Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

JOURNAL OF VIROLOGY, June 2005, p. 7217–7226 Vol. 79, No. 110022-538X/05/$08.00�0 doi:10.1128/JVI.79.11.7217–7226.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That BindsDouble-Stranded RNAs without Size Specificity‡

Zsuzsanna Merai,1 Zoltan Kerenyi,1 Attila Molnar,1† Endre Barta,1 Anna Valoczi,1 Gyorgy Bisztray,2Zoltan Havelda,1 Jozsef Burgyan,1 and Daniel Silhavy1*

Agricultural Biotechnology Center, Godollo, Hungary,1 and Department of Genetics and Horticultural Plant Breeding,Budapest University of Economic Sciences and Public Administration, Budapest, Hungary2

Received 1 September 2004/Accepted 17 January 2005

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determinedby small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requiresexpression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorlyunderstood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA(dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs(siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution ofsilencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closestrelatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is anefficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAsand double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppresssilencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machin-ery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibitsinverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAsaccumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing bysequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressorsdiverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the commonancestor of aureusvirus-tombusvirus movement proteins.

RNA silencing (also termed posttranscriptional gene silenc-ing in plants and RNA interference in animals) is a conservedeukaryotic gene inactivation system that plays regulatory rolesin many biological processes including development, mainte-nance of genome stability, and antiviral responses (2, 6, 12, 25,54). RNA silencing is induced by accumulation of double-stranded RNAs (dsRNAs). dsRNAs are first processed by anRNase III-like nuclease called DICER (in plants termedDICER-LIKE, or DCL) into short (21 to 25 nucleotide [nt])RNAs, and then these short RNAs incorporate and guidedifferent silencing effector complexes to homologous nucleicacids for suppression (2, 6, 12, 16, 25, 54). In plants, RNAsilencing acts at both single-cell (cell-autonomous silencing)and at whole-plant (systemic silencing) levels. Cell-autono-mous silencing inactivates genes in the cells in which dsRNAsaccumulated. Moreover, cell-autonomous silencing generatesmobile silencing signals that confer suppression of homologousmRNAs in neighboring cells (short distance) and in distanttissues (long-distance systemic silencing) (29, 31, 32, 56).

DICERs can process dsRNAs into two functionally differentsmall RNAs, micro-RNAs (miRNAs) and small interfering

RNAs (siRNAs). miRNAs are involved in the control of manyendogenous protein-encoding mRNAs, while siRNAs mainlyplay a role in suppression of molecular parasites such as trans-posons, transgenes, and viruses (2, 6, 12, 16, 25). In Arabidop-sis, matured miRNAs are 21- to 22-nt-long single-strandedRNAs (ssRNAs) that are excised from endogenous hairpinRNA precursors by DCL1 (60). siRNAs, which are generatedfrom long dsRNAs, accumulate as short (21 to 22 nt) or long(23 to 25 nt) double-stranded molecules having 2-nt 3� over-hangs.

miRNA- and short siRNA-mediated silencing pathwaysshare components. Both types of small RNAs are incorporatedinto and guide a multicomponent nuclease (RNA-induced si-lencing complex, or RISC) to homologous mRNAs for sup-pression. RISC cleaves targeted mRNA in the case of (near)perfect base-pairing between mRNA and guide RNA. Whenthe guide RNA is only partially complementary to the mRNA,RISC mediates translational repression (2, 6, 12). siRNAs alsoguide other silencing effector complexes. In addition to RISC,short siRNAs are supposed to provide sequence specificity fora host-encoded RNA-dependent RNA polymerase that trans-forms homologous mRNAs into dsRNAs, thus amplifying si-lencing. Moreover, short siRNAs could be involved in short-distance systemic silencing (15, 18). Long siRNAs would play arole in long-distance systemic silencing (15) and in transcrip-tional silencing by directing the histone/DNA methylation ofhomologous DNA (7, 15, 25, 51).

RNA silencing plays an antiviral role in plants, in insects,and perhaps in other eukaryotes (2, 13, 22, 37, 46, 59). DCL2and perhaps other DCL enzymes generate viral siRNAs from

* Corresponding author. Mailing address: Agricultural Biotechnol-ogy Center, Plant Science Institute, P.O. Box 411, H-2101 Godollo,Hungary. Phone: 36 28 526 194. Fax: 36 28 526 145. E-mail: [email protected].

† Present address: The Sainsbury Laboratory, John Innes Centre,Colney Lane, Norwich NR4 7UH, United Kingdom.

‡ Supplemental material for this article may be found at http://jvi.asm.org/.

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double-stranded replicative intermediates of RNA viruses (61)or from hairpins of viral mRNAs (48). Viral siRNAs couldtarget RISC to viral mRNAs for suppression. As RNA-depen-dent RNA polymerase mutant plants are more susceptible tocertain viruses, it is likely that silencing amplification is also animportant antiviral pathway against particular viruses (9, 11,30, 61). Importantly, virus-induced silencing acts as a short-distance systemic defense system. Viral siRNAs might spread10 to 15 cell layers and activate silencing in still noninvadedneighboring cells, thus limiting the extent of virus invasion (15,17, 18, 43).

To counteract RNA silencing, most plant viruses expresssilencing suppressor proteins. Viral suppressors target differ-ent steps of the silencing response (22, 39, 46, 57, 59). Al-though many suppressors have been identified, the molecularbasis of silencing inhibition and the evolution of suppressorsare poorly understood.

