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Antiviral RNA Silencing Initiated in the Absence of RDE-4, a Double-Stranded RNA Binding Protein, in Caenorhabditis elegans

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Stranded RNA Binding Protein, in

Caenorhabditis elegans

Xunyang Guo, Rui Zhang, Jeffrey Wang, Rui Lu

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

Small interfering RNAs (siRNAs) processed from double-stranded RNA (dsRNA) of virus origins mediate potent antiviral de-fense through a process referred to as RNA interference (RNAi) or RNA silencing in diverse organisms. In the simple inverte-brateCaenorhabditis elegans, the RNAi process is initiated by a single Dicer, which partners with the dsRNA binding protein RDE-4 to process dsRNA into viral siRNAs (viRNAs). Notably, inC. elegansthis RNA-directed viral immunity (RDVI) also re-quires a number of worm-specific genes for its full antiviral potential. One such gene isrsd-2(RNAi spreading defective 2), which was implicated in RDVI in our previous studies. In the current study, we first established an antiviral role by showing thatrsd-2 null mutants permitted higher levels of viral RNA accumulation, and that this enhanced viral susceptibility was reversed by ecto-pic expression of RSD-2. We then examined the relationship ofrsd-2with other known components of RNAi pathways and es-tablished thatrsd-2functions in a novel pathway that is independent ofrde-4but likely requires the RNA-dependent RNA poly-merase RRF-1, suggesting a critical role for RSD-2 in secondary viRNA biogenesis, likely through coordinated action with RRF-1. Together, these results suggest that RDVI in the single-Dicer organismC. elegansdepends on the collective actions of both RDE-4-dependent and RDE-4-independent mechanisms to produce RNAi-inducing viRNAs. Our study reveals, for the first time, a novel siRNA-producing mechanism inC. elegansthat bypasses the need for a dsRNA-binding protein.

I

n fungi, plants, insects, and nematodes, double-stranded RNAs

(dsRNAs), formed by complementary viral transcripts or through intramolecular base pairing, trigger potent antiviral de-fense through a process often referred to as RNA interference

(RNAi) (1). Increasing evidence suggested that this RNA-directed

viral immunity (RDVI) is initiated upon the processing of viral dsRNA into small interfering RNAs (siRNAs) by an RNase III-like

enzyme called Dicer (2). Subsequently, these virus-derived

siRNAs are incorporated into RNA-induced silencing complexes (RISCs), formed by Argonaute protein and cofactors, and serve as sequence guides for target viral transcript selection and

destruc-tion (1). In diverse organisms, dsRNA binding proteins are

essen-tial components of RDVI that contribute to the biogenesis or

function of virus-derived siRNAs (viRNAs) (3–8). In plants,

RDVI is further potentiated by secondary viRNAs processed from dsRNAs converted from cleaved viral transcripts by

RNA-depen-dent RNA polymerases (RdRPs) (9–12). Plant RDVI also involves

an intercellular signal that is believed to restrict systemic spread-ing of the invadspread-ing virus by primspread-ing an antiviral status prior to

virus arrival (13,14). Recently, siRNAs were found to serve as the

physical carrier of this mobile silencing signal in plants (15,16). In

the fruit fly RDVI also spreads systemically, although the

mecha-nism involved is different (17).

Since RNAi triggered by artificial dsRNA mechanistically reca-pitulates RDVI, previous studies on artificial dsRNA-triggered

RNAi inC. eleganshave significantly improved our understanding

of RDVI in nematode worms. Current models envision that the worm RDVI is initiated upon the processing of viral dsRNA into

primary viRNAs by the worm Dicer, called DCR-1 (4, 5, 18).

DCR-1 also processes precursor microRNAs (miRNAs) into ma-ture miRNAs, a class of endogenous small RNAs with important

roles in development (19). Whereas efficient processing of viral

dsRNA into primary viRNA requires a dsRNA binding protein, termed RDE-4 (RNAi defective 4), the processing of precursor miRNAs into mature miRNAs appears to be RDE-4 independent

(3,5,19–21). Interestingly, RDE-4, a key factor of RDVI, is also conserved in the fruit fly, whose genome is known to encode two Dicer proteins with dedicated function in the biogenesis of siRNA

and miRNA, respectively (7,20–23). So far, at least two closely

related AGO proteins, RDE-1 (RNAi defective 1) and C04F12.1,

have been found to play an important role in RDVI inC. elegans

(3–5,24). RDE-1 has the slicer activity that is required for the

maturation of RISC but not the cleavage of the target transcript

(25). Currently, how C04F12.1 contributes to RDVI remains

largely unknown. Efficient RDVI inC. elegansalso requires an

RdRP, termed RRF-1, which produces 22-nucleotide (nt)

single-stranded secondary siRNAs in a Dicer-independent manner (4,5,

26,27) (28). The fact that RDE-1 is required for production of

secondary siRNAs suggests that RRF-1 functions downstream of

RDE-1 in the same genetic pathway (28,29).

