size-independent and noncooperative recognition of … · size-independent and noncooperative...

doi:10.1016/j.jmb.2010.10.007 J. Mol. Biol. (2010) 404, 665–679

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Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

Size-Independent and Noncooperative Recognitionof dsRNA by the Rice Stripe Virus RNA SilencingSuppressor NS3

Mei Shen1,2, Yi Xu3, Ru Jia2, Xueping Zhou3 and Keqiong Ye2⁎1Graduate Program at Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100730, China2National Institute of Biological Sciences, Beijing 102206, China3State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China

Received 30 September 2010;accepted 6 October 2010Available online14 October 2010

Edited by D. E. Draper

Keywords:cooperativity;dsRNA-binding protein;RNA silencing;viral suppressor;plant–virus interactions

*Corresponding author. E-mail [email protected] used: VSR, viral su

silencing; dsRBS, dsRNA-binding sustripe virus; RHBV, Rice hoja blancainterfering RNA; EMSA, electrophoassays; sulfo-EGS, ethylene glycol b(sulfosuccinimidylsuccinate); RNP, Rcomplex; RDR, RNA-dependent RNdays post-infiltration; TRBP, trans-aRNA-binding protein; MW, molecul

0022-2836/$ - see front matter © 2010 E

Plant and animal viruses employ diverse suppressor proteins to thwart thehost antiviral reaction of RNA silencing. Many suppressors bind dsRNAwith different size specificity. Here, we examine the dsRNA recognitionmechanism of the Rice stripe virus NS3 suppressor using quantitativebiochemical approaches, as well as mutagenesis and suppression activityanalyses in plants. We show that dimeric NS3 is a size-independent, ratherthan small interfering RNA-specific, dsRNA-binding protein that recog-nizes a minimum of 9 bp and can bind to long dsRNA with two or morecopies. Global analysis using a combinatorial approach reveals that NS3dimer has an occluded site size of ∼13 bp on dsRNA, an intrinsic bindingconstant of 1×108 M−1, and virtually no binding cooperativity. This lack ofcooperativity suggests that NS3 is not geared to target long dsRNA. Thelarger site size of NS3, compared with its interacting size, indicates that theNS3 structure has a border region that has no direct contact with dsRNA butoccludes a ∼4-bp region from binding. We also develop a method to correctthe border effect of ligand by extending the lattice length. In addition, wefind that NS3 recognizes the helical structure and 2′-hydroxyl group ofdsRNA with moderate specificity. Analysis of dsRNA-binding mutantssuggests that silencing of the suppression activity of NS3 is mechanisticallyrelated to its dsRNA binding ability.

© 2010 Elsevier Ltd. All rights reserved.

ress:

ppressor of RNAppressor; RSV, Ricevirus; siRNA, small

retic mobility shiftisNA–proteinA polymerase; dpi,ctivation responsear weight.

lsevier Ltd. All rights reserve

Introduction

RNA silencing regulates gene expression in mosteukaryotes, with specificity determined by smallRNA molecules of 21–24 nucleotides (nt) inlength.1–4 In plants and invertebrates, RNA silenc-ing also functions as an adaptive antiviral immu-nity system.5 Virus infection often induces theappearance of viral dsRNAs that are generated bythe activities of viral and host RNA-dependentRNA polymerases (RDRs) or derived from struc-tured regions in viral RNAs. Viral dsRNAs areprocessed by the RNase III enzyme Dicer intosmall interfering RNAs (siRNAs). siRNA is

d.

666 Recognition of dsRNA by RSV NS3

composed of two strands of 21–24 nt in length thatform a 19- to 22-bp duplex with characteristic 2-nt3′-overhangs at both ends, as a result of RNase IIIdigestion. One strand of siRNA assembles into anArgonaute (AGO) protein to form an RNA-induced silencing complex, which cleaves viralRNAs that hybridize with the siRNA. The modelplant Arabidopsis thaliana encodes 4 Dicer-like(DCL) proteins, 10 AGOs, and 6 RDRs, whichfunction redundantly and specifically in differentRNA silencing pathways.6 Among them, DCL4,DCL2, AGO1, RDR6, and RDR1 have been shownto be involved in defending RNA viruses, asevidenced by their mutants becoming hypersensi-tive to the viruses.5

To counter RNA silencing-based antiviral defense,viruses produce diverse viral suppressors of RNAsilencing (VSRs) to inhibit the host antiviralreaction.7,8 A large number of VSRs have beenfound to bind non-sequence-specifically to siRNAand/or long dsRNA precursor to inhibit siRNAutilization and production.9–18 Recent studies havealso identified a fewVSRs that target theAGOproteinof the antiviral RNA silencing machinery.19–24dsRNA-binding suppressors (dsRBSs) can be

categorized into three types on the basis of theirspecificity for dsRNA size. Type I dsRBSs targetsiRNA in a size-dependent manner, such as theprototypical tombusvirus P19 suppressor.9 Struc-tural analysis showed that the dimeric P19 proteinmeasures the siRNA size by symmetrically contact-ing each end of the duplex.25,26 Type II dsRBSs bindto both siRNA and long dsRNA without sizespecificity. The B2 protein of Flock house virus is atypical size-independent dsRBS.11,12,27 In the crystalstructure of the B2–dsRNA complex, the dimeric B2protein contacts one face of the duplex withoutinteraction with the duplex ends, underlying itsnonselectivity for duplex size.12 The third type ofdsRBSs bind preferentially to long dsRNA, butpoorly to siRNA, as shown for the 1A protein ofDrosophila C virus (DCV-1A).16

Different specificities for dsRNA size could lead todifferent mechanisms of silencing suppression. TypeI siRNA-specific dsRBSs most likely sequester viralsiRNA and prevent its utilization, as shown forP19.29 The type III dsRBS DCV-1A specificallyinhibits Dicer processing of the dsRNA precursorwith no effect on siRNA-induced silencing.16 Incontrast, binding of both long dsRNA and siRNAcould theoretically allow type II dsRBSs to interferewith both the upstream Dicer processing step andthe downstream siRNA utilization step.11–13,15However, it is difficult to distinguish which typeof dsRNA is the primary target of type II dsRBS.Proteins that associate with long nucleic acid inmultiple copies often display positive bindingcooperativity, which enhances the contiguous asso-ciation of an otherwise weak ligand.30,31 Positive

cooperativity would thus be an indicator for size-independent dsRBS to naturally target long dsRNA.However, quantitative analysis of cooperativity hasnot been reported for any of size-independentdsRBSs.Rice stripe virus (RSV) is the type member of the

Tenuivirus genus, having a negative-sense single-stranded (ss) RNA genome. RSV infection causesserious problems for rice production in the EastAsian region, especially in China. Its genomic RNA iscomposed of four segmented parts, which togetherencode seven open reading frames. The nonstructur-al protein NS3, encoded in the sense strand of thethird largest part RNA3, has recently been shown tobe a VSR.32 RSV NS3 inhibits local silencing, beinginduced by either ssRNA or dsRNA, and also blockssystemic silencing when the protein is present in thespreading route of systemic signals.32 RSV istransmitted by a small plant hopper (Laodelphaxstriatellus) and also replicates in the insect vector.33,34

