silencing by the bantam
engrailed-GAL4, Tubulin.GFP-ban wing imaginal discs shown
in Fig. S2
B2 confocal set. Confocal slice sets of UAS.B2;
Found at: doi:10.1371/journal.pone.0005866.s003 (56.46 MB
engrailed.GAL4, Tubulin.GFP-ban wing imaginal discs shown
in Fig. S2
Found at: doi:10.1371/journal.pone.0005866.s004 (34.75 MB
P15 confocal set. Confocal slice set of UAS.P15;
We thank O. Voinnet, C. Vaury, C. Desbiez, H. Siomi, R. Carthew and S.
Cohen for providing materials, the Bestgene Inc for transgenesis, the PFID
for assistance on microscopy, and C. Saleh and M. Vignuzzi for discussions
and critical reading of the manuscript.
Conceived and designed the experiments: BB SD JLI CA. Performed the
experiments: BB SD. Analyzed the data: BB SD JLI CA. Contributed
reagents/materials/analysis tools: DK. Wrote the paper: BB CA.
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