Members of the Tombusviridae plant virus family have ico-sahedral particles and linear, small, single-stranded positive-sense RNA genomes (42). Different genera of Tombusviridaeexpress distinct suppressors. The coat protein (CP) of Turnipcrinkle virus (Tombusviridae, Carmovirus) has multiple func-tions; in addition to forming a capsid, it also suppresses silenc-ing (36, 52, 64). By contrast, the 19-kDa suppressor protein(P19) of tombusviruses (Tombusviridae, Tombusvirus) is appar-ently required only for silencing inhibition, as it is dispensablefor replication, movement, or virion formation (34, 35, 45, 57).P19 is a specific dsRNA binding protein, which binds dsRNAssize selectively (55, 62). P19 forms strong complexes withdsRNAs having 19-nt duplex regions, thus it binds siRNAs invitro (45, 55, 62) and in vivo (8, 14, 21). Importantly, it bindsshorter or longer dsRNAs with much weaker affinity. It isproposed that in tombusvirus-infected cells, P19 sequesterssilencing-generated siRNAs, thereby suppressing antiviral si-lencing responses (21, 45). Indeed, in Cymbidium ringspot virus(CymRSV; Tombusviridae, Tombusvirus)-infected plants, viralsiRNAs are present in complex with P19 (21). To better un-derstand the evolution of silencing suppression within Tom-busviridae, we wanted to identify and analyze the silencingsuppressor of Pothos latent virus (PoLV), the type species ofaureusviruses (Tombusviridae). The genome organization ofaureusviruses is identical to that of tombusviruses, but theAureusvirus genome is significantly smaller and the sequencesimilarity between the two genera is limited (26, 28, 41) (Fig.1A). The PoLV open reading frame (ORF) 5-encoded 14-kDaprotein (P14), like the tombusvirus ORF 5-encoded P19, in-creases the severity of viral symptoms (symptom determinant)(40). Infection of Nicotiana benthamiana plants with eitherPoLV�14, a mutant PoLV that fails to express P14 (40) (Fig.1B), or a mutant CymRSV that is unable to express P19(Cym19stop) leads to similar recovery phenotypes (48). As therecovery phenotype is supposed to be the manifestation ofvirus-induced systemic silencing (1, 17, 37, 48), it has beensuggested that P14, like P19, operates as a silencing suppressor(38, 40). Interestingly, although the genomic positions of P14and P19 are identical and both proteins are symptom determi-nant, no significant sequence homology has been detected be-tween P19 and P14 (40).

Here we report that PoLV P14 is an efficient suppressor ofboth virus- and transgene-induced silencing. P14 is a dsRNA

binding protein that binds dsRNA in vitro without obvious sizeselection. The potential mechanism of P14-mediated silencingsuppression and the evolution of P14 and P19 suppressors willbe discussed.

MATERIALS AND METHODS

Plant materials and Agrobacterium tumefaciens infiltration. Transgenic N.benthamiana carrying the green fluorescent protein (GFP) ORF was describedpreviously (4). The A. tumefaciens infiltration method was carried out as de-scribed previously (56). For coinfiltration, equal volumes of respective A. tume-faciens cultures (optical density at 600 nm, 0.25) were mixed before infiltration.

Silencing suppression assay and GFP imaging. The RNA silencing suppres-sion assay was carried out as described previously (58). Visual detection of GFPfluorescence was performed using a 100-W handhold long-wave UV lamp (BlackRay model B 100AP; UV Products, Upland, CA).

Plasmid constructs. The infectious cDNA clones of PoLV, PoLV�14 (40),CymRSV (10), and Cym19stop (48) were described previously.

Silencing suppressors for agroinfiltration assays were cloned into pBIN61S(45). P19 and Sigma3 binary constructs were described previously (24, 45). P14was PCR amplified with P14 5� and 3� primers corresponding to the first and last20 nt of P14. The P14 5� primer carried an additional BamHI site, while the P143� primer contained an additional SalI site. The PCR product was cloned inreverse orientation into SmaI-cleaved pBluescript KS vector (KS-P14). To createthe P14 binary construct, the BamHI-SalI fragment was cloned from KS-P14 intopBin61S. G-P14 was generated by cloning the BamHI fragment from KS-P14into Gex-2T, and then the sense orientation was selected. PVX-P14 was gener-ated by cloning a refilled BamHI-SalI fragment from KS-P14 into EcoRV-digested pP2C2S.

In vitro RNA transcription and plant inoculation. In vitro transcription fromPoLV, PoLV�14 (40), CymRSV, Cym19stop, and PVX cDNA clones and inoc-ulation of RNA transcripts onto plants were performed as described previously(10, 48).

FIG. 1. PoLV P14 is a symptom determinant. (A) Schematic rep-resentation of genome organization of a tombusvirus (CymRSV) andPoLV, the type species of aureusviruses. K, kilodalton. (B) PoLV P14increases symptom severity. N. benthamiana plants were infected withPoLV and PoLV�14, a mutant virus that fails to express P14.PoLV�14 infection results in a recovery phenotype characterized bylow virus titers and mild symptoms in the upper leaves. Plants weregrown at 21°C.

7218 MERAI ET AL. J. VIROL.

Protoplast preparation and inoculation. Protoplasts were isolated from N.benthamiana and transfected with in vitro transcripts of PoLV or PoLV�14 (10,20).

Protein separation and Western analysis. Proteins were separated in a 12%sodium dodecyl sulfate-polyacrylamide gel and than transferred onto a HybondC Extra filter (Amersham Pharmacia Biotech). Rabbit P14 polyclonal antibodies(anti-P14) raised against the GST-P14 fusion protein were used for Westernanalysis.

RNA gel blot analysis. The same total RNA extract was used for high- andlow-molecular-weight RNA gel blot analysis. RNA extraction and RNA gel blotanalysis were carried out as described previously (48). PCR fragments labeledwith the random priming method were used for Northern analyses of high-molecular-weight RNAs. Radioactively labeled in vitro transcripts correspondingto the positive strand of virus RNA and antisense strand of GFP were used asprobes for Northern analyses of low-molecular-weight RNAs. Labeling was car-ried out as described previously (48).

Gel mobility shift assay. Synthetic siRNAs were labeled with T4 PNK.[�-32P]UTP-labeled in vitro RNA transcripts were used as long RNA probes.Transcripts were produced from a T7-T3 Bluescript PCR fragment by the T7 andT3 RNA polymerases, respectively (48).