Hence, NS3 needs to suppress antiviral RNAsilencing in both plants and insects. Rice hoja blancavirus (RHBV) is another member of the Tenuivirusgenus, and its NS3 homolog is also a VSR.35 RHBVNS3 displays size-specific recognition of siRNA andsuppresses RNA silencing in plants and insects.17

The exclusive siRNA-binding property of RHBVNS3has been utilized to examine the siRNA-mediatedantiviral response in mammalian cells.36

To gain insight into themechanism of action of NS3and to better utilize it as a research tool, wecharacterize in detail the energetics of the RSV NS3interaction with dsRNA. We analyze the associationof NS3 with dsRNAs of various lengths using acombinatorial approach and derive several dsRNAbinding parameters, including the stoichiometry,intrinsic binding constant, minimal binding sitesize, occluded site size, and cooperativity. We findthat NS3 is a size-independent, rather than siRNA-specific, dsRNA binding protein with virtually nocooperativity when binding long dsRNA. In addi-tion, we investigate the structure feature of dsRNArecognized by NS3 and amino acid residues respon-sible for dsRNA binding. NS3 mutants display astrong correlation between dsRNA binding activityand silencing suppression activity, providing amechanistic link between the two activities.

Results

Expression and purification of the NS3 protein

The RSV NS3 protein was expressed in Escherichiacoli with a six-histidine tag at its C terminus andpurified through Ni-affinity and heparin chroma-tography (Fig. 1). Most of the NS3 protein wassoluble when expressed at 16 °C. The NS3 protein

Fig. 1. Purification of histidine-tagged RSV NS3. Thesamples were analyzed by SDS-PAGE and stained withCoomassie blue. Lane 1,MWmarker; lane 2, cell lysate afterinduction of the NS3 gene expression; lane 3, lysatesupernatant; lane 4, pellet after centrifugation; lanes 5–7,HisTrap chromatography; lane 5, flow-through; lane 6,fraction after washing with 50 mM imidazole; lane 7, eluateof 500mM imidazole; lanes 8–10, heparin chromatography;lane 8, flow-through; lane 9, fraction after washing with0.3 M KCl; lane 10, peak fractions in a salt gradient.

667Recognition of dsRNA by RSV NS3

bound tightly to a heparin column and eluted ataround 0.6 M KCl. However, about half of theprotein failed to bind to the heparin column (lane 8).This was not because the column was saturated, asNS3 protein in the flow-through still could not bindto a regenerated column. The unbound NS3 proteinwas probably different from the bound one in itsstructural and biochemical properties. Only theheparin-bound protein was used for subsequentbiochemical experiments. Purified NS3 protein wasfound to be highly homogeneous using SDS-PAGE(Fig. 1) and had a 280 nm/260 nm absorbance ratioof 1.8, indicating that it was free of nucleic acidcontamination.

NS3 dimer forms a 1:1 complex with a 16-bpdsRNA

To assess the oligomeric state of NS3, we treatedthe protein sample with the homobifunctional cross-linking agent ethylene glycol bis(sulfosuccinimidyl-succinate) (sulfo-EGS), which reacts with the freeamine group. Chemical cross-linking led to theappearance of a new species found by SDS-PAGEthat corresponded to the dimeric form (Fig. 2a),suggesting that NS3 predominantly forms a dimerin solution.We further analyzed the molecular size of NS3 in

the free state and dsRNA-bound state using size-exclusion chromatography (Fig. 2b and c). The freeNS3 protein eluted as a single peak, but the peakposition was dependent on the concentration ofloaded protein. The apparent molecular weight(MW) of NS3, calculated based on the calibrationcurve, shifted downward from 59.5 to 45.6 kDawhen the sample was diluted from 62 to 5 μM. Sincethe NS3 monomer has a MW of 24.7 kDa, theseresults suggest that NS3 forms a dimer in solution,

but that the dimer can further aggregate weakly in aconcentration-dependent manner.To assess the stoichiometry of the NS3–dsRNA

complex, NS3 in a dilute concentration of 6 μM wasmixed with a 16-bp dsRNA (SD-16B) and subjectedto size-exclusion chromatography. The resultantelution profile displayed two peaks at 14.4 and18.8 ml. These peaks should both contain RNA, asthe absorbance at 260 nmwas two times higher thanthat at 280 nm. The 14.4-ml peak corresponded to a54.1-kDa species, which was likely composed of aNS3 dimer (49.4 kDa) bound to a 16-bp RNA duplex(9.8 kDa). The 18.5-ml peak corresponded to a 17.4-kDa species, which was likely from unbounddsRNA with an elongated structure.

NS3 dimer is stable against subunit dissociation

To assess the stability of NS3 dimer, we attemptedto study the subunit exchange between two distin-guishable NS3 dimers. We prepared a maltosebinding protein fusion of NS3 (MBP-NS3,MW=67.6 kDa) that could be distinguished fromHis-tagged NS3 (His-NS3, MW=24.7 kDa) by theirMW.WhenMBP-NS3andHis-NS3were coexpressed,a single species composed of MBP-NS3 and His-NS3could be purified by tandem Ni–NTA and maltoseaffinity chromatography. This confirms that NS3forms a dimer rather than higher-order multimers,otherwise the tandem-affinity purification wouldyield heterogeneous NS3 complexes in the latter case.To examine the subunit exchange of NS3 dimer,

we mixed MBP-NS3 and His-NS3. If the subunitsdissociate and reassociate, the MBP-NS3/His-NS3heterodimer would result. The three types of NS3dimer can be quantified by the formation of siRNAcomplex and native gel separation (Fig. 2d).However, even after incubation up to 22 h, therewas little heterodimer formed when the proteinconcentrations were in the range of 0.1 to 1000 nM.This result shows that NS3 dimer is stable againstsubunit dissociation.