In addition to Dicer, AGO, and RdRP proteins, the worm RDVI also requires some unique components, such as DRH-1 (Dicer-related RNA helicase 1) and RSD-2 (RNAi spreading

de-fective 2) (5). DRH-1 encodes a putative DEAD box RNA helicase

that shares significant sequence homology with RIG-I (retinoic acid-inducible gene 1), a cytosolic virus sensor that initiates inter-feron-mediated viral immunity upon virus detection in mammals

(30). Intriguingly, although essential to RDVI, DRH-1 appears to

be dispensable in RNAi triggered by artificial dsRNAs, suggesting

a dedicated role of DRH-1 in antiviral defense (5). Previously, we

reported that viRNAs detected indrh-1mutants became

undetect-able indrh-1;rde-4double mutants (5), suggesting that DRH-1,

Received15 May 2013Accepted18 July 2013

Published ahead of print24 July 2013 Address correspondence to Rui Lu, ruilu@lsu.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01305-13

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together with RDE-1 and RRF-1, functions in the same genetic pathway as RDE-4.

RSD-2, together with SID-1 (systemic RNA interference defi-cient 1), was originally identified as one of the key factors that

enable systemic spreading of RNAi (31–33). A recent study

sug-gested that RSD-2 contributes to the accumulation of secondary

siRNAs in both exogenous and endogenous RNAi pathways (34).

Sincersd-2mutants displayed defects in transposon silencing

un-der unfavorable conditions, it is believed that RSD-2 plays an

im-portant role in maintaining chromosome integrity (35). SID-1

contains multiple putative transmembrane domains and has been

shown to be required for dsRNA intake (33,36,37). Interestingly,

a recent study suggested that at least two classes of silencing

dsRNAs that move betweenC. eleganstissues can act as or generate

the systemic silencing signal (38). Currently, whether SID-1

con-tributes to systemic RNAi by transporting these two classes of silencing RNAs remains largely unknown.

Previously, high concentrations of dsRNAs were found to

trig-ger efficient RNAi in the absence of RDE-4 (39). Currently, the

biological significance of this observation remains largely un-known. By analyzing viral replication and viRNA accumulation in

worm single and double mutants defective inrsd-2, we show here

that RSD-2, as an important component of worm RDVI, func-tions downstream of primary viRNA biogenesis. Most impor-tantly, with an increase in viral replication, primary viRNAs

accu-mulated to a higher level inrsd-2;rde-4double mutants than in

rsd-2single mutants, suggesting an rde-4-independent

mecha-nism for the biogenesis of primary viRNAs inC. elegans. We

fur-ther showed that RRF-1, togefur-ther with RDE-1, also contributed to therde-4-indepedent RDVI. Notably, although playing an essen-tial role in facilitating the spreading of RNAi triggered by artificial dsRNAs, SID-1 appears dispensable in RDVI targeting a natural

viral pathogen ofC. elegans.

MATERIALS AND METHODS

Worm genetics.The Bristol isolate ofC. elegans, N2, was used as the reference strain in this study. All other strains used in this study are de-rived from N2 and includedrh-1(tm1329),drh-2(ok951),rde-1(ne300), rde-4(ne337),rsd-2(tm1429andpk3307),sid-1(qt2), andrrf-1(pk1417) mutants. The genotypes ofdrh-1,drh-2,rsd-2, andrrf-1mutants were confirmed using PCR. The primers used to confirmtm1329anddrh-2 alleles were described previously (5). The primer pairs used to confirm the alleles are tm1429intf (CTACTAAACCGGTTACGTGT) and tm1429intb (CCACAGGGATTTTGTAGGGA) fortm1429and 1417EcoRV (AGGAG AGCATAGAAGGATATCA) and 1417PfoI (GTCACGGGGAGCTATTG TGAA) forpk1417. The genotypes ofrde-1,sid-1, andrde-4mutants were confirmed usingskn-1feeding RNAi combined with genomic DNA se-quencing. NGM plates seeded withEscherichia colistrain OP50 were used to maintain all worm strains. Single and double mutants containing var-ious transgenes were created through standard genetic crosses.

Plasmid constructs and transgenic worms. The construction of FR1gfp replicon was described previously (5). Psur5::RSD-2 was devel-oped by inserting RSD-2 coding sequence, amplified through reverse transcription-PCR (RT-PCR), into the LR50 vector described previously (40). The RSD-2 coding sequence in the resulting construct was con-firmed by DNA sequencing. The plasmid construct used to drivegfp dsRNA expression inE. coliwas described previously (40). The chromo-somal integrant for Psur5::RSD-2 was generated using a protocol de-scribed previously (40).

