doi:10.1016/j.jmb.2010.10.007
J. Mol. Biol. (2010) 404, 665–679
Contents lists available at www.sciencedirect.com
Journal of Molecular Biology
j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b
Size-Independent and Noncooperative Recognition
of dsRNA by the Rice Stripe Virus RNA Silencing
Suppressor NS3
Mei Shen 1,2 , Yi Xu 3 , Ru Jia 2 , Xueping Zhou 3 and Keqiong Ye 2 ⁎
1
Graduate Program at Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100730, China
2
National Institute of Biological Sciences, Beijing 102206, China
3
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China
Received 30 September 2010;
accepted 6 October 2010
Available online
14 October 2010
Edited by D. E. Draper
Keywords:
cooperativity;
dsRNA-binding protein;
RNA silencing;
viral suppressor;
plant–virus interactions
Plant and animal viruses employ diverse suppressor proteins to thwart the
host antiviral reaction of RNA silencing. Many suppressors bind dsRNA
with different size specificity. Here, we examine the dsRNA recognition
mechanism of the Rice stripe virus NS3 suppressor using quantitative
biochemical approaches, as well as mutagenesis and suppression activity
analyses in plants. We show that dimeric NS3 is a size-independent, rather
than small interfering RNA-specific, dsRNA-binding protein that recognizes a minimum of 9 bp and can bind to long dsRNA with two or more
copies. Global analysis using a combinatorial approach reveals that NS3
dimer has an occluded site size of ∼13 bp on dsRNA, an intrinsic binding
constant of 1 × 108 M− 1, and virtually no binding cooperativity. This lack of
cooperativity suggests that NS3 is not geared to target long dsRNA. The
larger site size of NS3, compared with its interacting size, indicates that the
NS3 structure has a border region that has no direct contact with dsRNA but
occludes a ∼ 4-bp region from binding. We also develop a method to correct
the border effect of ligand by extending the lattice length. In addition, we
find that NS3 recognizes the helical structure and 2′-hydroxyl group of
dsRNA with moderate specificity. Analysis of dsRNA-binding mutants
suggests that silencing of the suppression activity of NS3 is mechanistically
related to its dsRNA binding ability.
© 2010 Elsevier Ltd. All rights reserved.
Introduction
*Corresponding author. E-mail address:
yekeqiong@nibs.ac.cn.
Abbreviations used: VSR, viral suppressor of RNA
silencing; dsRBS, dsRNA-binding suppressor; RSV, Rice
stripe virus; RHBV, Rice hoja blanca virus; siRNA, small
interfering RNA; EMSA, electrophoretic mobility shift
assays; sulfo-EGS, ethylene glycol bis
(sulfosuccinimidylsuccinate); RNP, RNA–protein
complex; RDR, RNA-dependent RNA polymerase; dpi,
days post-infiltration; TRBP, trans-activation response
RNA-binding protein; MW, molecular weight.
RNA silencing regulates gene expression in most
eukaryotes, with specificity determined by small
RNA molecules of 21–24 nucleotides (nt) in
length.1–4 In plants and invertebrates, RNA silencing also functions as an adaptive antiviral immunity system.5 Virus infection often induces the
appearance of viral dsRNAs that are generated by
the activities of viral and host RNA-dependent
RNA polymerases (RDRs) or derived from structured regions in viral RNAs. Viral dsRNAs are
processed by the RNase III enzyme Dicer into
small interfering RNAs (siRNAs). siRNA is
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.