NS3 interaction with dsRNAs of various lengths

Previous studies have already shown that the NS3proteins from RHBV and RSV bind the siRNAduplex.17,32 To further define the dsRNA bindingmode, we used electrophoresis mobility shift assays(EMSA) to characterize the interaction of RSV NS3with a series of dsRNAs with lengths ranging from 6to 100 bp. These dsRNAs labeled with 32P at the 5′-end were titrated with increasing concentrations ofNS3 protein and resolved in native polyacrylamidegels. Only a single RNA–protein complex (RNP)was observed for blunt-ended dsRNAs with lengthsup to 21 bp (Figs. 3a and 4a–b). For these RNAs, thefraction of bound RNA was fit to a one-site bindingmodel (Eq. 1) to obtain the apparent macroscopic

Fig. 2. NS3 forms a stable dimer. (a) SDS-PAGE of NS3 protein cross-linked with sulfo-EGS. (b) Gel-filtration profiles ofNS3 and its complex with a 16-bp dsRNA SD-16B. The continuous lines 1–3 are the profiles of free NS3 at 62, 25, and 5 μM,respectively. Dashed line 4 is the profile of NS3 (6 μM) in complex with dsRNA SD-16B. (c) Calibration curve of theSuperdex 200 10/30 column and the derived apparent MW for each sample. MW is shown in the logarithmic scale. Thestandards are lysozyme (14 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and bovine serum albumin(67 kDa). (d) Subunit exchange of NS3 dimer. MBP-NS3 and His-NS3 at 0.1, 1, 10, 100, and 1000 nM were mixed andincubated for 0.5–22 h on ice. All samples were adjusted to 10 nM, assembled with 32P-labeled siRNA, and resolved in anative gel. However, the final protein concentrations of the 0.1 and 1 nM samples were less than 10 nM because of proteinloss during the concentrating process. The siRNA complexes formed byMBP-NS3 homodimer, His-NS3 homodimer, andMBP-NS3/His-NS3 heterodimer were indicated.


dissociation constantKd (seeMaterials andMethods).The representative autoradiograph and fitting curvefor a 9-bp blunt-ended dsRNA are shown in Fig. 3.The obtained Kd values for various dsRNAs are listedin Table 1.Notably, the RNA binding curve at the transition

zone appears more cooperative than the one-sitebinding model predicts, even for dsRNAs that onlyaccommodate a single NS3 dimer. It seems thatadditional protein concentration-dependent eventsoccur at the transition zone. One possibility is the

dissociation of NS3 dimer. However, the abovesubunit exchange assay shows that NS3 dimerremains stable at the protein concentration rangeof 0.1–1000 nM. Hence, we can exclude thecontribution of the NS3 monomer–dimer transitionto the RNA binding process. Alternatively, proteinaggregation and protein sticking to the tube maycontribute to the deviation of the binding data froman ideal two-state transition.RSV NS3 binds strongly to a 21-nt siRNA duplex

with overhangs, with a Kd value of 2.4±0.9 nM. This

image of Fig. 2


value is very similar to that measured for RHBVNS3.17 The affinity was found to decrease gradually(Kd=0.7–11 nM) as the length of a series of blunt-ended duplexes was reduced from 21 to 9 bp.However, the affinity decreased abruptly by∼20-fold when the duplex size was changed from 9to 8 bp, and no binding was detected for a 6-bpduplex. We conclude that the NS3 dimer contactsminimally a 9-bp region in dsRNA and does notspecifically recognize the size of dsRNA as long as itcontains a full binding site. The increased affinity inlonger dsRNA is ascribed to the non-sequence-specific nature of the NS3–dsRNA interaction (seefollowing section).The size-independent interaction of NS3 with

dsRNA suggests that dsRNAs having a sufficientlength can simultaneously accommodate more thanone NS3 dimer. Indeed, a second slow-migratingspecies, RNP2, was observed for a 21-bp duplex with2-nt 3′-overhangs at protein concentrations greaterthan 62 nM (Fig. 4c). The gradual appearance of theRNP2 species was concomitant with the disappear-ance of the RNP1 species as the NS3 proteinconcentration was increased. Apparently, the formerwas converted from the latter as a result of its

Fig. 3. NS3 interaction with a 9-bp dsRNA. (a)Representative autoradiography of an EMSA. The 9-bpdsRNA was formed by annealing RNAs Si-9a and Si-9b.After being labeled with 32P at the 5′-end, the dsRNA wastitrated with NS3 protein of increasing concentrations, asindicated above each lane, in a 10-μl solution containing100 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NP-40, and25 mMHepes-K (pH 7.5). The reactions were incubated onice for 20 min and resolved on a 5% native gel. (b) Analysisof the EMSA data with a one-site binding model. Thecontinuous line is the best fit to Eq. 1 with Kd=7.1 nM.

association with a second NS3 dimer. The RNP1-to-RNP2 transition occurred at lower protein concentra-tions when the duplex was elongated (Fig. 4d and e).We observed a successive binding pattern for a

100-bp dsRNA, although the complexes bound withmore than twoNS3 dimers were not well resolved inthe native gel (Fig. 4f). Increasing amounts ofcomplex were retained at the wells at higherconcentrations of the protein likely because of non-specific aggregation. The association of more thantwo NS3 dimers indicates that NS3 can bind to theinternal region of long dsRNAwithout requiring theterminal structure of the duplex.

Quantitative analysis of NS3–dsRNA bindingisotherms

RSVNS3 does not recognize the sequence or size ofdsRNA. Classic models describing the non-sequence-specific binding of large ligands to a homogenousone-dimensional lattice could be applied to quanti-tatively analyze this type of interaction.30,37 McGheeand von Hippel derived a closed expression based onthe conditional probability approach to describe thecooperative and noncooperative binding of ligandsto an infinite lattice.30 As the short dsRNAs used inour experiments could not be treated as infinitelattices, we employed the exact combinatorialexpression for finite lattices to analyze the bindingisotherms of dsRNAs that accommodate two NS3dimers.37–39 This type of ligand–lattice interactingsystem is characterized by the lattice length M, thetotal site size of the ligand n, the interaction site size c,the intrinsic binding constant Kint, and the coopera-tivity parameter ω. Each base pair of dsRNA isregarded as a repeating unit of the lattice. The totalsite size of a ligand refers to the number of base pairsoccluded from further binding upon binding of aligand. The lattice length that is actually interactingwith the ligand may be less than the total occludedsite size and is termed the interaction site size c. Theintrinsic binding constant Kint describes the interac-tion between the ligand and an isolated singlebinding site. The cooperativityω is a positive unitlessfactor that describes the relative change in bindingconstant when a ligand is bound next to an alreadybound ligand compared with an isolated site. Thebinding is regarded as cooperative when ωN1,anticooperative when ωb1, and noncooperativewhen ω=1.Non-sequence-specific ligand–lattice interactions

are complicated by the overlap of potential bindingsites on the lattice. For example, an M-site latticecontainsM-c+1 binding sites for a ligand that has aninteraction site size of c. As a result, the observedmacroscopic binding constantKa (the inverse ofKd) isthe product of the intrinsic binding constant Kint anda statistical factor that is equal to the number ofbinding sitesM-c+1 (Eq. 2).37,40 A fit of the Kd values

image of Fig. 3

Fig. 4. Interaction of NS3 with long dsRNAs. Repre-sentative EMSA gels of NS3 with (a) 20-bp blunt-endedRNA SD-20B, (b) 21-bp blunt-ended dsRNA Si-1/Si-4, (c)21-bp dsRNA Si-42/Si-43 with 3′-overhangs, (d) 22-bpdsRNA Si-29 with 3′-overhangs, (e) 30-bp RNA Si-19/Si-20 with 3′-overhangs, and (f) 100-bp dsRNA. Theconcentrations of NS3 protein are indicated above eachlane. RNP1 and RNP2 refer to the RNA complexes boundby one or two NS3 dimers, respectively.