To generate double-stranded siRNAs, 5�-phosphorylated complementarystrand siRNAs in 5 times molar excesses were added to labeled single-strandedsiRNAs, and then siRNAs were heated and annealed. To generate long dsRNAs,a 1:1 mixture of labeled T7 and T3 in vitro transcripts were heated and annealed.GST, G-P14, and G-P19 proteins were expressed and purified according to themanufacturer’s protocols (Amersham Pharmacia Biotech).

To prepare protein extract, 0.25 g leaf tissue was grinded in 1 ml band shiftbuffer (83 mM Tris-HCl [pH 7.5], 0.8 mM MgCl2, 66 mM KCl, 100 mM NaCl,and 10 mM dithiothreitol), and then this crude extract was centrifuged twice for15 min at 15,000 � g. The supernatant was frozen in aliquots at �70°C. In abinding reaction, labeled dsRNA (in a 1 nM concentration) was incubated withextract containing �2 �g total protein. Binding reaction and mobility shift assayswere carried out as described previously (48), except that 8 U RNasin was addedto each 10-�l reaction mixture. For long dsRNA direct competition assays,0.02% Tween 20 was added to the binding buffer.

Computer analysis. Multiple alignments of RNA and deduced protein sequenceswere carried out with ClustalX (53). Relationships among proteins were analyzed bythe bootstrap parsimony (47) and maximum-likelihood methods (44).

RESULTS

P14 is a silencing suppressor. Expression of a silencingsuppressor from a heterologous virus intensifies viral symp-

toms (57). To test whether P14 is a silencing suppressor, weinfected N. benthamiana and Nicotiana clevelandii plants withPotato virus X (PVX) that expressed P14 (PVXP14) and withPVX as a control. While PVX infection caused only mildsymptoms on both hosts, PVXP14-infected plants showedstrong symptoms including stunting and necrosis along theveins (Fig. 2A and data not shown). Findings that P14 in-creased PoLV and PVX symptoms suggest that P14 is a sup-pressor of virus-induced silencing.

Viral silencing suppressors can be identified in sense trans-gene-induced silencing assays (58). Infiltration of the leaves ofan N. benthamiana plant with an Agrobacterium carrying aplasmid that expresses GFP (35SGFP) leads to strong, tran-sient GFP expression, but it also triggers GFP silencing (4, 58).Cell-autonomous GFP silencing is manifest as a weakening ofgreen fluorescence, a decrease in the level of GFP mRNA, andan accumulation of both short (21 to 22 nt) and long (23 to 25nt) GFP-specific siRNAs in the infiltrated patches (Fig. 2B andC). However, GFP silencing is partially or fully inhibited if35SGFP is coinfiltrated with a second Agrobacterium express-ing a silencing suppressor. To determine whether P14 sup-presses sense transgene-induced silencing, N. benthamianaplants were coinfiltrated with 35SGFP and with a secondAgrobacterium expressing P14 (P14). As the green fluorescencewas much stronger and lasted longer in coinfiltrated patchesthan in leaves infiltrated with 35SGFP alone (Fig. 2B), weconcluded that P14 inhibited sense transgene-induced cell-autonomous silencing.

The effect of silencing suppressors on accumulation of shortand long siRNAs depends on the targeted step of a particularsuppressor (15, 18). To investigate which step of silencing istargeted by P14, we studied the accumulation of GFP mRNAand the GFP-derived siRNAs in 35SGFP- and P14-coinfil-trated leaves. As Fig. 2C shows, in coinfiltrated leaves, GFPmRNAs accumulated to much higher levels than in leaves that

FIG. 2. P14 is an RNA silencing suppressor. (A) P14 increases the symptoms of PVX. N. benthamiana plants were infected with PVX orPVXP14, a modified PVX that expressed P14. (B) P14 suppresses sense transgene-induced RNA silencing. Leaves of N. benthamiana plants wereinfiltrated with an Agrobacterium (35SGFP) expressing GFP (�) or were coinfiltrated with 35SGFP and a second Agrobacterium expressing P14(P14). Photos were taken at 6 d.p.i. (C) Effect of P14 on accumulation of GFP mRNAs and GFP-derived siRNAs (siRNS).

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were infiltrated with 35SGFP alone. Moreover, neither shortnor long GFP-specific siRNAs could be detected in coinfil-trated leaves. These data indicate that P14 interferes withsense transgene-induced cell-autonomous silencing by prevent-ing the accumulation of siRNAs. The effects of a silencingsuppressor on transgene-induced short- and long-distance sys-temic silencing can be also studied in coinfiltration assays. If aGFP-transgenic N. benthamiana is infiltrated with 35SGFP,cell-autonomous GFP silencing generates mobile signals,which lead to systemic GFP silencing. Since P14 prevents theaccumulation of either short or long siRNAs in coinfiltratedpatches and siRNAs are supposed to play role in systemicsilencing, we postulated that P14 also inhibits the developmentof systemic silencing. Indeed, coinfiltration of P14 preventedthe development of both short- and long-distance systemicGFP silencing (data not shown).

Taken together, the findings that P14 increases viral symp-toms and inhibits silencing in agroinfiltration assays indicatethat P14 is an efficient suppressor of both virus and transgene-induced silencing.