666
composed of two strands of 21–24 nt in length that
form a 19- to 22-bp duplex with characteristic 2-nt
3′-overhangs at both ends, as a result of RNase III
digestion. One strand of siRNA assembles into an
Argonaute (AGO) protein to form an RNAinduced silencing complex, which cleaves viral
RNAs that hybridize with the siRNA. The model
plant Arabidopsis thaliana encodes 4 Dicer-like
(DCL) proteins, 10 AGOs, and 6 RDRs, which
function redundantly and specifically in different
RNA silencing pathways.6 Among them, DCL4,
DCL2, AGO1, RDR6, and RDR1 have been shown
to be involved in defending RNA viruses, as
evidenced by their mutants becoming hypersensitive to the viruses.5
To counter RNA silencing-based antiviral defense,
viruses produce diverse viral suppressors of RNA
silencing (VSRs) to inhibit the host antiviral
reaction.7,8 A large number of VSRs have been
found to bind non-sequence-specifically to siRNA
and/or long dsRNA precursor to inhibit siRNA
utilization and production.9–18 Recent studies have
also identified a few VSRs that target the AGO protein
of the antiviral RNA silencing machinery.19–24
dsRNA-binding suppressors (dsRBSs) can be
categorized into three types on the basis of their
specificity for dsRNA size. Type I dsRBSs target
siRNA in a size-dependent manner, such as the
prototypical tombusvirus P19 suppressor.9 Structural analysis showed that the dimeric P19 protein
measures the siRNA size by symmetrically contacting each end of the duplex.25,26 Type II dsRBSs bind
to both siRNA and long dsRNA without size
specificity. The B2 protein of Flock house virus is a
typical size-independent dsRBS.11,12,27 In the crystal
structure of the B2–dsRNA complex, the dimeric B2
protein contacts one face of the duplex without
interaction with the duplex ends, underlying its
nonselectivity for duplex size.12 The third type of
dsRBSs bind preferentially to long dsRNA, but
poorly to siRNA, as shown for the 1A protein of
Drosophila C virus (DCV-1A).16
Different specificities for dsRNA size could lead to
different mechanisms of silencing suppression. Type
I siRNA-specific dsRBSs most likely sequester viral
siRNA and prevent its utilization, as shown for
P19.29 The type III dsRBS DCV-1A specifically
inhibits Dicer processing of the dsRNA precursor
with no effect on siRNA-induced silencing.16 In
contrast, binding of both long dsRNA and siRNA
could theoretically allow type II dsRBSs to interfere
with both the upstream Dicer processing step and
the downstream siRNA utilization step. 11–13,15
However, it is difficult to distinguish which type
of dsRNA is the primary target of type II dsRBS.
Proteins that associate with long nucleic acid in
multiple copies often display positive binding
cooperativity, which enhances the contiguous association of an otherwise weak ligand.30,31 Positive
Recognition of dsRNA by RSV NS3
cooperativity would thus be an indicator for sizeindependent dsRBS to naturally target long dsRNA.
However, quantitative analysis of cooperativity has
not been reported for any of size-independent
dsRBSs.
Rice stripe virus (RSV) is the type member of the
Tenuivirus genus, having a negative-sense singlestranded (ss) RNA genome. RSV infection causes
serious problems for rice production in the East
Asian region, especially in China. Its genomic RNA is
composed of four segmented parts, which together
encode seven open reading frames. The nonstructural protein NS3, encoded in the sense strand of the
third largest part RNA3, has recently been shown to
be a VSR.32 RSV NS3 inhibits local silencing, being
induced by either ssRNA or dsRNA, and also blocks
systemic silencing when the protein is present in the
spreading route of systemic signals.32 RSV is
transmitted by a small plant hopper (Laodelphax
striatellus) and also replicates in the insect vector.33,34
Hence, NS3 needs to suppress antiviral RNA
silencing in both plants and insects. Rice hoja blanca
virus (RHBV) is another member of the Tenuivirus
genus, and its NS3 homolog is also a VSR.35 RHBV
NS3 displays size-specific recognition of siRNA and
suppresses RNA silencing in plants and insects.17
The exclusive siRNA-binding property of RHBV NS3
has been utilized to examine the siRNA-mediated
antiviral response in mammalian cells.36
To gain insight into the mechanism of action of NS3
and to better utilize it as a research tool, we
characterize in detail the energetics of the RSV NS3
interaction with dsRNA. We analyze the association
of NS3 with dsRNAs of various lengths using a
combinatorial approach and derive several dsRNA
binding parameters, including the stoichiometry,
intrinsic binding constant, minimal binding site
size, occluded site size, and cooperativity. We find
that NS3 is a size-independent, rather than siRNAspecific, dsRNA binding protein with virtually no
cooperativity when binding long dsRNA. In addition, we investigate the structure feature of dsRNA
recognized by NS3 and amino acid residues responsible for dsRNA binding. NS3 mutants display a
strong correlation between dsRNA binding activity
and silencing suppression activity, providing a
mechanistic link between the two activities.
Results
Expression and purification of the NS3 protein
The RSV NS3 protein was expressed in Escherichia
coli with a six-histidine tag at its C terminus and
purified through Ni-affinity and heparin chromatography (Fig. 1). Most of the NS3 protein was
soluble when expressed at 16 °C. The NS3 protein
Recognition of dsRNA by RSV NS3
Fig. 1. Purification of histidine-tagged RSV NS3. The
samples were analyzed by SDS-PAGE and stained with
Coomassie blue. Lane 1, MW marker; lane 2, cell lysate after
induction of the NS3 gene expression; lane 3, lysate
supernatant; 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, eluate
of 500 mM imidazole; lanes 8–10, heparin chromatography;
lane 8, flow-through; lane 9, fraction after washing with
0.3 M KCl; lane 10, peak fractions in a salt gradient.
bound tightly to a heparin column and eluted at
around 0.6 M KCl. However, about half of the
protein failed to bind to the heparin column (lane 8).