Table 1. Apparent macroscopic disassociation constant(Kd) for RSV NS3 and oligonucleotides

Structurea Oligob Kd (nM)c

6-bp dsRNA SD-6B No binding8-bp dsRNA SD-8B 235±36.89-bp dsRNA Si-9a/Si-9b 11.0±4.610-bp dsRNA SD-10B 8.8±5.311-bp dsRNA SD-11B 3.5±1.312-bp dsRNA SD-12B 3.5±1.216-bp dsRNA SD-16B 2.7±1.020-bp dsRNA SD-20B 2.3±0.421-bp dsRNA Si-1/Si-4 0.7±0.219-bp+ov dsRNA Si-1/Si-2 2.4±0.919-bp+ov dsDNAd Dsi-1/Dsi-2 50.2±27.919-bp+ov DNA/RNAd Si-1/Dsi-2 4.8±1.221-nt ssRNA Si-1 28.8±11.921-nt ssDNA Dsi-3 No binding

a Duplexes are blunt-ended by default, and those having 2-nt3′-overhangs are indicated by “+ov”.

b The sequences of oligos are listed in Table 2. One oligo isindicated for self-complementary duplexes, whereas two oligosare indicated for non-self duplexes.

c The reportedKd is themean of three or twomeasurements±SD.d Although the EMSA gels of the dsDNA and RNA/DNA

hybrid showed clear association of a second NS3 dimer at highprotein concentrations, the reported Kd values were derivedwithout taking into account the multiple binding. The amount ofbound RNA was calculated simply as the sum of one-NS3 andtwo-NS3 bound complexes.


for 9- to 21-bp dsRNAs as a function of duplex lengthto Eq. 2 yielded Kint=(8.0±0.8)×10

7 M−1 (Fig. 5).The minimal length of dsRNA required for NS3

binding suggests that its interaction site size c is 9.The total site size n must be greater than c becausethe 21-bp blunt-ended duplex, which has more thantwice the minimal binding size, was not capable ofbinding a second NS3 dimer (Fig. 4b). This indicatesthat part of the NS3 structure, hereafter termed theborder, does not directly contact dsRNA butoccludes a number of nucleotide residues forbinding. As an example of ligand with borderregion, E. coli helicase PriA also has an occluded

site size larger than its minimal binding size onssDNA.39,41 When such a ligand binds at the end ofa lattice, the border region protrudes out, reducingthe effective site size. For the purpose of quantitativeanalysis, we assumed that the border regions of NS3dimer are evenly partitioned around its centrallattice-interacting region, with a size of b on eachside (Fig. 6a). It follows that n= c+2b. In addition,the combinatorial analysis requires that the total sitesize is an integer value.We attempted to estimate the total site size n of the

NS3 dimer based on the EMSA results for dsRNAs ofvariable lengths. If n=12, then a 21-bp blunt-endedduplex would be able to accommodate two NS3dimers on consideration of the border effect. How-ever, the second RNP was not observed (Fig. 4b),indicating that n should be greater than 12. We foundthat a 21-bp duplex with 2-nt 3′-overhangs wascapable of binding a second NS3 dimer. Calculationof the lattice length of this RNA was not straightfor-ward because of its single-stranded overhangs. Theoverhanging regions totaled 4 nt in length but couldnot be literally counted as 4 lattice sites. This isbecause NS3 binds ssRNA with a much weakeraffinity (see following section). We assumed that theoverhang was equivalent to v sites (v=1, 2, 3, or 4). Agrid search showed that the overhangs were opti-mally accounted for by one site and that the site sizeof NS3 dimer was around 13 bp. After correction ofthe overhang effect, the 21-bp duplex with 2-nt 3′-

image of Fig. 4

Fig. 5. Macroscopic Kd values of one-NS3 bindingdsRNAs as a function of duplex length. The continuousline is the best nonlinear fit to Eq. 2 withKint=(8.0±0.8)×10

7.The interaction site size of NS3 dimer was fixed at c=9.


overhangs was equivalent to a 22-bp blunt-endedduplex. Fig. 6b illustrates the scenario in which adsRNA lattice of 22 sites exactly fits two NS3 ligandsof n=13 and b=2.The border region present in NS3 dimer also

complicated the analysis of binding data. A ligandwith border would display a smaller effective sitesize (n−b) when bound at the end of a lattice thanwhen bound internally (Fig. 6c). The border effectneeds be corrected for short lattices that accom-modate only a few ligands. An early approachmodified the site size to n−2b in the case of one-ligand binding and to n−b in the case of two-ligand binding.39,41 Such a correction cannot bereadily applied to lattices that can accommodatemore than two ligands. In the present work, we

Fig. 6. Schematic of the NS3 ligand with border regions andrepresented by a box and two protruding borders. The box denthe borders refer to the parts of the NS3 structure that do not coside of the ligand for further binding. (b) Illustration showingand b=2. Black bar represents the dsRNA lattice. (c) Ligand wbinding at the end of the lattice. The ligand border effect couldsites at both ends. The extensions are represented as gray bar

took a different approach to correct the ligandborder effect by extending the nominal length ofthe lattice at each end by b sites while keeping theligand site size constant along the whole lattice(Fig. 6c). The lattice extensions were created tovirtually contact the border region of the ligandbound at the lattice end, so that the ligand couldbe treated as if it did not have a border. Theconsequence of extending a lattice is equivalent tothe previous correction made for the ligand sitesize for the cases of one- and two-ligand binding,but our approach should be applicable to anynumber of bound ligands and is also uniform intreatment.For the lattice that can accommodate two, but not

three, ligands, Eqs. 3–6 can be derived to relatefractions of the free RNA, one-ligand complex, andtwo-ligand complex to the concentration of freeligand L and binding parameters M, n, Kint, and ω(see Materials and Methods).37,39 We applied theseequations to globally fit the binding data of 21-, 22-,and 30-bp dsRNAs with overhangs to derive theparameters Kint and ω. The lattice length M wascorrected for the ligand border effect and the dsRNAoverhang effect, as discussed above. M and n werefixed in the fit and their optimal values were foundusing a grid search. The minimal fitting residual wasachieved when n=13 and v=1. The global fittingresults, shown in Fig. 7, yielded Kint=1.0×10

8 M−1

and a cooperativity parameter of ω=0.5. This Kintvalue agreed with that obtained from the analysis ofone-NS3 binding isotherms for 9- to 21-bp dsRNAs(Kint=8.0×10

7 M−1). More importantly, this analysisindicates that there is virtually no or a slightlynegative cooperativity among adjacently boundNS3 dimers.

its interaction with dsRNA lattice. (a) The NS3 structure isotes the core region that directly contacts dsRNA, whereasntact dsRNA but occlude a length of b base pairs on eitherthat a 22-bp dsRNA exactly fits two NS3 dimers with c=9ith border regions having a smaller site-size (n−b) whenbe corrected by increasing the nominal lattice length by b

s at the end of the lattice.

image of Fig. 5

Fig. 7. Global fitting of the EMSA data of dsRNAsbound by twoNS3 dimers. The binding data of the 21-, 22-,and 30-bp dsRNAs with overhangs were analyzed.Experimental measurements of the fraction of free RNA,one-NS3 bound RNA, and two-NS3 bound RNA aredrawn as circles, squares, and inverted triangles, respec-tively. The continuous lines are global nonlinear leastsquare fits of Eqs. 3–6, with Kint=1.0×10

8 M−1 and ω=0.5.The total site size was fixed at n=13, and the effectivelattice length was fixed at M=26, 27, and 35 for the threedsRNAs, respectively, after correcting for the border effect(2b=4) and the overhang effect (v=1).