P14 is a dsRNA binding protein. In a GFP coinfiltration(sense transgene-induced silencing) assay, P14 acts like the P19suppressor of closely related tombusviruses (18, 34, 35, 45).Both proteins prevent the accumulation of GFP-specific shortand long siRNAs in the coinfiltrated patches, thus inhibitingthe development of cell-autonomous and systemic silencing.These data open up the possibility that P14 and P19 suppres-sors target an identical step in the silencing pathway. P19inhibits silencing by sequestering double-stranded siRNAs. Totest whether P14 could also suppress silencing by binding dou-ble-stranded siRNAs, we studied the RNA binding activity ofP14 in gel mobility shift assays. P14 was expressed and purifiedas a GST fusion protein (G-P14), and then G-P14 was probedwith labeled, synthetic single-stranded and double-strandedsiRNAs. A GST fusion version of the previously characterizedCarnation Italian ringspot tombusvirus (CIRV) P19 (G-P19)was used as a control (55). As expected, G-P19 did not shiftssRNAs, while it bound 21-nt double-stranded siRNAs. Im-portantly, G-P14 also failed to form complexes with ssRNAsbut bound 21-nt double-stranded siRNA (Fig. 3A). These datasuggest that G-P14, like G-P19, is a double-stranded siRNAbinding protein. However, G-P14 forms complexes with dou-ble-stranded siRNAs with less efficiency than G-P19, since asimilar shift required a much higher G-P14 concentration. Thestriking feature of P19-mediated dsRNA binding is its strongsize selectivity. To test whether P14 is also a size-selectivedsRNA binding protein, G-P14 was also probed with long (144nt) dsRNAs. Interestingly, we found that unlike G-P19, G-P14formed complexes with long dsRNAs (Fig. 3A). These datasuggest that G-P14 is a dsRNA binding protein that lacks sizespecificity. Unfortunately, we failed to release functional P14from G-P14 with thrombin cleavage; therefore, we cannotcharacterize the RNA binding activity of Escherichia coli-ex-pressed native P14.

Although in vitro G-P14 fusion protein inefficiently formscomplexes with double-stranded siRNAs, it is possible thatnative P14 efficiently binds double-stranded siRNAs in PoLV-infected cells. Moreover, full double-stranded siRNA bindingactivity of P14 might require plant-specific posttranslationalmodification and/or the presence of host/viral factors. There-

fore we wanted to directly analyze the double-stranded siRNAbinding activity of PoLV-expressed P14. To study whetherPoLV-expressed P14 binds double-stranded siRNAs, we com-pared the double-stranded siRNA binding activity of crudeextracts prepared from PoLV (PoLV extract)- and PoLV�14(PoLV�14 extract)-inoculated N. benthamiana leaves. Impor-tantly, PoLV extract efficiently bound 21-nt double-strandedsiRNAs, while PoLV�14 extract failed to form complexes with21-nt double-stranded siRNAs (Fig. 3B). As P14 protein wasexpressed only in PoLV-infected leaves (see Materials S1 andFig. S1A in the supplemental material), it is likely that P14provided the double-stranded siRNA binding capacity for thePoLV extract. Moreover, PoLV extract causes almost as stronga shift on 21-nt double-stranded siRNAs as the extract that wasprepared from CymRSV tombusvirus-inoculated leaves(CymRSV extract) (Fig. 3B). This result suggests that PoLV-expressed P14 effectively forms complexes with double-stranded siRNA.

To investigate whether P14 can bind double-strandedsiRNAs in the absence of viral factors, we analyzed the double-stranded siRNA binding activity of crude extract preparedfrom P14-agroinfiltrated N. benthamiana leaves (P14 extract).Extract prepared from 35SGFP-infiltrated leaves was used as anegative control (GFP extract). We could not detect double-stranded siRNA binding activity in the GFP extract (data notshown), while P14 extract caused a strong shift on 21-nt dou-ble-stranded siRNAs (Fig. 3D, second lane). Moreover, asboth GFP and P14 extracts weakly and identically bound sin-gle-stranded siRNAs (data not shown), it is likely that P14 doesnot bind ssRNAs. Therefore, we conclude that in planta ex-pressed P14 is a double-stranded siRNA binding protein whichdoes not require viral factors for dsRNA binding.

In vitro, P19 binds dsRNAs size selectively, while G-P14binds dsRNAs without size specificity. To test whether inplanta-expressed P14 and P19 proteins also differ in dsRNApreference, we defined the relative affinity of plant-producedP14 and P19 for 21-nt and 26-nt double-stranded siRNAs. Toaim this, direct competition assays were carried out with P14and P19 extracts prepared from agroinfiltrated leaves. Labeled21-nt double-stranded siRNAs were incubated with plant ex-tracts and with increasing molar concentrations of cold 21-ntand 26-nt double-stranded siRNA competitors (Fig. 3D andF). Competition experiments were repeated with labeled 26-ntdouble-stranded siRNAs (Fig. 3E and G). In line with resultsobtained with P19 expressed in E. coli, the P19 extract showeda much higher affinity for 21-nt double-stranded siRNAs thanfor 26-nt double-stranded siRNAs (55, 62). A large molarexcess of 26-nt double-stranded siRNAs (320�) was requiredfor detectable competition when 21-nt double-stranded siRNAwas labeled (Fig. 3F), while 21-nt double-stranded siRNAsoutcompeted 26-nt double-stranded siRNAs even at a low(20�) molar excess (Fig. 3G). By contrast, the P14 extract hadno obvious size specificity because approximately the samemolar excess of cold 21-nt double-stranded siRNAs and 26-ntdouble-stranded siRNAs was required for outcompeting eitherlabeled 21- or 26-nt double-stranded siRNAs (Fig. 3D and E).These results suggest that both suppressors bind double-stranded siRNAs efficiently but with different selectivities. P19might be more specific for small double-stranded siRNAs,


whereas P14 could bind short and long double-strandedsiRNAs with comparable affinity.

In vitro G-P14 fusion protein binds long dsRNAs. To testwhether in planta-expressed P14 also binds long dsRNAs, la-beled 144-nt dsRNA was incubated with PoLV and with P14extract. As negative controls, PoLV�14 and GFP extract wereused, respectively. As Fig. 3C shows, a long dsRNA bindingactivity could be detected in both PoLV and P14 extracts thatwas lacking in either the PoLV�14 or GFP extract. Therefore,it is very likely that P14 provided the long dsRNA bindingactivity for PoLV and for P14 extracts. Findings that longdsRNA binding of P14 extract could be outcompeted with colddsRNA but not with ssRNA further support the notion that inplanta-expressed P14 is a dsRNA binding protein (see Mate-

rials S2 and Fig. S1B in the supplemental material). Moreover,in line with results obtained with G-P19 fusion protein, labeledlong dsRNA was not bound by CymRSV-expressed P19 (Fig.3C).