This was not because the column was saturated, as
NS3 protein in the flow-through still could not bind
to a regenerated column. The unbound NS3 protein
was probably different from the bound one in its
structural and biochemical properties. Only the
heparin-bound protein was used for subsequent
biochemical experiments. Purified NS3 protein was
found to be highly homogeneous using SDS-PAGE
(Fig. 1) and had a 280 nm/260 nm absorbance ratio
of 1.8, indicating that it was free of nucleic acid
contamination.
NS3 dimer forms a 1:1 complex with a 16-bp
dsRNA
To assess the oligomeric state of NS3, we treated
the protein sample with the homobifunctional crosslinking agent ethylene glycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), which reacts with the free
amine group. Chemical cross-linking led to the
appearance of a new species found by SDS-PAGE
that corresponded to the dimeric form (Fig. 2a),
suggesting that NS3 predominantly forms a dimer
in solution.
We further analyzed the molecular size of NS3 in
the free state and dsRNA-bound state using sizeexclusion chromatography (Fig. 2b and c). The free
NS3 protein eluted as a single peak, but the peak
position was dependent on the concentration of
loaded protein. The apparent molecular weight
(MW) of NS3, calculated based on the calibration
curve, shifted downward from 59.5 to 45.6 kDa
when the sample was diluted from 62 to 5 μM. Since
the NS3 monomer has a MW of 24.7 kDa, these
results suggest that NS3 forms a dimer in solution,
667
but that the dimer can further aggregate weakly in a
concentration-dependent manner.
To assess the stoichiometry of the NS3–dsRNA
complex, NS3 in a dilute concentration of 6 μM was
mixed with a 16-bp dsRNA (SD-16B) and subjected
to size-exclusion chromatography. The resultant
elution profile displayed two peaks at 14.4 and
18.8 ml. These peaks should both contain RNA, as
the absorbance at 260 nm was two times higher than
that at 280 nm. The 14.4-ml peak corresponded to a
54.1-kDa species, which was likely composed of a
NS3 dimer (49.4 kDa) bound to a 16-bp RNA duplex
(9.8 kDa). The 18.5-ml peak corresponded to a 17.4kDa species, which was likely from unbound
dsRNA with an elongated structure.
NS3 dimer is stable against subunit dissociation
To assess the stability of NS3 dimer, we attempted
to study the subunit exchange between two distinguishable NS3 dimers. We prepared a maltose
binding protein fusion of NS3 (MBP-NS3,
MW = 67.6 kDa) that could be distinguished from
His-tagged NS3 (His-NS3, MW = 24.7 kDa) by their
MW. When MBP-NS3 and His-NS3 were coexpressed,
a single species composed of MBP-NS3 and His-NS3
could be purified by tandem Ni–NTA and maltose
affinity chromatography. This confirms that NS3
forms a dimer rather than higher-order multimers,
otherwise the tandem-affinity purification would
yield heterogeneous NS3 complexes in the latter case.
To examine the subunit exchange of NS3 dimer,
we mixed MBP-NS3 and His-NS3. If the subunits
dissociate and reassociate, the MBP-NS3/His-NS3
heterodimer would result. The three types of NS3
dimer can be quantified by the formation of siRNA
complex and native gel separation (Fig. 2d).
However, even after incubation up to 22 h, there
was little heterodimer formed when the protein
concentrations were in the range of 0.1 to 1000 nM.
This result shows that NS3 dimer is stable against
subunit dissociation.