RSD-2 function rescue assay.The RSD-2 function rescue assay uti-lized ectopic expression of RSD-2 coding sequence inrsd-2null mutants (tm1429) carrying a nuclear transgene corresponding to the FR1gfp

rep-licon. Thesur-5promoter, known to be active in most of the worm cells (41), was used to drive the expression of RSD-2. A construct that directs mCherry expression in the pharynx tissue was used to produce a visual mark for the Psur5::RSD-2 transgene. The RSD-2 function rescue assay began with microinjection of Psur5::RSD-2 construct into the gonad of rsd-2;FR1gfp worms together with the mCherry reporter construct. Be-cause of the nature of worm transformation through gonad injection, most of the transgenic extrachromosomal arrays are randomly passed on to the next generations. As a result, some progenies are free of the extra-chromosomal arrays and can serve as internal negative control. Upon heat induction, which will initiate the replication of the FR1gfp replicon, trans-genic worms marked with red fluorescence in the head will show no or decreased green fluorescence if the Psur5::RSD-2 transgene is able to re-store the function of RSD-2 in thersd-2mutants.

RNA preparation and Northern analysis.Total RNA was prepared 48 h after heat induction using TRI reagent by following the manufacturer’s instructions (Sigma-Aldrich, Inc.). Small RNA species in the total RNA samples were further enriched using the mirVana kit (Ambion). For the detection of high-molecular-weight viral RNAs, 4␮g total RNA of each sample was fractionated in 1.2% agarose gel. For the detection of small RNA, including viRNAs and miRNAs, 10 to 20␮g of small RNA per sample was resolved in 15% acrylamide denaturing gel. After electropho-resis, all RNA samples were transferred onto a Hybond N⫹membrane (GE Healthcare Inc.) and UV cross-linked using 1.8⫻105J/cm2as the output power (SpectroLinker).

The blots for the detection of high-molecular-weight viral RNAs were hybridized with alkaline phosphatase-labeled cDNA probes prepared us-ing an AlkPhos direct labelus-ing module (GE Healthcare). The probes for FR1gfp transcript detection were prepared using cDNA fragments ampli-fied from the green fluorescent protein (GFP) coding sequence within FR1gfp. The probes used to detect Orsay virus RNA1 were prepared using cDNA fragments amplified, through RT-PCR, from the 3=end of Orsay virus RNA1 genome. The hybridization was performed at 65°C overnight. After washing at 65°C three times, the labeled cDNA probes were detected using CDP-Star detection reagent by following the manufacturer’s in-structions (GE Healthcare).

The detection of siRNAs and miRNAs adopted a protocol described previously (40). For the detection of FR1gfp-derived siRNAs, DNA oligo-nucleotides covering the entire subgenomic RNA sequence were labeled using the digoxigenin (DIG) oligonucleotide tailing kit (Roche Applied Science). DNA oligonucleotides with the sequence ATTGCCGTACTGA ACGATCTCA were labeled and used for the detection of miR-58. Four DNA oligonucleotides with sizes of 19, 21, 23, and 25 nt, respectively, were detected using DIG-labeled DNA oligonucleotides and, together with miR-58, served as size references.

Infectious filtrate preparation and Orsay virus inoculation.Orsay virus was maintained using the JU1580 isolate ofC. elegansat room tem-perature by following a protocol described previously (6). To prepare Orsay virus inoculum, JU1580 worms infected with Orsay virus were washed off, using M9 buffer, from slightly starved 10-cm agar plates. After spinning at low speed, the virus-containing supernatant was filtered through a 0.22-␮m filter unit (Millipore), and the filtrate was used to resuspend pelleted OP50E. colifor NGM plate seeding.

RNAi experiments.Feeding RNAi targeting the endogenous gene skin-1or the transgenegfpwas performed using a bacterial feeding pro-tocol described previously (42). NGM agar plates containing 5 mM iso-propyl-␤-D-thiogalactopyranoside (IPTG) and 50 mg/ml carbenicillin were used to prepare the feeding RNAi food. All feeding RNAi experi-ments were carried out at room temperature.

Imaging microscopy.The green and red fluorescence images were recorded using the same exposure for each set of images. A Nikon digital camera p7000 mounted on a Nikon SMZ1500 microscope was used to record all images.

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RESULTS

rsd-2is not essential for the biogenesis of primary viRNAs.rsd-2

ofC. eleganswas first implicated in RDVI through an RNAi screen that selected for genes whose downregulation by dsRNA feeding

led to loss of RDVI (5). To confirm that the loss of RDVI in worms

fed withrsd-2-targeting dsRNA was indeed caused by partial loss

ofrsd-2function, we examined the replication of FR1gfp replicon

in the mutant worms containing thetm1429orpk3307allele of

rsd-2. Thetm1429allele contains a 251-nt deletion, spanning from

nt 1376 to 1626 ofrsd-2cDNA, and is predicted to produce a

truncated RSD-2 that contains only the first 458 amino acid (aa)

residues of the 1,266-aa wild-type RSD-2. Conversely, thepk3307

allele contains a premature stop codon in the RSD-2 coding se-quence that caused the truncation of the last 542 aa of RSD-2.