Collectively, mobility shift assays revealed that P14 and P19suppressors are different dsRNA binding proteins: P19 is astrict size-specific dsRNA binding protein, while P14 bindsdsRNAs without strong size preference.

P14 and P19 act differently in hairpin-induced agroinfiltra-tion assays. As dsRNAs play a key role in silencing and P14 isa dsRNA binding protein, we postulated that P14-mediatedsilencing is based on sequestering a dsRNA component of thesilencing machinery. P14 might sequester long dsRNAs, theinducers of silencing machinery, or double-stranded siRNAs,

FIG. 3. P14 is a dsRNA binding protein. (A) RNA binding activity of P14 (G-P14) and P19 (G-P19) expressed as a GST fusion protein werestudied in gel mobility shift assays. Shift assays with expressed GST or without additional protein (�) were used as negative controls. Synthetic21-nt RNAs were labeled and used as ssRNA probes, while labeled 21-nt RNAs, which annealed to dsRNAs having a 19-nt duplex with 2-nt 3�overhangs (21 ds siRNA) were used as double-stranded siRNA probes; 144-nt-long, labeled, complementary in vitro transcripts were annealed andused as long dsRNA probes (144 dsRNA). Concentrations of G-P14 of 15 �M and 1.5 �M were used for the double-stranded siRNA and for thelong dsRNA binding experiments, respectively. A 1.5 �M concentration of G-P19 was used for both double-stranded siRNA and long dsRNAbinding tests. Free probes and protein-probe complexes are referred as F and C, respectively. (B) PoLV-expressed P14 efficiently bindsdouble-stranded siRNAs. dsRNA binding activity of extracts prepared from PoLV- or PoLV�14-inoculated leaves were probed with labeled 21-ntdouble-stranded siRNAs. Extracts prepared from leaves inoculated with CymRSV and Cym19stop (C19stop), a mutant CymRSV that was unableto express P19, were used as controls. Extracts were isolated at 3 d.p.i. from N. benthamiana leaves inoculated with the corresponding viruses. (C) Inplanta-expressed P14 binds long dsRNA. Extracts prepared from mock (�)-, PoLV-, PoLV�14-, CymRSV-, and C19stop-inoculated N. benthami-ana leaves (left panel) were probed with labeled 144-nt dsRNA. Extracts prepared from mock (�)-, 35SGFP (GFP)-, and P14-infiltrated N.benthamiana leaves (right panel) were also probed with labeled 144-nt dsRNA. Each extract was obtained at 3 d.p.i. An asterisk indicates anonspecific binding activity that is not associated with P14 or P19 suppressors because it is present in all extracts prepared from PoLV�14,CymRSV, and C19stop virus-inoculated leaves. Similar nonspecific long dsRNA binding activity can be occasionally detected in extracts preparedfrom mock- or 35SGFP-infiltrated leaves. (D to G) Plant-expressed P14 binds double-stranded siRNAs without size selectivity. P14 and P19extracts were obtained from P14- and P19-infiltrated N. benthamiana leaves at 3 d.p.i. Direct competition assays were carried out with labeled 21-nt(D and F) and 26-nt double-stranded siRNAs (E and G) and with cold competitors added in the indicated molar excesses. A 0 indicates that theshift assay was conducted in the absence of competitor.


the specificity determinants of the silencing system. To distin-guish between these two possibilities, we studied the suppres-sor activity of P14 in hairpin transcript-induced silencing as-says.

Agroinfiltration with an inverted repeat GFP construct(GFP IR) leads to expression of hairpin GFP RNAs (GFP-ir).As hairpin transcripts are rapidly processed into siRNAs by thesilencing machinery, GFP-ir transcripts are barely detectable,while GFP-ir-derived siRNAs accumulate to high levels in theinfiltrated leaves (Fig. 4C). Moreover, coinfiltration of GFP IRwith 35SGFP (GFP IR plus 35SGFP) prevents transient GFPactivity (Fig. 4A and B) because GFP-ir-derived siRNAs directearly degradation of GFP mRNAs (19). However, coinfiltra-tion of GFP IR plus 35SGFP with dsRNA binding proteinssuch as reovirus Sigma3 (24) or P19 (50) result in strong greenfluorescence and accumulation of GFP mRNAs (Fig. 4A andB). Sigma3 and P19 suppress hairpin-induced silencing at dif-ferent steps. Sigma3 is a strong dsRNA binding protein thatforms complexes only with dsRNAs longer than �30 nt (63).Sigma3 is proposed to suppress silencing by sequestering hair-pin transcripts (24) because, in coinfiltrated leaves, GFP-irtranscripts accumulate to high levels, while siRNAs could not

be detected (Fig. 4B). By contrast, in P19-coinfiltrated leavesGFP-ir transcripts could not be detected, while siRNAs areeasily detected (50) (Fig. 4B). These data are interpreted tomean that the siRNA-specific dsRNA binding protein P19inhibits hairpin-induced silencing by sequestering double-stranded siRNAs (50).

To test whether P14 can suppress hairpin-induced silencing,we coinfiltrated leaves with GFP IR plus 35SGFP and withP14. We found that GFP mRNAs accumulated to high levelsand green fluorescence was strong in P14 coinfiltrated leaves,indicating that P14 suppressed hairpin-induced silencing effi-ciently (Fig. 4A). Surprisingly, we failed to detect either hair-pin transcripts or siRNAs in P14-coinfiltrated leaves (Fig. 4B).To prove that P14 directly affects on hairpin-derived siRNAaccumulation, we infiltrated leaves with GFP IR or coinfil-trated leaves with GFP IR and P14. As expected, in GFPIR-infiltrated samples, siRNA accumulated to high levels,while hairpin transcripts could not be detected. By contrast, inGFP IR- and P14-coinfiltrated leaves, neither hairpin tran-scripts nor siRNAs could be found (Fig. 4C).