NS3 interaction with dsRNAs of various lengths
Previous studies have already shown that the NS3
proteins from RHBV and RSV bind the siRNA
duplex.17,32 To further define the dsRNA binding
mode, we used electrophoresis mobility shift assays
(EMSA) to characterize the interaction of RSV NS3
with a series of dsRNAs with lengths ranging from 6
to 100 bp. These dsRNAs labeled with 32P at the 5′end were titrated with increasing concentrations of
NS3 protein and resolved in native polyacrylamide
gels. Only a single RNA–protein complex (RNP)
was observed for blunt-ended dsRNAs with lengths
up to 21 bp (Figs. 3a and 4a–b). For these RNAs, the
fraction of bound RNA was fit to a one-site binding
model (Eq. 1) to obtain the apparent macroscopic
668
Recognition of dsRNA by RSV NS3
Fig. 2. NS3 forms a stable dimer. (a) SDS-PAGE of NS3 protein cross-linked with sulfo-EGS. (b) Gel-filtration profiles of
NS3 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 the
Superdex 200 10/30 column and the derived apparent MW for each sample. MW is shown in the logarithmic scale. The
standards 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 and
incubated for 0.5–22 h on ice. All samples were adjusted to 10 nM, assembled with 32P-labeled siRNA, and resolved in a
native gel. However, the final protein concentrations of the 0.1 and 1 nM samples were less than 10 nM because of protein
loss during the concentrating process. The siRNA complexes formed by MBP-NS3 homodimer, His-NS3 homodimer, and
MBP-NS3/His-NS3 heterodimer were indicated.
dissociation constant Kd (see Materials and Methods).
The representative autoradiograph and fitting curve
for a 9-bp blunt-ended dsRNA are shown in Fig. 3.
The obtained Kd values for various dsRNAs are listed
in Table 1.
Notably, the RNA binding curve at the transition
zone appears more cooperative than the one-site
binding model predicts, even for dsRNAs that only
accommodate a single NS3 dimer. It seems that
additional protein concentration-dependent events
occur at the transition zone. One possibility is the
dissociation of NS3 dimer. However, the above
subunit exchange assay shows that NS3 dimer
remains stable at the protein concentration range
of 0.1–1000 nM. Hence, we can exclude the
contribution of the NS3 monomer–dimer transition
to the RNA binding process. Alternatively, protein
aggregation and protein sticking to the tube may
contribute to the deviation of the binding data from
an 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
Recognition of dsRNA by RSV NS3
value is very similar to that measured for RHBV
NS3.17 The affinity was found to decrease gradually
(Kd = 0.7–11 nM) as the length of a series of bluntended duplexes was reduced from 21 to 9 bp.
However, the affinity decreased abruptly by
∼20-fold when the duplex size was changed from 9
to 8 bp, and no binding was detected for a 6-bp
duplex. We conclude that the NS3 dimer contacts
minimally a 9-bp region in dsRNA and does not
specifically recognize the size of dsRNA as long as it
contains a full binding site. The increased affinity in
longer dsRNA is ascribed to the non-sequencespecific nature of the NS3–dsRNA interaction (see
following section).
The size-independent interaction of NS3 with
dsRNA suggests that dsRNAs having a sufficient
length can simultaneously accommodate more than
one NS3 dimer. Indeed, a second slow-migrating
species, RNP2, was observed for a 21-bp duplex with
2-nt 3′-overhangs at protein concentrations greater
than 62 nM (Fig. 4c). The gradual appearance of the
RNP2 species was concomitant with the disappearance of the RNP1 species as the NS3 protein
concentration was increased. Apparently, the former
was 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-bp
dsRNA was formed by annealing RNAs Si-9a and Si-9b.
After being labeled with 32P at the 5′-end, the dsRNA was
titrated with NS3 protein of increasing concentrations, as
indicated above each lane, in a 10-μl solution containing
100 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NP-40, and
25 mM Hepes-K (pH 7.5). The reactions were incubated on
ice for 20 min and resolved on a 5% native gel. (b) Analysis
of the EMSA data with a one-site binding model. The
continuous line is the best fit to Eq. 1 with Kd = 7.1 nM.
669
association with a second NS3 dimer. The RNP1-toRNP2 transition occurred at lower protein concentrations when the duplex was elongated (Fig. 4d and e).
We observed a successive binding pattern for a
100-bp dsRNA, although the complexes bound with
more than two NS3 dimers were not well resolved in
the native gel (Fig. 4f). Increasing amounts of
complex were retained at the wells at higher
concentrations of the protein likely because of nonspecific aggregation. The association of more than
two NS3 dimers indicates that NS3 can bind to the
internal region of long dsRNA without requiring the
terminal structure of the duplex.