Specificity for the dsRNA structure

To examine the structural features of dsRNArecognized by NS3, we compared its bindingaffinity to dsRNA, DNA/RNA hybrid, dsDNA,ssRNA, and ssDNA, which are all 21 nt in length(Fig. 8; Table 1). The ssRNA was bound ∼10-foldweaker (Kd=28.8±11.9 nM) than the dsRNA,suggesting that the helical structure of dsRNAwas recognized. The 2′-hydroxyl group of ssRNAappeared to be critical, as the ssDNA was barelybound (Fig. 8e). The dsDNA was also bound∼20-fold weaker than the siRNA duplex. This waslikely due to the lack of the 2′-hydroxyl group inDNA and their different helical structures (B-form inDNA versus A-form in RNA). Interestingly, theDNA/RNA hybrid duplex had a similar bindingaffinity (Kd=4.8±1.2 nM) compared with thedsRNA duplex. Two factors likely account for thisobservation. First, the RNA/DNA hybrid anddsRNA duplex share an A-form helical structurethat is recognized by NS3. Second, NS3 mightprimarily contact only the RNA strand in theRNA/DNA hybrid duplex and is hence less affectedby the other strand being DNA. This prediction isconsistent with a significant interaction betweenNS3 and ssRNA. Notably, a two-NS3 boundcomplex was observed for the 21-nt dsDNA andDNA/RNA hybrid, but not for the 21-nt dsRNA.This suggests that the total site size of NS3 is smallerfor duplexes that have at least one DNA strandcompared with dsRNA.

dsRNA binding mutants of NS3

Nucleic acid-binding proteins frequently use basicarginine and lysine residues to contact negativelycharged phosphate groups through electrostaticinteractions. To identify the dsRNA-binding resi-dues of NS3, we replaced a few arginine and lysineresidues with glycine or positively charged aspartateand glutamate residues. Their binding affinities witha 21-nt siRNA duplex were measured using EMSA(Fig. 9a). The single-site mutants R94G, K127G,K165G, and R169E had Kd values (within experi-mental uncertainty) similar to that of the wild-typeprotein, suggesting that these respective residues arenot involved in dsRNA interaction. In contrast, thesingle-site mutants R50G, K77G, K112G, R124G, andR190G and the double mutant K173G/K174G dis-played an 11- to 25-fold decrease in binding affinity,suggesting that these respective residues contacteddsRNA via electrostatic interactions. A triple mutantwith a stretch of basic residues K173–K174–R175replaced by negatively charged Glu–Asp–Glushowed the most dramatic reduction (∼1000-fold)in affinity. This was likely due to the repulsive forcebetween the introduced acidic residues and thephosphate group of RNA, as well as the additive

effect of the mutation on residue R175. Thesemutants also provide a useful tool for assessing thefunctional role of the dsRNA-binding ability of NS3.

RNAsilencing suppressor activity of NS3mutants

To examine the relationship between the dsRNA-binding activity and suppressor activity of NS3, weanalyzed these mutants for their suppression ofdsRNA-induced GFP silencing using an agrofiltra-tion assay (Fig. 9b). Nicotiana benthamiana plantswere co-infiltrated with strains of Agrobacteriumcontaining three vectors that encoded a reportergene, a silencing trigger, and a silencing suppressor.The first vector expressed a reporter GFP proteindriven by the 35S promoter. The second vector

image of Fig. 7


contained part of the antisense sequence of the GFPgene followed by the sense sequence of GFP. In thiscase, the transcript would fold back into a longhairpin that would be processed into GFP-targetingsiRNA. The third vector encoded an RNA silencingsuppressor for testing. The infiltrated leaves werephotographed under UV illumination at 3 dayspost-infiltration (dpi). Regions with GFP expressiondisplayed green fluorescence under UV light, whilethose from unfiltrated regions appeared red owingto chlorophyll autofluorescence.Infiltration of the 35S-GFP vector with the two other

empty vectors induced GFP expression at 3 dpi. Co-infiltration with the GFP-dsRNA expressing vectorblocked GFP expression, indicating an active RNAsilencing mechanism in the plant. The expression ofGFP was efficiently restored in the presence of wild-type NS3 and another RNA silencing suppressor, P19,of Cymbidium ringspot virus, as expected.9,32The suppressor activities of 11 NS3 mutants were

examined and found to generally correlate withtheir dsRNA-binding affinities. Mutants that werenormal in dsRNA binding (R94G, K127G, K165G,and R169E) maintained their ability to efficiently

Fig. 8. EMSA of NS3 with different nucleic acidstructures. Representative autoradiograms are shown for(a) 21-nt dsRNA Si-1/Si-2, (b) 21-nt dsDNA Dsi-1/Dsi-2,(c) 21-nt RNA/DNA hybrid Si-1/Dsi-2, (d) 21-nt ssRNASi-1, and (e) 21-nt ssDNADsi-3. All nucleic acid constructswere 21 nt in length, and their sequences are shown inTable 2.

inhibit GFP silencing. On the other hand, mutantsthat were defective in dsRNA binding (R50G, K77G,K112G, R124G, K173G/K174G, K173E/K174D/R175E, and R190G) were also defective in thesuppression of GFP silencing. We conclude thatthe in vivo suppressor activity of NS3 is correlated toits dsRNA binding ability, providing a mechanisticlink between the two activities.