Collectively, GFP IR coinfiltration studies revealed thatP14-mediated suppression of hairpin-induced silencing ismechanistically different than either Sigma3- or P19-mediatedsilencing inhibition. We suggest that these differences are theconsequences of the different dsRNA binding preferences ofthe Sigma3, P19, and P14 proteins (see Discussion).

P14 fails to prevent accumulation of viral siRNAs. The ob-servation in agroinfiltration assays that P14 prevents the accu-mulation of hairpin transcript-derived siRNAs suggests thatP14 suppresses virus-induced silencing by preventing the accu-mulation of viral siRNAs. To test this model, we monitored theaccumulation of viral RNAs and siRNAs in PoLV- andPoLV�14-inoculated N. benthamiana leaves. By 1 day postin-oculation (d.p.i.), PoLV and PoLV�14 viral RNAs accumu-lated to detectable levels, while by 2 d.p.i., PoLV andPoLV�14 RNAs were abundant in the inoculated leaves (Fig.5A). Surprisingly, virus-specific siRNAs could be identified inboth PoLV- and PoLV�14-inoculated leaves (Fig. 5A). As theP14 protein could already be detected at 1 d.p.i. in PoLV-infected leaves (Fig. 5A bottom panel), we concluded that P14fails to prevent the accumulation of viral siRNAs. However,viral siRNA/viral genomic RNA ratios were higher inPoLV�14-inoculated leaves than in PoLV-inoculated ones(Fig. 5A). These data indicate that virus-induced silencing op-erated less efficiently in PoLV-inoculated leaves than inPoLV�14-infected tissues, confirming that P14 acts as an effi-cient suppressor of aureusvirus-induced silencing.

Suppressors of aureusviruses and tombusviruses derivefrom a common ancestor protein. Findings that both P14 andP19 proteins bind double-stranded siRNAs and suppress si-lencing suggest that these proteins evolved from a commonancestor. However, the nonrelated P21 suppressor of Beet yel-lows virus (Closteroviridae, Closterovirus) also binds double-stranded siRNAs (8), indicating that dsRNA binding silencingsuppressors evolved more than once. Therefore, it is also pos-sible that P14 and P19 suppressors evolved independently.

To clarify whether P14 and P19 proteins evolved indepen-dently or have a common ancestor, multiple-sequence align-ments were carried out with many tombusvirus and aureusvirussequences. In both genera, ORF 5 is completely nested within

FIG. 4. P14 suppresses hairpin-induced RNA silencing. (A) N.benthamiana leaves were infiltrated with 35SGFP and a secondAgrobacterium (GFP IR) expressing hairpin GFP transcripts (�) orcoinfiltrated with GFP IR plus 35SGFP and P14, P19, and Sigma3suppressors, respectively. Photos were taken at 3 d.p.i. (B) Effect ofP14 on accumulation of hairpin transcripts, GFP mRNAs, and siR-NAs. GFP-ir indicates hairpin transcripts derived from GFP IR, whileGFP refers to GFP mRNA transcribed from 35SGFP (upper panel).RNA samples were isolated at 3 d.p.i. from GFP IR plus 35SGFP-infiltrated (�) or from GFP IR plus 35SGFP and dsRNA bindingprotein-coinfiltrated leaves. Note that the probe we used for siRNAhybridization (bottom panel) detected both hairpin transcript and GFPmRNA-derived siRNAs (GFP siRNA). (C) P14 prevents the genera-tion of siRNAs from hairpin transcripts. RNA samples were isolated at3 d.p.i. from GFP IR-infiltrated (�) or from GFP IR plus dsRNAbinding protein-coinfiltrated leaves.


ORF 4 but it is translated in the third frame related to ORF 4(Fig. 1A). Therefore, we first defined the biologically mean-ingful gaps that optimized both the alignment of ORF 4-en-coded movement proteins (MP) and the alignment of suppres-sor proteins (for details regarding defining gaps and creatingprotein alignments, see Materials S4 and Fig. S2 in the sup-plemental material).

Multiple-sequence alignments of proteins deduced from

tombusvirus and aureusvirus ORF 4 (see Materials S4 and Fig.S3 in the supplemental material) and ORF 5 (Fig. 6) RNAsequences revealed that similarity was strong for both move-ment and suppressor proteins within a genus but weak betweenaureusviruses and tombusviruses (see Fig. S4 in the supple-mental material). Amino acids that were identical or similarbetween aureusviruses and tombusviruses could be found allalong the MPs except in the C-terminal region (see MaterialsS4 and Fig. S3 in the supplemental material). By contrast,conserved amino acid positions were limited to short regions inthe suppressor alignment (Fig. 6). Interestingly, the regions ofsimilarity in the suppressor sequences coincide with previouslyidentified secondary structural elements (1, 2, 3, 4, andthe last �-helix) that play key roles in double-stranded siRNAbinding by P19. 4 and the last �-helix contribute to the P19homodimer formation, while the four -strands form a concave sheet that makes contact with the sugar-phosphate backboneof double-stranded siRNA (3, 55, 62). No similarity was foundbetween aureusvirus and tombusvirus suppressors in the regioncorresponding to the P19 �2 helix (reading head) that interactswith the end of siRNAs (3, 55, 62) (Fig. 6).

As tombusvirus and aureusvirus suppressors show similarityin conserved regions that are important for P19-mediated sup-pression, we suggest that these proteins have evolved from anancient suppressor, which was nested in the common ancestorof the MPs of tombusviruses and aureusvirures. Tombusvirusand aureusvirus MPs belong to the 30-kDa MP superfamily(27). To explore the evolution of tombusvirus-aureusvirus sup-pressors, we sought to determine whether related MPs alsoencoded a nested suppressor. The closest relatives to tombus-virus and aureusvirus MPs are the umbro, tobra (27), andtrichovirus MPs (E. Barta, unpublished data). Importantly,none of these MP encodes a nested protein (data not shown).

DISCUSSION

Previously P14 was identified as a symptom determinant(40). Here we demonstrate that P14 is a dsRNA binding pro-tein that inhibits virus- and transgene-induced silencing.