Quantitative analysis of NS3–dsRNA binding
isotherms
RSV NS3 does not recognize the sequence or size of
dsRNA. Classic models describing the non-sequencespecific binding of large ligands to a homogenous
one-dimensional lattice could be applied to quantitatively analyze this type of interaction.30,37 McGhee
and von Hippel derived a closed expression based on
the conditional probability approach to describe the
cooperative and noncooperative binding of ligands
to an infinite lattice.30 As the short dsRNAs used in
our experiments could not be treated as infinite
lattices, we employed the exact combinatorial
expression for finite lattices to analyze the binding
isotherms of dsRNAs that accommodate two NS3
dimers.37–39 This type of ligand–lattice interacting
system is characterized by the lattice length M, the
total site size of the ligand n, the interaction site size c,
the intrinsic binding constant Kint, and the cooperativity parameter ω. Each base pair of dsRNA is
regarded as a repeating unit of the lattice. The total
site size of a ligand refers to the number of base pairs
occluded from further binding upon binding of a
ligand. The lattice length that is actually interacting
with the ligand may be less than the total occluded
site size and is termed the interaction site size c. The
intrinsic binding constant Kint describes the interaction between the ligand and an isolated single
binding site. The cooperativity ω is a positive unitless
factor that describes the relative change in binding
constant when a ligand is bound next to an already
bound ligand compared with an isolated site. The
binding is regarded as cooperative when ω N 1,
anticooperative when ω b 1, and noncooperative
when ω = 1.
Non-sequence-specific ligand–lattice interactions
are complicated by the overlap of potential binding
sites on the lattice. For example, an M-site lattice
contains M-c + 1 binding sites for a ligand that has an
interaction site size of c. As a result, the observed
macroscopic binding constant Ka (the inverse of Kd) is
the product of the intrinsic binding constant Kint and
a statistical factor that is equal to the number of
binding sites M-c + 1 (Eq. 2).37,40 A fit of the Kd values
670
Recognition of dsRNA by RSV NS3
site size larger than its minimal binding size on
ssDNA.39,41 When such a ligand binds at the end of
a lattice, the border region protrudes out, reducing
the effective site size. For the purpose of quantitative
analysis, we assumed that the border regions of NS3
dimer are evenly partitioned around its central
lattice-interacting region, with a size of b on each
side (Fig. 6a). It follows that n = c + 2b. In addition,
the combinatorial analysis requires that the total site
size is an integer value.
We attempted to estimate the total site size n of the
NS3 dimer based on the EMSA results for dsRNAs of
variable lengths. If n = 12, then a 21-bp blunt-ended
duplex would be able to accommodate two NS3
dimers on consideration of the border effect. However, the second RNP was not observed (Fig. 4b),
indicating that n should be greater than 12. We found
that a 21-bp duplex with 2-nt 3′-overhangs was
capable of binding a second NS3 dimer. Calculation
of the lattice length of this RNA was not straightforward because of its single-stranded overhangs. The
overhanging regions totaled 4 nt in length but could
not be literally counted as 4 lattice sites. This is
because NS3 binds ssRNA with a much weaker
affinity (see following section). We assumed that the
overhang was equivalent to v sites (v = 1, 2, 3, or 4). A
grid search showed that the overhangs were optimally accounted for by one site and that the site size
of NS3 dimer was around 13 bp. After correction of
the overhang effect, the 21-bp duplex with 2-nt 3′-
Table 1. Apparent macroscopic disassociation constant
(Kd) for RSV NS3 and oligonucleotides
Fig. 4. Interaction of NS3 with long dsRNAs. Representative EMSA gels of NS3 with (a) 20-bp blunt-ended
RNA 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-bp
dsRNA Si-29 with 3′-overhangs, (e) 30-bp RNA Si-19/Si20 with 3′-overhangs, and (f) 100-bp dsRNA. The
concentrations of NS3 protein are indicated above each
lane. RNP1 and RNP2 refer to the RNA complexes bound
by one or two NS3 dimers, respectively.
for 9- to 21-bp dsRNAs as a function of duplex length
to Eq. 2 yielded Kint = (8.0 ± 0.8) × 107 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 because
the 21-bp blunt-ended duplex, which has more than
twice the minimal binding size, was not capable of
binding a second NS3 dimer (Fig. 4b). This indicates
that part of the NS3 structure, hereafter termed the
border, does not directly contact dsRNA but
occludes a number of nucleotide residues for
binding. As an example of ligand with border
region, E. coli helicase PriA also has an occluded
Structurea
6-bp dsRNA
8-bp dsRNA
9-bp dsRNA
10-bp dsRNA
11-bp dsRNA
12-bp dsRNA
16-bp dsRNA
20-bp dsRNA
21-bp dsRNA
19-bp+ov dsRNA
19-bp+ov dsDNAd
19-bp+ov DNA/RNAd
21-nt ssRNA
21-nt ssDNA
Oligob
Kd (nM)c
SD-6B
SD-8B
Si-9a/Si-9b
SD-10B
SD-11B
SD-12B
SD-16B
SD-20B
Si-1/Si-4
Si-1/Si-2
Dsi-1/Dsi-2
Si-1/Dsi-2
Si-1
Dsi-3
No binding
235 ± 36.8
11.0 ± 4.6
8.8 ± 5.3
3.5 ± 1.3
3.5 ± 1.2
2.7 ± 1.0
2.3 ± 0.4
0.7 ± 0.2
2.4 ± 0.9
50.2 ± 27.9
4.8 ± 1.2
28.8 ± 11.9
No binding
a
Duplexes are blunt-ended by default, and those having 2-nt
3′-overhangs are indicated by “+ov”.