Thus, bothrsd-2alleles are considered null alleles. The FR1gfp

replicon was developed from flock house virus (FHV) RNA1 by replacing the coding sequence of B2, the FHV-encoded RNAi sup-pressor, with that of green fluorescence protein (GFP). As a result, the replication of FR1gfp is subdued by RDVI in wild-type N2

worms but rescued in RDVI-defective worms, such as therde-1

mutants. As shown inFig. 1A, similar to therde-1mutants, both

rsd-2mutants supported elevated accumulation of FHV RNAs (Fig. 1A, compare lanes 3 and 4 to lanes 1 and 2), confirming a critical role of wild-type RSD-2 in FHV-targeting RDVI.

En-hanced accumulation was also observed in thersd-2mutants for

Orsay virus, a natural viral pathogen ofC. elegansthat is closely

related to FHV (Fig. 1B, compare lanes 2 and 3 to lane 1) (6). These

results together strongly suggested that RSD-2 plays an important role in RDVI.

To reconfirm that the loss of RDVI inrsd-2mutants indeed

resulted from thersd-2null alleles but not any other closely linked

genetic alleles, we checked if ectopic expression of wild-type

RSD-2 restores RDVI inrsd-2mutants (tm1429) containing the

FR1gfp replicon. The ectopic expression of wild-type RSD-2 cod-ing sequence was achieved through gonad microinjection of plas-mid construct containing RSD-2 coding sequence driven by the

sur-5promoter (Fig. 1C) (41). A plasmid construct that directs mCherry expression in pharynx tissue was coinjected to produce a

visual reporter for the transgene. As shown inFig. 1C, FR1gfp

replication, manifested as green fluorescence, was suppressed in

rsd-2mutants containing the Psur-5::RSD-2 transgene (compare the worms that showed red fluorescence in the heads to worms that did not). Consistent with this observation, FR1gfp transcripts were detected at a reduced level in worms containing the Psur-5::

RSD-2 transgene (Fig. 1D, compare lane 2 to lane 3). These results

confirmed that RSD-2 indeed plays an important role in worm RDVI.

rsd-2is known to contribute to the accumulation of secondary,

but not primary, endogenous siRNAs (34). To find out whether

RSD-2 contributes to RDVI through a similar mechanism, we

checked the accumulation of viRNAs inrsd-2mutants (tm1429)

using Northern blotting. Bothrde-1andrde-4play essential roles

in worm RDVI. However, virus-derived siRNAs accumulate to

readily detectable levels inrde-1 but not rde-4 mutants (3, 5).

Thus, as a control, the accumulation of viRNAs inrde-1andrde-4

mutants was also detected in this test. As shown inFig. 1E,

al-though not detectable in wild-type N2 worms orrde-4mutants,

FR1gfp-derived viRNAs were detected at high levels inrde-1

mu-tants, with a major band detected at the position corresponding to

23 nt. FR1gfp-derived siRNAs were also detected inrsd-2mutants

with a pattern similar to that inrde-1mutants but at a visibly lower

level. Previously, it has been shown that primary siRNAs pro-duced by worm Dicer are predominantly 23 nt in size and

accu-mulate at high levels inrde-1mutants (18,29,43,44). This result

suggested thatrsd-2is not essential for the biogenesis of primary

viRNAs.

rsd-2contributes torde-4-independent antiviral silencing.

We previously reported that FR1gfp-derived siRNAs detected in

drh-1mutants become undetectable indrh-1;rde-4double

mu-tants, suggesting thatdrh-1andrde-4function in a linear genetic

pathway (5). To find out whetherrsd-2also functions in the same

genetic pathway, we compared the accumulation of primary

viRNAs in double mutants corresponding todrh-1;rde-4,rsd-2;

rde-4, andrsd-2;drh-1. As shown inFig. 2A, although not

detect-able in drh-1;rde-4 double mutants, FR1gfp-derived primary

siRNAs were detected in bothrsd-2;drh-1andrsd-2;rde-4double

mutants (compare lane 3 to lanes 1 and 4). Most importantly, an enhanced replication for both Orsay virus and FR1gfp was

ob-served inrsd-2;rde-4andrsd-2;drh-1double mutants compared to

rde-4anddrh-1single mutants, respectively (Fig. 2BandC, left).