P14 is a dsRNA binding protein. P14 is a dsRNA bindingprotein which forms complexes with 21- or 26-nt double-stranded siRNAs and with long dsRNAs (Fig. 3). It was anunexpected finding because the related P19 preferentiallybound 21-nt double-stranded siRNAs (55, 62). Sequence align-ments might explain the molecular basis of different double-stranded siRNA binding of these two suppressors. Regionsthat play roles in forming the dsRNA binding surface areconserved between aureusviruses and tombusviruses, while thereading head, which is involved in size-specific binding of P19,cannot be identified in aureusviruses (Fig. 6). Therefore, it ispossible that P14 and P19 suppressors bind double-strandedsiRNAs with similar structure, except that P14 does not inter-act with the 5� ends of RNAs.

P14-mediated silencing suppression. Previous results haveshown that dsRNA binding proteins could act as silencingsuppressors. P19 and P21 suppressors bind double-strandedsiRNAs in vivo (8, 14, 21), indicating that these proteins inhibitsilencing by sequestering double-stranded siRNAs. It is pro-posed that the influenza Ns1 protein also inactivates silencingin plant and insect cells by binding double-stranded siRNAs (5,

FIG. 5. P14 does not prevent accumulation of PoLV-derivedsiRNAs. (A) Accumulation of high- and low-molecular-weight viralRNAs (viral siRNA) in the inoculated leaves of PoLV- and PoLV�14-infected N. benthamiana plants. G indicates genomic virus RNAs,while Sg1 and Sg2 refer to subgenomic 1 and subgenomic 2 RNAs,respectively. Note that P14 was already detectable at 1 d.p.i. and thatthe level of PoLV Sg2 declines. Numbers below the bottom panel referto the ratios of viral siRNA/viral genomic RNAs. The sample loaded inthe first lane was taken as 100% (1), and others were normalized to it.�-P14 refers to polyclonal antibody raised against P14. (B) Virus-induced cell-autonomous silencing fails to control accumulation ofsuppressorless PoLV. RNA samples were isolated from N. benthami-ana protoplasts transfected with in vitro transcripts of PoLV orPoLV�14. A Western blot assay with the available polyclonal antibodyfailed to detect P14 protein in PoLV-transfected cells, therefore con-clusions about the role of P14 in suppression of virus induced cell-autonomous silencing cannot be drawn (see Materials S3 in the sup-plemental material).


23). Moreover, as Sigma3 suppresses transgene-induced silenc-ing efficiently, it is likely that binding to long dsRNAs couldalso inhibit certain silencing pathways (24). Although we can-not exclude that P14-mediated silencing suppression dependson its interaction with a host protein, the simplest explanationis that P14 targets silencing by binding long dsRNAs and/ordouble-stranded siRNAs. We suggest that P14 inhibits hairpin-induced silencing by binding long dsRNAs, while it suppressesvirus-induced silencing by sequestering double-strandedsiRNAs or by delaying the generation of viral siRNAs.

Coinfiltration of P14 with GFP IR plus 35SGFP prevents theaccumulation of both hairpin transcript- and hairpin-derivedsiRNA (Fig. 4). These findings can be explained if P14 does notinterfere with the generation of siRNAs, instead it acceleratesthe degradation of them. However, viral siRNAs accumulate tohigh levels in PoLV-infected cells (Fig. 5). Therefore, we pre-fer the alternative explanation that P14 binds hairpin tran-scripts (long dsRNAs); thus, it inhibits DCL-mediated process-ing of hairpin transcripts but allows their degradation byalternative decay systems. Sigma3 might bind long dsRNAsmore strongly than P14; therefore, it protects hairpin tran-scripts. P19, which does not bind long dsRNAs, fails to preventthe generation of siRNAs from hairpin transcripts, instead itinhibits silencing by sequestering hairpin-derived double-stranded siRNAs.

Findings that P14 increases PVX and PoLV symptoms andthat virus-induced silencing is more intense in PoLV�14-inoc-

ulated leaves than in PoLV-inoculated ones strongly indicatethat P14 acts as an efficient suppressor of virus-induced silenc-ing. Viral siRNAs can be easily detected in PoLV-infectedleaves, suggesting that P14 inhibits virus-induced silencing bytargeting a step downstream of siRNA generation. As in vitroP14 binds double-stranded siRNAs, we postulate that in virus-infected cells P14, like P19, suppresses silencing by sequester-ing double-stranded siRNAs. However, we failed to coimmu-noprecipitate viral siRNAs from PoLV-infected leaves withP14 polyclonal antibody (Z. Merai, unpublished data). It couldbe due to technical difficulties (for instance, the P14 double-stranded siRNA complex is weak and dissociates during ma-nipulation or the P14 antibody is not suitable for coimmuno-precipitation), and we cannot exclude that in vivo P14 does notbind double-stranded siRNAs. An alternative model for P14-mediated suppression of virus-induced silencing could be thatP14 binds the precursors of viral double-stranded siRNAs;thus, it slightly delays the accumulation of siRNAs. Impor-tantly, observation that viral siRNAs are relatively more abun-dant in PoLV�14-infected leaves than in PoLV-infected onesis consistent with both suppression models.