b
The sequences of oligos are listed in Table 2. One oligo is
indicated for self-complementary duplexes, whereas two oligos
are indicated for non-self duplexes.
c
The reported Kd is the mean of three or two measurements ± SD.
d
Although the EMSA gels of the dsDNA and RNA/DNA
hybrid showed clear association of a second NS3 dimer at high
protein concentrations, the reported Kd values were derived
without taking into account the multiple binding. The amount of
bound RNA was calculated simply as the sum of one-NS3 and
two-NS3 bound complexes.
Recognition of dsRNA by RSV NS3
Fig. 5. Macroscopic Kd values of one-NS3 binding
dsRNAs as a function of duplex length. The continuous
line is the best nonlinear fit to Eq. 2 with Kint =(8.0 ± 0.8) ×107.
The interaction site size of NS3 dimer was fixed at c =9.
overhangs was equivalent to a 22-bp blunt-ended
duplex. Fig. 6b illustrates the scenario in which a
dsRNA lattice of 22 sites exactly fits two NS3 ligands
of n = 13 and b = 2.
The border region present in NS3 dimer also
complicated the analysis of binding data. A ligand
with border would display a smaller effective site
size (n − b) when bound at the end of a lattice than
when bound internally (Fig. 6c). The border effect
needs be corrected for short lattices that accommodate only a few ligands. An early approach
modified the site size to n − 2b in the case of oneligand binding and to n − b in the case of twoligand binding.39,41 Such a correction cannot be
readily applied to lattices that can accommodate
more than two ligands. In the present work, we
671
took a different approach to correct the ligand
border effect by extending the nominal length of
the lattice at each end by b sites while keeping the
ligand site size constant along the whole lattice
(Fig. 6c). The lattice extensions were created to
virtually contact the border region of the ligand
bound at the lattice end, so that the ligand could
be treated as if it did not have a border. The
consequence of extending a lattice is equivalent to
the previous correction made for the ligand site
size for the cases of one- and two-ligand binding,
but our approach should be applicable to any
number of bound ligands and is also uniform in
treatment.
For the lattice that can accommodate two, but not
three, ligands, Eqs. 3–6 can be derived to relate
fractions of the free RNA, one-ligand complex, and
two-ligand complex to the concentration of free
ligand L and binding parameters M, n, Kint, and ω
(see Materials and Methods).37,39 We applied these
equations to globally fit the binding data of 21-, 22-,
and 30-bp dsRNAs with overhangs to derive the
parameters Kint and ω. The lattice length M was
corrected for the ligand border effect and the dsRNA
overhang effect, as discussed above. M and n were
fixed in the fit and their optimal values were found
using a grid search. The minimal fitting residual was
achieved when n = 13 and v = 1. The global fitting
results, shown in Fig. 7, yielded Kint = 1.0 × 108 M− 1
and a cooperativity parameter of ω = 0.5. This Kint
value agreed with that obtained from the analysis of
one-NS3 binding isotherms for 9- to 21-bp dsRNAs
(Kint = 8.0 × 107 M− 1). More importantly, this analysis
indicates that there is virtually no or a slightly
negative cooperativity among adjacently bound
NS3 dimers.
Fig. 6. Schematic of the NS3 ligand with border regions and its interaction with dsRNA lattice. (a) The NS3 structure is
represented by a box and two protruding borders. The box denotes the core region that directly contacts dsRNA, whereas
the borders refer to the parts of the NS3 structure that do not contact dsRNA but occlude a length of b base pairs on either
side of the ligand for further binding. (b) Illustration showing that a 22-bp dsRNA exactly fits two NS3 dimers with c = 9
and b = 2. Black bar represents the dsRNA lattice. (c) Ligand with border regions having a smaller site-size (n − b) when
binding at the end of the lattice. The ligand border effect could be corrected by increasing the nominal lattice length by b
sites at both ends. The extensions are represented as gray bars at the end of the lattice.