These observations together suggested an rde-4-independent

mechanism that directs antiviral silencing in anrsd-2-dependent

manner inC. elegans. RDE-1 specifically recruits primary siRNAs,

but not secondary siRNAs, for target RNA selection and is

be-lieved to function downstream of RDE-4 (29). Thus, we expected

to see enhanced viral replication inrsd-2;rde-1double mutants

compared torde-1single mutants ifrsd-2indeed contributes to

RDVI in anrde-4-independent manner. As shown inFig. 2Band

C, right, enhanced viral replication was observed, although to a

lesser extent, inrsd-2;rde-1 double mutants compared torde-1

single mutants. As expected, FR1gfp-derived siRNAs accumulated inrsd-2;rde-1double mutants (Fig. 2A, lane 2). Taken together,

these results strongly suggest that there is anrde-4-independent

butrsd-2-dependent pathway contributing to RDVI inC. elegans.

rsd-2andrrf-1function in the same RDVI pathways.rrf-1

encodes an RdRP that initiatesde novosynthesis of secondary

siRNAs using the targeted transcript as the template in RNAi (26,

27,45,46). To find out whetherrsd-2functions in the same genetic

pathway asrrf-1, which is known to function downstream ofrde-4

andrde-1(28,29), we checked viral replication inrsd-2;rrf-1 dou-ble mutants compared to the single mutants. We expected to see

enhanced viral replication in the double mutants ifrsd-2andrrf-1

function in separate genetic pathways. However, as shown inFig.

3A, an enhancement in the replication of FR1gfp or Orsay virus

was not observed in the double mutants compared to the single mutants (compare lane 4 to lanes 2 and 3 in the left panel and lane 5 to lanes 3 and 4 in the right panel). Consistently, FR1gfp-derived 23-nt primary siRNAs accumulated to comparable levels in the

rsd-2;rrf-1double mutants and in the single mutants (Fig. 3B, compare lanes 1 and 2 to lane 7). This result, together with the

results shown inFig. 2, suggest that bothrrf-1andrsd-2function

in two independent genetic pathways that mediaterde-4

-depen-dent andrde-4-independent antiviral silencing, respectively.

rrf-1contributes torde-4-independent antiviral silencing.

To confirm thatrrf-1indeed contributes torde-4-independent

RDVI, we first compared the replication of both FR1gfp and Orsay

virus inrrf-1;rde-4andrrf-1;drh-1double mutants to that in the

single mutants corresponding torrf-1,drh-1, andrde-4. We

ex-pected to see enhanced viral replication in the double mutants if

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FIG 1rsd-2is not essential for the biogenesis of primary viRNA. (A) Northern blot showing the accumulation of FR1gfp genomic and subgenomic transcripts in different genetic backgrounds. Total RNA was extracted from heat-induced worms 48 h after heat induction. The viral transcripts were detected using probes prepared from full-lengthgfpcDNA. RNA1, genomic RNA of FR1gfp; RNA3, subgenomic RNA of FR1gfp; rRNA, methylene blue-stained rRNA serving as an equal loading control. (B) Accumulation of Orsay virus RNA1 (ovRNA1) in wild-type N2 worms andrsd-2mutants carrying thetm1429orNL3307allele as indicated. Total RNA was prepared 72 h after viral inoculation. Orsay virus RNA1 was detected using cDNA probes prepared using a cDNA fragment amplified from the 3=end of Orsay virus RNA1. (C) The upper panel shows the structure of Psur-5::rsd-2. Psur-5, the promoter for the endogenous genesur-5; RSD-2, the coding sequence of wild-typersd-2; UTR, the 3=-end untranslated region ofunc-54. The lower panel shows visualization of green fluorescence inrsd-2mutants (tm1429allele) carrying the FR1gfp replicon transgene and extrachromosmal array corresponding to Psur-5::RSD-2 48 h after heat induction. Shown here are merged images recorded, using the same exposure, under red fluorescence and green fluorescence. Worms showing red color are transgenic for Psur-5::RSD-2. (D) Accumulation of FR1gfp transcripts in wild-type N2 worms,drh-1mutants, andrsd-2mutants (tm1429) with or without the extrachromosomal assay derived from the Psur-5::RSD-2 construct. RSD-2wtdenotes the Psur-5::RSD-2 transgenic array. (E) Accumulation of FR1gfp-derived siRNAs in wild-type N2 worms and RDVI-defective mutants as indicated. Lane M, four DNA oligonucleotides with sizes of 19, 21, 23, and 25 nt detected and used, together with miR-58, as size references.

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rrf-1indeed contributes torde-4-independent RDVI. As shown in

Fig. 4AandB, both FR1gfp and Orsay virus replicated to higher levels in the double mutants than in the single mutants. Most importantly, we detected FR1gfp-derived primary siRNAs in the

rrf-1;rde-4andrrf-1;drh-1double mutants, in contrast to what was

seen indrh-1;rde-4double mutants (Fig. 4C, compare lanes 2 and

4 to lane 1). As shown inFig. 4AandB, an increase in FR1gfp

replication was observed in therrf-1;rde-1double mutants

com-pared to the single mutants, although such an increase was not clear for Orsay virus. As expected, FR1gfp-derived primary

siRNAs also accumulated inrde-1;rrf-1double mutants (Fig. 4C,

lane 3). These observations confirmed thatrrf-1plays a role in

rde-4-independnet RDVI.