Discussion

The divergent sequences among dsRBSs suggestthat they each adopt a unique structure andrecognize dsRNA in different ways. Elucidationof the dsRNA binding mode of dsRBSs isimportant to understand their specific mechanismof silencing suppression and other physiologicalfunctions. In this study, we have shown bychemical cross-linking, gel-filtration chromatogra-phy, and subunit exchange assay that RSV NS3 isa stable dimer in solution, and that one dimerbinds to a 16-bp dsRNA (Fig. 2). EMSA on a seriesof dsRNA probes allowed us to define the minimalbinding size of NS3 to be 9 bp. We show thatdsRNAs of 9 to 21 bp in length accommodate onlya single NS3 dimer, and dsRNAs longer than21 bp can associate simultaneously with two ormore NS3 dimers (Fig. 4). The thermodynamicparameters of the NS3–dsRNA interaction havebeen derived from a global analysis of two-NS3binding data using the combinatorial model. Ouranalysis shows that NS3 has an intrinsic bindingconstant of 1.0×108 M−1, an occluded site size of∼13 bp, and no cooperativity in dsRNA binding.The analysis assumes that NS3 dimer binds with

equal affinity to overlapped binding sites in adsRNA lattice. NS3 should not recognize the RNAsequence, as many short dsRNAs of diversesequences tested for binding show consistent Kdvalues. Although NS3 can bind to the middle of theduplex as shown for a 100-bp dsRNA, current datacannot exclude the possibility that NS3 has adifferent affinity towards the end versus the middleof the duplex.Our results demonstrate that RSV NS3 does not

specifically recognize the size of dsRNA. The ability tobind long dsRNA suggests that RSV NS3 has thepotential to protect dsRNA from Dicer cleavage, ashas already been demonstrated in vitro for B2.11,12 ThedsRNA binding of RHBV NS3 has been previouslyanalyzed as a C-terminal fusion to themaltose bindingprotein.17 RHBV NS3 and RSV NS3 are both dimericproteins and bind the 21-nt siRNA duplex with nearlyidentical affinities (Kd=∼2 nM). However, the previ-ous study concluded that RHBV NS3 specificallytargets the 21-nt siRNA duplex, as a 26-nt siRNAduplex was observed to bind poorly in comparison.17

In the previous EMSA gel, we noticed that the 26-nt

Table 2. Name, size, and sequence of the oligos used in this study

Namea Size (nt) Sequenceb

Si-1 21 5′-CGUACGCGGAAUACUUCGAUU-3′Si-2 21 5′-UCGAAGUAUUCCGCGUACGUU-3′Dsi-1 21 5′-CGTACGCGGAATACTTCGATT-3′Dsi-2 21 5′-TCGAAGTATTCCGCGTACGTT-3′Dsi-3 21 5′-AAAGGTGGAAAAGGTGGAAAA-3′SD-6B 6 5′-CCCGGG-3′SD-8B 8 5′-CCCCGGGG-3′Si-9a 9 5′-AGCGUGACU-3′Si-9b 9 5′-AGUCACGCU-3′SD-10B 10 5′-CGCGGCCGCG-3′SD-11B 11 5′-CGCGGUCCGCG-3′SD-12B 12 5′-CGACGGCCGUCG-3′SD-16B 16 5′-CGUAGCGGCCGCUACG-3′SD-20B 20 5′-CGUUAGGCGGCCGCCUAACG-3′Si-4 21 5′-AAUCGAAGUAUUCCGCGUACG-3′Si-42 23 5′-GAAGUAGUAAUUGUCGCUCUCCU-3′Si-43 23 5′-GAGAGCGACAAUUACUACUUCUG-3′Si-29 24 5′-CGUACGUAAGCGCUUACGUACGUU-3′Si-19 32 5′-CGUACUCGAGAUAUCCUAGCUGGACUCUGAUU-3′Si-20 32 5′-UCAGAGUCCAGCUAGGAUAUCUCGAGUACGUU-3′

a All oligos are RNA except for Dsi-1, Dsi-2, and Dsi-3, which are DNA.b Self-pairing regions are underlined.


siRNA formed two RNP complexes at high concen-trations of the RHBV NS3 protein. However, this typeof binding pattern was not interpreted as a successionof two binding events.17 In light of our result, RHBVNS3 is likely also a size-independent dsRNA bindingprotein that can associate with the 26-nt siRNA in twocopies. It would be surprising to find that RSV NS3and RHBV NS3, which share 43% sequence identityand 61% similarity, have different RNA bindingproperties.Gaining an understanding of the action mecha-

nism of size-independent dsRBSs is complicated bytheir dual ability to bind both siRNA and longdsRNA. The latter activity could interfere with anupstream step in the Dicer processing of longdsRNA. The presence of cooperativity in a dsRBSwould strongly suggest that it naturally targets longdsRNA, as this character is of no use for bindingshort siRNA that only accommodates a singleprotein molecule. The correlation between coopera-tivity and dsRNA binding targets has recently beendemonstrated in a comparison between twodsRNA-binding proteins in the RNAi pathway.42,43

Most Dicer enzymes require a dsRNA-bindingprotein as a partner, such as RDE-4 in C. elegansand trans-activation response RNA-binding protein(TRBP) in mammals. However, RDE-4 and TRBPplay different roles in siRNA processing. RDE-4 isessential for Dicer to cleave long dsRNA into siRNA,while TRBP functions downstream to load siRNAinto the RNA-induced silencing complex. Consistentwith their different roles, RDE-4 preferentially bindslong dsRNA with cooperativity, while TRBP bindssiRNAwith high affinity and has no cooperativity inbinding long dsRNA.42,43

We have carried out the first quantitative coop-erativity analysis of a size-independent dsRBS.However, we find that NS3 lacks cooperativity.One simple interpretation of this result is that NS3 isnot designed to target long dsRNA, and siRNA ismore likely the primary target of NS3. The highaffinity to siRNA (Kd=2.4 nM) would allow NS3 tosequester siRNA. Alternatively, the high intrinsicaffinity of NS3 might make cooperativity less criticalfor NS3 to bind long dsRNA. Lack of cooperativitymeans that it is difficult for NS3 to form acontinuous protein cluster on long dsRNA. This isbecause, in this case, random association of theligand will create unoccupied gaps on the lattice thatare not large enough to accommodate a newligand.30,31 It is unknown whether or not othersize-independent dsRBSs bind cooperatively to longdsRNA.11–13,15,18,28 Two dsRBSs, B2 and DCV-1A,have been shown to bind long dsRNA with higheraffinities than siRNA,11,16 suggesting that theirbinding may be cooperative.We identified eight residues that are important for

dsRNA binding: R50, K77, K112, R124, K173, K174,R175, and R190 (Fig. 9). These basic residues likelyconstitute the RNA-binding surface and makecontact with the phosphate group of RNA. Thesemutants are defective in dsRNA binding and arealso impaired in the suppression of dsRNA-inducedGFP silencing in plants, indicating that dsRNAbinding of NS3 is responsible for its suppressionactivity. In previous work, we have shown that atriple-alanine mutation of residues K174, K175, andR175 (NS3/3A) displays reduced activity in sup-pressing both local and systemic GFP silencing. Wehave also shown that these three basic residues are

Fig. 9. The siRNA binding activity and silencing suppression activity of NS3 mutants. (a) Gel-shift titrations of a 21-ntsiRNA duplex, Si-1/Si-2, with NS3 mutants. The concentrations of NS3 proteins were 0.25, 2.5, 25, 250, and 2500 nM. Thebinding buffer contained 25 mMHepes-K (pH 7.5), 300 mMKCl, 2 mMMgCl2, 1 mMDTT, 0.01% NP-40, and 5% glycerolin a 10-μl volume. The derived Kd values are indicated. (b) Silencing suppression activity of NS3mutants. Leaves of theN.benthamiana plant were co-agroinfiltrated with a GFP-expressing vector (GFP); a vector encoding GFP-targeting dsRNA(dsGFP); and a vector encoding P19 (as a positive control), wild-type NS3, or mutant NS3. Empty vectors are denoted as“V”. The leaves were photographed at 3 dpi under a hand-held long-wavelength UV illuminator.