672
Recognition of dsRNA by RSV NS3
Specificity for the dsRNA structure
To examine the structural features of dsRNA
recognized by NS3, we compared its binding
affinity 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-fold
weaker (Kd = 28.8 ± 11.9 nM) than the dsRNA,
suggesting that the helical structure of dsRNA
was recognized. The 2′-hydroxyl group of ssRNA
appeared to be critical, as the ssDNA was barely
bound (Fig. 8e). The dsDNA was also bound
∼ 20-fold weaker than the siRNA duplex. This was
likely due to the lack of the 2′-hydroxyl group in
DNA and their different helical structures (B-form in
DNA versus A-form in RNA). Interestingly, the
DNA/RNA hybrid duplex had a similar binding
affinity (Kd = 4.8 ± 1.2 nM) compared with the
dsRNA duplex. Two factors likely account for this
observation. First, the RNA/DNA hybrid and
dsRNA duplex share an A-form helical structure
that is recognized by NS3. Second, NS3 might
primarily contact only the RNA strand in the
RNA/DNA hybrid duplex and is hence less affected
by the other strand being DNA. This prediction is
consistent with a significant interaction between
NS3 and ssRNA. Notably, a two-NS3 bound
complex was observed for the 21-nt dsDNA and
DNA/RNA hybrid, but not for the 21-nt dsRNA.
This suggests that the total site size of NS3 is smaller
for duplexes that have at least one DNA strand
compared with dsRNA.
dsRNA binding mutants of NS3
Nucleic acid-binding proteins frequently use basic
arginine and lysine residues to contact negatively
charged phosphate groups through electrostatic
interactions. To identify the dsRNA-binding residues of NS3, we replaced a few arginine and lysine
residues with glycine or positively charged aspartate
and glutamate residues. Their binding affinities with
a 21-nt siRNA duplex were measured using EMSA
(Fig. 9a). The single-site mutants R94G, K127G,
K165G, and R169E had Kd values (within experimental uncertainty) similar to that of the wild-type
protein, suggesting that these respective residues are
not involved in dsRNA interaction. In contrast, the
single-site mutants R50G, K77G, K112G, R124G, and
R190G and the double mutant K173G/K174G displayed an 11- to 25-fold decrease in binding affinity,
suggesting that these respective residues contacted
dsRNA via electrostatic interactions. A triple mutant
with a stretch of basic residues K173–K174–R175
replaced by negatively charged Glu–Asp–Glu
showed the most dramatic reduction (∼ 1000-fold)
in affinity. This was likely due to the repulsive force
between the introduced acidic residues and the
phosphate group of RNA, as well as the additive
Fig. 7. Global fitting of the EMSA data of dsRNAs
bound by two NS3 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 are
drawn as circles, squares, and inverted triangles, respectively. The continuous lines are global nonlinear least
square fits of Eqs. 3–6, with Kint = 1.0 × 108 M− 1 and ω = 0.5.
The total site size was fixed at n = 13, and the effective
lattice length was fixed at M = 26, 27, and 35 for the three
dsRNAs, respectively, after correcting for the border effect
(2b = 4) and the overhang effect (v = 1).
effect of the mutation on residue R175. These
mutants also provide a useful tool for assessing the
functional role of the dsRNA-binding ability of NS3.
RNA silencing suppressor activity of NS3 mutants
To examine the relationship between the dsRNAbinding activity and suppressor activity of NS3, we
analyzed these mutants for their suppression of
dsRNA-induced GFP silencing using an agrofiltration assay (Fig. 9b). Nicotiana benthamiana plants
were co-infiltrated with strains of Agrobacterium
containing three vectors that encoded a reporter
gene, a silencing trigger, and a silencing suppressor.
The first vector expressed a reporter GFP protein
driven by the 35S promoter. The second vector
673
Recognition of dsRNA by RSV NS3
contained part of the antisense sequence of the GFP
gene followed by the sense sequence of GFP. In this
case, the transcript would fold back into a long
hairpin that would be processed into GFP-targeting
siRNA. The third vector encoded an RNA silencing
suppressor for testing. The infiltrated leaves were
photographed under UV illumination at 3 days
post-infiltration (dpi). Regions with GFP expression
displayed green fluorescence under UV light, while
those from unfiltrated regions appeared red owing
to chlorophyll autofluorescence.