Antiviral silencing operates as a cell-autonomous and sys-temic response. Because viral RNAs accumulate to high lev-els in PoLV�14-transfected protoplasts (40) (Fig. 5B) eventhough viral siRNAs are present (Fig. 5B), we conclude thatcell-autonomous antiviral silencing is unable to limit the accu-mulation of the rapidly replicating virus. By contrast, infection

FIG. 6. P14 and P19 suppressors appear to be evolutionarily related. Multiple alignments of amino acid sequences deduced from many availableaureusvirus and tombusvirus ORF5 sequences. Defined gaps (see Materials S4 in the supplemental material) were incorporated into the deducedprotein sequences. PoLV (indicated as PLV), Cucumber leaf spot virus (CLSV), and Johnsongrass chlorotic stripe mosaic virus (JCSMV) areaureusviruses, and other viruses included in the alignments are tombusviruses. CIRV refers to the P19 protein of CIRV, whereas CRSV showsCymRSV P19. List of viruses, which were not used in this study but were included in the comparison are available in the supplemental material(see Materials S4 and Table S1 in the supplemental material). Different colors show different groups of amino acids. Asterisks indicate amino acidsthat are perfectly conserved in each aligned protein, while colons and periods refer to conservative and semiconservative substitutions, respectively.Secondary structural elements defined for CIRV P19 are shown at the top.


of PoLV�14 leads to a recovery phenotype (40) (Fig. 1A),indicating that colonization of the host plant requires the sup-pression of systemic silencing (1, 17, 18, 46, 49). We suggestthat, in wild-type infection, P14 inhibits the development ofsystemic silencing by sequestering 21-nt double-strandedsiRNAs and/or by delaying the generation of siRNAs; thus,PoLV spreads more quickly than the silencing signal and col-onizes the plant. These models, in which P14 suppresses virus-induced systemic silencing by binding double-stranded siRNAsand/or precursor dsRNAs, predict that it inhibits systemic si-lencing in a dose-dependent manner. Indeed, at a high tem-perature (27°C) where siRNA generation is efficient (49), evenPoLV infection leads to a recovery phenotype, indicating thatat 27°C P14 fails to completely inhibit systemic silencing (Me-rai, unpublished).

Evolution of dsRNA binding silencing suppressors of au-reusviruses and tombusviruses. Both MP and suppressor pro-teins of aureusviruses are homologous to the correspondingtombusvirus proteins, indicating that the common ancestor ofthese two genera already carried an ancestral silencing sup-pressor nested in the MP. Because nested protein could not befound in any related MP, we suggest that this ancestral sup-pressor has evolved within the common ancestor of aureusvi-rus-tombusvirus MP after it diverged from other 30-kDa MPsbut before the branching of Aureusvirus and Tombusvirus gen-era. Moreover, as P19 and P14 suppressors are dsRNA bindingproteins and as the conserved suppressor regions are impor-tant in forming the dsRNA binding structure of P19, it is likelythat the ancestral suppressor was a dsRNA binding protein.P19 is a unique dsRNA binding protein because it bindsdsRNAs size selectively, while all other characterized dsRNAbinding proteins binds dsRNAs without strict size specificity.Therefore, we speculate that the common ancestor of the au-reusvirus-tombusvirus suppressor was a size-independentdsRNA binding protein.

Suppressors have evolved to target antiviral responses, butthey have also been selected for causing as little damage aspossible to the host. It is proposed that, as size-specific double-stranded siRNA binding is a result of such a dual selection, P19can efficiently bind siRNAs that play a role in antiviral re-sponse, while it might not interfere with long double-strandedsiRNA programmed silencing pathways such as RNA-medi-ated epigenetic gene regulation (3, 33, 55). It is conceivablethat expression of a size-independent dsRNA binding proteinwould cause additional damages, for instance, P14 might in-terfere with RNA-directed epigenetic regulation or bind struc-tured host mRNAs. Interestingly, the level of Sg2 RNA, fromwhich P14 is translated, declines after 2 to 3 d.p.i. in PoLV-infected plants (40) (Fig. 5A), while CymRSV Sg2 is abundanteven at 10 d.p.i (48). It has been suggested that decreasedexpression of PoLV Sg2 is a viral control measure to reducethe toxicity of P14 (40). Indeed, infection with a PoLV mutantthat constitutively expresses Sg2 RNA results in rapid plantdeath (40). It is appealing to speculate that the common an-cestor suppressor might have evolved by two ways to reducethe damage to the host. In tombusviruses, it has evolved into asize-specific double-stranded siRNA binding suppressor, whilein PoLV, it could have evolved into a temporally/spatially con-trolled suppressor.

Is dsRNA binding a frequent suppressor strategy? P14 sup-pressed hairpin-induced silencing by preventing the accumula-tion of hairpin transcript and siRNAs. Interestingly, transgenicexpression of peanut clump virus P15, potato virus X P25, andturnip crinkle virus CP silencing suppressors in an Arabidopsisline that expressed hairpin transcripts of chalcone synthasealso lead to similar inhibition of hairpin-induced silencing (14).In all three cases, hairpin-derived siRNA levels were dramat-ically reduced even though hairpin transcripts were not pro-tected (14). These findings suggest that (some of) thesesuppressors target silencing like P14. They might be size-inde-pendent dsRNA binding proteins, which inhibit hairpin-in-duced silencing by preventing DCL-mediated processing. Ifthese proteins bind dsRNAs like P14, they also form complexeswith double-stranded siRNAs; hence, they could suppress si-lencing by sequestering double-stranded siRNAs. Indeed, P15and CP suppressed siRNA-mediated silencing efficiently inHeLa cells (14). Since these proteins are nonrelated (8), wespeculate that dsRNA binding is a frequent suppression strat-egy, which has evolved independently many times. In thisstudy, we described a rapid mobility shift assay using crudeextracts prepared from either virus-infected or agroinfiltratedleaves that is suitable for recognition and characterization ofdsRNA binding proteins. We think that this convenientmethod could facilitate the identification of other dsRNAbinding viral suppressors.

ACKNOWLEDGMENTS

We are grateful to M. Russo for kindly providing full-length cDNAclones of PoLV and PoLV�14 and to D. Baulcombe for the GFP plantand 35SGFP constructs. We are especially grateful to J. Vargason andT. Tanaka Hall for providing siRNAs and for help with conducting andanalyzing competition assays. We thank T. Tanaka Hall, G. Szittya,and L. Lakatos for useful comments on the manuscript, G. Takacs forhelp with figure preparations, and Edina Kapuszta for excellent tech-nical assistance.

This research was supported by grants from the Hungarian ScientificResearch Fund (OTKA) (T15042787). D.S. was financed by the Bolyaischolarship.

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Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

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