image of Fig. 9


critical for nuclear localization of the GFP-NS3fusion protein.32 Our new results suggest that thethree basic residues play an additional role indsRNA binding. In homologous RHBV NS3, theequivalent residues have also been found to becritical for siRNA binding and RNA silencing.44

NS3 exhibits only moderate structural specificityfor dsRNA with significant affinity to 21-nt ssRNA(Kd=28.8±11.9 nM), dsDNA (Kd=50.2±27.9 nM),and RNA/DNA hybrid (Kd=4.8±1.2 nM) (Table 1).In contrast, the dsRBSs P19, B2, and 2b all possessstrict specificity for the dsRNA structure and cannotbind ssRNA.9,12,18,25,26 B2 also distinguishes stronglyagainst dsDNA and RNA/DNA hybrid.12 In termsof structural specificity, NS3 is similar to P21, whichalso interacts with various nucleic acid structures.28

Further understanding of the size-independent andunique recognition mode between NS3 and dsRNAmust await the determination of their complexstructure.

Materials and Methods

Plasmid construction

The RSV NS3 gene was PCR-amplified from plasmidpBin438-35S-NS345 with the primers RSVNS3_NcoI_N1(5′-TATCCATGGGCAACGTGTTCACATCGTC-3′, therestriction site is underlined) and RSVNS3_C6H_EcoRI_L211 (5′-CCGGAATTCTTAATGATGATGAT-GATGATGCAGCACAGCTGG-3′). The PCR productwas digested by NcoI and EcoRI restriction enzymesand then purified and cloned into pET28a, resulting in thepET28a-NS3 plasmid. Owing to the NcoI cloning, an extraglycine residue was introduced after the starting Metresidue, but the residues were numbered as in the wild-type sequence (211 residues). In addition, six histidineresidues encoded in the reverse primer were added to theC terminus of NS3 to facilitate affinity purification. Site-directed mutations were generated on pET28a-NS3 withQuikChange (Stratagene), using the appropriate primers.The NS3 gene was also cloned into a modified pMal-c2xplasmid (New England Biolabs), in which NS3 wasfused to the C terminus of MBP by a linker containinga PreCission cleavage site, yielding the pMal-NS3plasmid.

Protein expression and purification

His-tagged NS3 was expressed in E. coli Rossetta2 (DE3)cells at 16 °C after induction with 0.4 mM isopropyl-β-D-thiogalactopyranoside. Cells harvested from a 4-L culturewere resuspended in 100 ml of buffer H300 [0.3 M KCl, 5%glycerol, 20 mM Hepes-K (pH 7.6)] and 25 mM imidazole.This solutionwas then lysed using sonication. The cell lysatewas centrifuged at 30,000g for 60 min at 4 °C. Thesupernatant was loaded onto a 5-ml HisTrap column andthen washed with buffer H300 and 50 mM imidazole inH300. The bound protein was eluted with 500 mM

imidazole in H300. Fractions containing NS3 were pooledand loaded directly onto a 5-ml heparin column (GEHealthcare) equilibrated in buffer H100 [0.1 M KCl, 5%glycerol, 20 mM Hepes-K (pH 7.6)]. The bound protein waswashed with buffer H300 and eluted at ∼0.6 M KCl in alinear 0.3–1MKCl gradient in buffer H [5% glycerol, 20 mMHepes-K (pH 7.6)]. The purified protein was supplementedwith 5 mM dithiothreitol, divided into aliquots, flash-frozenin liquid nitrogen, and stored at −80 °C. The final yield wasabout 3–4 mg per liter of culture. His-tagged NS3 was usedin biochemical assay by default.MBP-NS3 was expressed in a similar way as His-NS3.

The clarified cell lysate was loaded into a maltose affinitycolumn and eluted with 20 mM maltose in buffer H300.The protein was further purified by heparin chromatog-raphy. For the His-NS3/MBP-NS3 heterodimer, plasmidspET28a-NS3 and pMal-NS3 were cotransformed into E.coli Rosetta2 (DE3) which was cultured in mediumcontaining ampicillin and kanamycin. The heterodimerwas coexpressed and copurified through Ni–NTA affinity,amylose affinity, and heparin chromatography. Theprotein concentration of NS3, expressed in the monomericform, was determined by absorbance at 280 nm and amolar extinction coefficient of 24,410 M−1 cm−1 for His-NS3 and 90,760 M−1 cm−1 for MBP-NS3.

Size-exclusion chromatography

The apparent molecular masses of NS3 and its RNAcomplex were analyzed with a Superdex 200 10/30 GLcolumn (GE Healthcare). The column was equilibrated at4 °C in a running buffer containing 300 mM KCl, 5%glycerol, and 20 mM Hepes-K (pH 7.6). The calibrationcurve was based on the following standard proteins:lysozyme (14 kDa), chymotrypsinogen A (25 kDa), oval-bumin (43 kDa), and bovine serum albumin (67 kDa). Thelogarithm of molecular mass of the standards was fit to alinear function of elution volume using OriginPro 8. Theinitial NS3 sample had a concentration of 62 μM (1.5 mg/ml) and was diluted to 25 and 5 μM with the runningbuffer. The NS3–RNA complex was formed by mixing3 nmol NS3 and 2 nmol SD-16B RNA. Each sample wasloaded in 500 μl.

Cross-linking

NS3 samples of 0.1 mg/ml were incubated with 0, 0.2,0.4, and 0.8 mM sulfo-EGS (Pierce) in 20-μl reactionscontaining 25 mM Hepes-K (pH 7.6) and 300 mM KCl for30 min at room temperature. The reactions were quenchedby adding 1 μl of 1 M Tris–HCl (pH 7.5) and resolved in a4–20% gradient SDS-PAGE gel.

Subunit exchange assay of NS3 dimer

Equal molar amounts of MBP-NS3 and His-NS3, 0.1, 1,10, 100, and 1000 nM, were mixed in a buffer containing100 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NP-40, and25 mMHepes-K (pH 7.6) and incubated on ice. Samples ofappropriate volume were frozen in liquid nitrogen at 0.5,1, 2, 4, 8, and 22 h. The 0.1 and 1 nM samples wereconcentrated to ∼10 nM, whereas the 100 and 1000 nM


samples were diluted to 10 nM. Twenty microliters ofthese 10 nM samples was mixed with 32P-labeled 21-ntsiRNA (Si-1/Si-2). After incubation on ice for 20 min, thereactions were resolved in a native gel and visualized byautoradiography.