rde-1plays a role inrde-4-independent RDVI.Previously,

en-hanced viral replication was observed indrh-1;rde-1, but not in

drh-1;rde-4, double mutants compared to the single mutants (5),

suggesting a role forrde-1inrde-4-independent RDVI. To find

out whether rde-1 indeed contributes to rde-4-independent

RDVI, we compared both viral replication and primary viRNA

accumulation inrde-1;rde-4double mutants and the single

mu-tants. We expected to see enhanced viral replication and

accumu-lation of primary viRNAs ifrde-1 indeed plays a role inrde-4

-independent RDVI. As shown inFig. 5A, both Orsay virus and

FR1gfp replicated to higher levels in the double mutants than the single mutants. Most importantly, FR1gfp-derived primary

siRNAs, although not detectable in thedrh-1;rde-4double

mu-tants, were readily detected inrde-1;rde-4double mutants (Fig.

5B, compare lane 3 to lane 2). These results together suggested that

rde-1also plays a role inrde-4-independent RDVI.

sid-1is not required for RDVI targeting Orsay virus.InC. elegans, RNAi triggered by artificial long dsRNA spreads systemi-cally and causes sequence-specific silencing in distant tissues that

are not exposed to the dsRNA trigger (32). This observation raised

the question of whether, like that in plants (13,14), worm RDVI

involves an intercellular signal that prevents viruses from spread-ing systemically. Previously, we have shown that RDVI triggered

by an FHV replicon was not compromised insid-1mutants (40)

defective in RNAi spreading (33,36,47). However, since potent

RDVI will have been triggered in every individual cell that con-tains the replicon transgene, a systemic silencing signal may not be needed to silence such a viral replicon that is not known to move systemically. To find out whether worm RDVI indeed spreads systemically to restrict virus spread under natural conditions, we compared Orsay virus infection between wild-type N2 worms and

worms defective insid-1. As shown inFig. 6, an increase in Orsay

virus replication was not detected insid-1mutants compared to

that in wild-type N2 worms (compare lane 4 to lane 2). Based on

FIG 2rsd-2contributes torde-4-independent antiviral silencing. (A) Accu-mulation of FR1gfp-derived siRNAs in double mutants defective in RDVI. Experimental details are the same as those described in the legend toFig. 1E. (B) Accumulation of Orsay virus RNA1 (ovRNA1) in wild-type N2 worms and RDVI-defective mutants carrying single or double mutations as indicated. See the legend toFig. 1Bfor experimental details. (C) Accumulation of FR1gfp transcripts in various genetic backgrounds as indicated. See the legend toFig. 1Afor experimental details.

FIG 3rsd-2andrrf-1function in the same antiviral silencing pathways. (A) Accumulation of FR1gfp and Orsay virus transcripts in various genetic back-grounds as indicated. See the legend toFig. 1Afor experimental details. (B) The accumulation of FR1gfp-derived siRNAs in single or double mutants con-tainingrsd-2and/orrrf-1null alleles. See the legend toFig. 1Efor experimental details.

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this observation, we concluded that sid-1-mediated systemic

RNAi is not required for restricting Orsay virus infection inC.

elegans.

DISCUSSION

rsd-2was first identified as a gene that enables RNAi spreading

from soma to germ line (31). A role ofrsd-2in RDVI was then

revealed in a genetic screening that utilized RNAi-mediated gene

knockdown to phenocopy genetic mutants (5). Recently, a genetic

study further suggested thatrsd-2helps maintain normal

chromo-somal function, such as transposon control, under unfavorable

conditions (35). Currently, howrsd-2contributes to those

biolog-ical processes remains poorly understood.rsd-2is unique to

nem-atode worms, such asC. elegans, which are known to encode the

single Dicer required for the biogenesis of both siRNAs and

miRNAs. Thus, the functional and mechanistic study ofrsd-2is

expected not only to shed light on the mechanism by whichrsd-2

helps maintain chromosomal integrity in response to environ-mental stresses but also to reveal the uniqueness of RDVI in

single-Dicer invertebrates. Here, we show thatrsd-2functions in two

parallel genetic pathways that mediate antiviral silencing inrde-4

-dependent and rde-4-independent manners. In Arabidopsis,

dsRNA binding protein regulates RDVI by facilitating the

process-ing of viral dsRNA into siRNAs by one of the four Dicers (8,48). In

Drosophila, the RDE-4 homologue R2D2 contributes to the

func-tion of viRNAs produced by one of the two drosophila Dicers (7,

22,49). Therefore, by demonstrating therde-4-indpendent

mech-anism for primary viRNA biogenesis, our study, for the first time, suggested that RDVI in single-Dicer invertebrates can be initiated in the absence of a dsRNA binding protein.