RNA preparation

Short RNAs were chemically synthesized by Dharmaconor Takara. DNA oligos were purchased from Invitrogen. Thetwo strands of 100-bp dsRNA were prepared by in vitrotranscription following standard protocols. The transcriptiontemplate for the sense strand RNA was amplified by PCRwith the primers S100-T7F (5′-CGCGTAATACGACTCAC-TATAGGGCATGGATATTCTCATCATTAGTTTG-3′) andS100-R (5′-CGGCTCCGGCTACTGC-3′) using the P25 geneas template.46 The template for the antisense strand RNAwas prepared with the primers AS100-F (5′-ATGGA-TATTCTCATCATTAGTTTG-3′) and AS100-T7R (5′-CGCGTAATACGACTCACTATAGGGCCGGCTCCGGC-TACTGC-3′). Transcribed RNAs were dephosphorylatedprior to 5′-labeling.

Electrophoretic mobility shift assay

Oligonucleotides were 5′-end labeled with T4 polynu-cleotide kinase (New England Biolabs) and [γ-32P]ATP(Furui Biotech, Beijing) in a 20- μl reaction at 37°C for40min and purified through aMicroSpin G-25 column (GEHealthcare). In EMSA, ∼0.1 nM of labeled oligos wasmixed with various amounts of NS3 in a 10-μl reactioncontaining binding buffer [100 mM KCl, 2 mM MgCl2,1 mM DTT, 0.01% NP-40, and 25 mM Hepes-K (pH 7.6)].The presence of NP-40 in the binding buffer was importantfor RNAbinding. In the binding reactions forNS3mutants,the binding buffer contained additionally 300mMKCl and5% glycerol to increase the solubility of somemutants. Thebinding reactions were incubated on ice for 20 min andresolved in a 5% native polyacrylamide gel run in 1× Tris–glycine buffer (pH 8.3) at 4 °C or room temperature. Thegels were dried and autoradiographed using a TyphoonPhosphorImager (GEHealthcare). The amounts of free andbound RNA molecules were measured by integrating thevolumes of the corresponding bands with ImageQuant(Molecular Dynamics). The fraction of each RNA specieswas calculated in comparison with the total amount ofRNA in that lane.

Analysis of one-NS3 binding data

For oligos that accommodated only one NS3 dimer, thefractions of bound RNA, Θ1, were fitted to the function:

Q1 = L= L + Kdð Þ ð1Þwhere Kd is the apparent macroscopic dissociationconstant and L is the concentration of free NS3 molecules(as monomer). Since the concentration of labeled RNAwas ∼0.1 nM in our experimental conditions, the amountof bound ligand was negligible and the free ligandconcentration L could be well approximated by theknown total concentration of ligand LT.

The macroscopic binding constant Ka (the inverse ofKd) is related to the microscopic intrinsic bindingconstant Kint by

37,40:

Ka = 1= Kd = M − c + 1ð ÞKint ð2Þwhere M is the lattice length and c is the interaction sitesize of NS3, which was fixed at 9 for NS3.

Analysis of two-NS3 binding data

We consider the case where the lattice containingM siteis capable of binding two n-site ligands, but is not longenough to bind three ligands (i.e., 2n≤Mb3n). Accordingto the exact combinatorial expression,37,39 the fraction ofRNA bound to i ligands, Θi (i=0, 1, 2), is given by:

Q0 = 1 =Z ð3Þ

Q1 = M − n + 1ð ÞKintL= Z ð4Þ

Q2 = ½0:5 M − 2n + 1ð Þ M − 2nð Þ KintLð Þ2

+ M − 2n + 1ð Þ KintLð Þ2x�= Z ð5Þwhere Kint is the intrinsic microscopic binding constantbetween the ligand and a single binding site, ω is thecooperativity factor between adjacent interacting ligands,L is the concentration of free ligand, and Z is thenormalization factor:

Z = 1 + M − n + 1ð ÞKintL

+ ½0:5 M − 2n + 1ð Þ M − 2nð Þ KintLð Þ2

+ M − 2n + 1ð Þ KintLð Þ2N� ð6ÞThe parameters Kint and ω were determined by

nonlinear global fit to Eqs. 3–6 of experimentallymeasured values Θi (i=0,1,2) for the 21-, 22-, and 30-bpdsRNAs with overhangs. L was approximated by LT. Thelattice length M was corrected as M0+2b+v, where M0 isthe length of the dsRNA duplex region, 2b is the correctionfactor for the ligand border effect, and v is the correctionfactor for the dsRNA overhang. The lattice length M andsite size n were fixed, but were subjected to a grid searchby varying the values of n and v. The site size nwas testedat values of 13, 14, and 15, which resulted in bordercorrections (2b) of 4, 6, and 8, respectively. We found thatwhen n=13, 14, and 15, the overhang correction factor vwas necessarily 1, 2, and 3, respectively. This ensured thatM≥2n and that cooperativity had a nonnegative value.The fitting residuals reached a minimum when n=13 andv=1. The nonlinear fit was carried out in MATLAB 6.1using home-written scripts.

RNA silencing suppression activity in plants

The RSVNS3wild-type and 11mutant genes were PCR-amplified from the corresponding pET28a-NS3 plasmidwith the primers NS3_BamHI-F (5′-GGATCCAT-GAACGTGTTCACATCGTCT-3′) and NS3_SalI-R (5′-GTCGACCTACAGCACAGCTGGAGA-3′) and thencloned into vector pGEM-T (Promega). The constructintegrity was confirmed by sequencing. The NS3 geneswere digested with BamHI and SalI and inserted into the


binary vector pBin438 between the 35S promoter andnopaline synthase terminator.32 Plasmids expressing 35S-GFP, an inverted repeat sequence of GFP (35S-dsGFP),and the Cymbidium ringspot virus P19 gene (35S-P19) weredescribed previously.32 All constructs were electroporatedinto A. tumefaciens strain C58C1 with a Gene Pulser IIsystem (Bio-Rad). For co-infiltration assays, A. tumefacienscontaining various plasmids were grown individually toan OD600 of 0.6–0.8. The cultures were pelleted andresuspended in an infiltration medium containing 20 mMMgCl2, 10 mM MES (pH 5.6), and 100 μM acetosyringoneto give a final OD600 of 1.0. Equal volumes of three A.tumefaciens cultures harboring the plasmids VSR, 35S-GFP,and 35S-dsGFP were mixed and infiltrated into leaf tissuesof 4-week-old N. benthamiana plants by using 1-mlsyringes. The leaves were photographed under UVillumination (UV Products) at 3 dpi using a Canon 400Ddigital camera with a 58-mm yellow filter.

Acknowledgements

We are grateful to Xinxing Yang for help inMATLAB programming. K. Ye was supported bythe Chinese Ministry of Science and Technologythrough the 863 and 973 projects and the BeijingMunicipal Government. X. Zhou was supported bythe National Natural Science Foundation of China(grant 30870110).

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