Previously, it has been shown that dsRNA of high

concentra-tion triggers RNAi inC. elegansin the absence of RDE-4 (39). This

observation suggests that viruses, as powerful replicators that rap-idly produce large amounts of dsRNA replication intermediates, trigger RDVI in the absence of RDE-4. Here, we show that with an increase in viral replication level, primary viRNAs were detected at

high levels inrde-4mutants containing eitherrsd-2or therrf-1

null allele, suggesting anrde-4-independent mechanism for the

FIG 4rrf-1contributes torde-4-independent antiviral silencing. (A) Accu-mulation of Orsay virus RNA1 (ovRNA1) in wild-type N2 worms and RDVI-defective mutants carrying single or double mutations as indicated. (B) Accu-mulation of FR1gfp transcripts in wild-type N2 worms and RDVI-defective mutants carrying single or double mutations as indicated. (C) Accumulation of FR1gfp-derived siRNAs in double mutants, as indicated, defective in RDVI.

FIG 5rde-1contributes torde-4-independent antiviral silencing. (A) Viral replication is further enhanced inrde-1;rde-4double mutants compared to the single mutants. Shown here is the accumulation of Orsay virus (left panel) and FR1gfp transcripts in single or double mutants containingrde-1and/orrde-4 null alleles. (B) Accumulation of FR1gfp-derived primary siRNAs in single or double mutants containingrde-1and/orrde-4null alleles.

FIG 6sid-1is not required for RDVI inC. elegans. Shown is the accumulation of Orsay virus RNA1 in wild-type N2 worms andsid-1mutants. See the legend toFig. 1Bfor experimental details.

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generation of primary viRNAs (Fig. 2and4). Apparently such an increase in viral replication and viRNA accumulation cannot be

attributed to residualrde-4function in therde-4allele used in this

study, since FR1gfp-derived siRNAs were not detected inrde-4

single ordrh-1;rde-4double mutants under the same conditions

(5) (Fig. 1E,2A,4C, and5B). Thus, by demonstrating the

exis-tence of anrde-4-independent pathway for antiviral silencing, we

explained why dsRNAs of high levels triggers RNAi in the absence

of RDE-4 inC. elegans(39). Notably, although readily detectable

in worms, such asrde-1,rrf-1, andrsd-2mutants, that are defective

in secondary viRNA biogenesis, virus-derived primary siRNAs were hardly detectable in wild-type N2 worms or worms defective inrde-4(Fig. 1E,2A,3C, and4C). This may simply reflect the fact

that antiviral silencing in bothrde-4-dependent andrde-4

-inde-pendent mechanisms is amplified by the secondary viRNAs and, as a result, much less viral dsRNA will be processed into primary viRNAs by Dicer in the presence of secondary viRNAs.

Notably, our observations suggested thatrde-1also plays a role

inrde-4-independent RDVI similar to that ofrsd-2(Fig. 5).

Inter-estingly, an increase in FHV replication level was observed in

rde-1;rsd-2double mutants compared torde-1single mutants.rde-1

probably plays a major role inrde-4-dependent RDVI but a minor

role inrde-4-independent RDVI, and as a result, antiviral silencing

inrde-1mutants is further compromised in the presence of an

rsd-2null allele. Nevertheless, by analyzing the accumulation of viral transcripts and primary viRNAs in various worm single and double mutants defective in RDVI, our study demonstrated, for

the first time, that in addition to the conventionalrde-4

-depen-dent pathway, there is anrde-4-independent pathway for antiviral

silencing inC. elegans(Fig. 7). Previously, we showed that another

RDE-1-related worm AGO, termed C04F12.1, plays a role in RDVI. It would be interesting to test whether C04F12.1 plays a

role inrde-4-independent RDVI.C. elegansis known to encode a

large number of AGO proteins. Thus, testing therde-4

depen-dence of C04F12.1 may allow us to address the question of whether different worm AGO proteins specifically function in

mechanistically distinct RDVI pathways inC. elegans.

rsd-2is known to be required for the accumulation of

second-ary, but not primsecond-ary, endogenous siRNAs (34). Currently, it

re-mains unclear whetherrsd-2contributes to RDVI through a

sim-ilar mechanism. Here, we show that FR1gfp-deirved primary

siRNAs accumulated inrsd-2with a pattern similar to that inrrf-1

orrde-1single mutants, which are known to accumulate primary

viRNAs (Fig. 1E,2A, and3B). This finding suggests that, similar to

that in endogenous RNAi,rsd-2enhances cell-autonomous

anti-viral silencing by contributing to the accumulation of secondary,

but not primary, viRNAs. This finding explained why rsd-2

mutants are resistant to low doses but sensitive to high doses of

dsRNAs (34). Presumably, when the trigger dsRNAs are present at

high concentrations, sufficient primary siRNAs will be produced to trigger efficient RNAi in the absence of secondary siRNAs. However, when the trigger dsRNAs are introduced at low levels, efficient RNAi will require those dose-sensitive RNAi factors, such as RSD-2, RDE-10, and RDE-11, which amplify RNAi by

promot-ing the production/accumulation of secondary siRNAs (34).

No-tably, the replication of both FHV and Orsay virus was further

enhanced inrsd-2mutants compared to that inrrf-1mutants,

suggesting thatrsd-2also contributes to the function of primary

viRNAs. RSD-2 contains 3 tandem domains of unknown function at the N terminus. Functional characterization of these domains

may yield further insight into the mechanistic basis of RSD-2 in RDVI.

Like that in plants, RNAi inC. elegansinvolves an intercellular

signal that is responsible for systemic spread of RNAi (32,50,51).

A recent study further suggested that long dsRNAs or their pro-cessed intermediates are the physical carrier of this intercellular

signal (38). Consistent with this finding, systemic spreading of the

intercellular signal requires the transmembrane protein SID-1 that transports dsRNAs without a size preference and may

facili-tate the intercellular transportation of the silencing signal (33,36,

47). Previously, it has been shown that SID-1 is dispensable for

RDVI triggered by artificial viruses, such as vesicular stomatitis

virus and FHV (4,40). Currently, whether SID-1 contributes to

RDVI under natural conditions remains largely unknown. To ad-dress this question, we compared Orsay virus infection between

wild-type N2 worms andsid-1mutants and found that Orsay virus

infection was not further enhanced in thesid-1mutants (Fig. 6).

Worm RDVI probably involves an intercellular silencing signal that is transported through an SID-1-independent mechanism. Consistent with this hypothesis, SID-1 was shown to be

dispens-able for the export of the mobile silencing signal inC. elegans(52).

Alternatively, SID-1, as a key component of systemic RNAi, may be active in nonintestinal tissues, and as a result its role in systemic

FIG 7Working model forrde-4-dependent andrde-4-independent antiviral silencing inC. elegans. WAGO, worm-specific AGO (29). Here, we propose that the processing of viral dsRNAs into primary viRNAs occurs in an rde-4-dependent andrde-4-independent manner.drh-1contributes to the rde-4-dependent, but notrde-4-independent, antiviral silencing. We believe that rsd-2andrrf-1contribute to bothrde-4-dependent andrde-4-independent antiviral silencing, whilerde-1plays a major function in therde-4-dependent antiviral silencing and a minor function inrde-4-independent antiviral silenc-ing. We also believe that whereas the major function ofrrf-1is to direct the synthesis of secondary viRNAs,rsd-2contributes to the function of primary viRNAs, thereby initiating the synthesis of secondary viRNAs. As proposed previously, some WAGOs may direct the cleavage of cognate viral transcripts using the secondary viRNAs as a sequence guide (29).

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RDVI cannot be identified using Orsay virus, which mainly targets

the intestine cells for replication (6). Moreover, probably due to

the lack of an RDVI suppression activity, Orsay virus is known to

be highly sensitive to RDVI (28). Thus, it is possible that the

rep-lication of Orsay virus insid-1mutants is suppressed by

intracel-lular antiviral silencing, making the intercelintracel-lular antiviral silenc-ing redundant in keepsilenc-ing this particular virus under control.

Unlike RDE-4, which plays an important role in the biogenesis of endogenous siRNAs, DRH-1 appears to be a dedicated factor of

worm RDVI (5,20). DRH-2 shares significant sequence homology

with DRH-1 but appears to negatively regulate worm RDVI (5).

These observations together raised the interesting question of whether DRH-2 specifically targets and regulates the function of

DRH-1, thereby negatively regulating therde-4-dependent RDVI.

Apparently, by addressing this question we will be able to find out whether worms have evolved such a mechanism that would allow for specific regulation of RDVI without affecting the normal cel-lular function under unfavorable environments.

ACKNOWLEDGMENTS

We thank the Caenorhabditis Genetics Center (CGC) for some of the worm strains used in this study. This work would not have been possible without the JU1580 worm strain and the Orsay virus from M.-A. Félix, E. A. Miska, and D. Wang.

This work was supported by the Board of Regents, Louisiana [LEQSF(2011-14)-RD-A-09 and LEQSF-EPS(2012)-PFUND-277], and the NIH (1R03AI092159-01A1).

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Figure

FIG 1 rsd-2transgene and extrachromosmal array corresponding to Psur-5::RSD-2 48 h after heat induction
FIG 3 rsd-2The accumulation of FR1gfp-derived siRNAs in single or double mutants con-tainingAccumulation of FR1gfp and Orsay virus transcripts in various genetic back-grounds as indicated
FIG 4 rrf-1mulation of Orsay virus RNA1 (ovRNA1) in wild-type N2 worms and RDVI-defective mutants carrying single or double mutations as indicated
FIG 7 Working model for rde-4-dependent and rde-4-independent antiviralsilencing in C

References

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