Infiltration of the 35S-GFP vector with the two other
empty vectors induced GFP expression at 3 dpi. Coinfiltration with the GFP-dsRNA expressing vector
blocked GFP expression, indicating an active RNA
silencing mechanism in the plant. The expression of
GFP was efficiently restored in the presence of wildtype NS3 and another RNA silencing suppressor, P19,
of Cymbidium ringspot virus, as expected.9,32
The suppressor activities of 11 NS3 mutants were
examined and found to generally correlate with
their dsRNA-binding affinities. Mutants that were
normal in dsRNA binding (R94G, K127G, K165G,
and R169E) maintained their ability to efficiently
Fig. 8. EMSA of NS3 with different nucleic acid
structures. 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 ssRNA
Si-1, and (e) 21-nt ssDNA Dsi-3. All nucleic acid constructs
were 21 nt in length, and their sequences are shown in
Table 2.
inhibit GFP silencing. On the other hand, mutants
that were defective in dsRNA binding (R50G, K77G,
K112G, R124G, K173G/K174G, K173E/K174D/
R175E, and R190G) were also defective in the
suppression of GFP silencing. We conclude that
the in vivo suppressor activity of NS3 is correlated to
its dsRNA binding ability, providing a mechanistic
link between the two activities.
Discussion
The divergent sequences among dsRBSs suggest
that they each adopt a unique structure and
recognize dsRNA in different ways. Elucidation
of the dsRNA binding mode of dsRBSs is
important to understand their specific mechanism
of silencing suppression and other physiological
functions. In this study, we have shown by
chemical cross-linking, gel-filtration chromatography, and subunit exchange assay that RSV NS3 is
a stable dimer in solution, and that one dimer
binds to a 16-bp dsRNA (Fig. 2). EMSA on a series
of dsRNA probes allowed us to define the minimal
binding size of NS3 to be 9 bp. We show that
dsRNAs of 9 to 21 bp in length accommodate only
a single NS3 dimer, and dsRNAs longer than
21 bp can associate simultaneously with two or
more NS3 dimers (Fig. 4). The thermodynamic
parameters of the NS3–dsRNA interaction have
been derived from a global analysis of two-NS3
binding data using the combinatorial model. Our
analysis shows that NS3 has an intrinsic binding
constant 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 a
dsRNA lattice. NS3 should not recognize the RNA
sequence, as many short dsRNAs of diverse
sequences tested for binding show consistent Kd
values. Although NS3 can bind to the middle of the
duplex as shown for a 100-bp dsRNA, current data
cannot exclude the possibility that NS3 has a
different affinity towards the end versus the middle
of the duplex.
Our results demonstrate that RSV NS3 does not
specifically recognize the size of dsRNA. The ability to
bind long dsRNA suggests that RSV NS3 has the
potential to protect dsRNA from Dicer cleavage, as
has already been demonstrated in vitro for B2.11,12 The
dsRNA binding of RHBV NS3 has been previously
analyzed as a C-terminal fusion to the maltose binding
protein.17 RHBV NS3 and RSV NS3 are both dimeric
proteins and bind the 21-nt siRNA duplex with nearly
identical affinities (Kd = ∼2 nM). However, the previous study concluded that RHBV NS3 specifically
targets the 21-nt siRNA duplex, as a 26-nt siRNA
duplex was observed to bind poorly in comparison.17
In the previous EMSA gel, we noticed that the 26-nt
674
Recognition of dsRNA by RSV NS3
Table 2. Name, size, and sequence of the oligos used in this study
Namea
Size (nt)
Sequenceb
Si-1
Si-2
Dsi-1
Dsi-2
Dsi-3
SD-6B
SD-8B
Si-9a
Si-9b
SD-10B
SD-11B
SD-12B
SD-16B
SD-20B
Si-4
Si-42
Si-43
Si-29
Si-19
Si-20
21
21
21
21
21
6
8
9
9
10
11
12
16
20
21
23
23
24
32
32
5′-CGUACGCGGAAUACUUCGAUU-3′
5′-UCGAAGUAUUCCGCGUACGUU-3′
5′-CGTACGCGGAATACTTCGATT-3′
5′-TCGAAGTATTCCGCGTACGTT-3′
5′-AAAGGTGGAAAAGGTGGAAAA-3′
5′-CCCGGG-3′
5′-CCCCGGGG-3′
5′-AGCGUGACU-3′
5′-AGUCACGCU-3′
5′-CGCGGCCGCG-3′
5′-CGCGGUCCGCG-3′
5′-CGACGGCCGUCG-3′
5′-CGUAGCGGCCGCUACG-3′
5′-CGUUAGGCGGCCGCCUAACG-3′
5′-AAUCGAAGUAUUCCGCGUACG-3′
5′-GAAGUAGUAAUUGUCGCUCUCCU-3′
5′-GAGAGCGACAAUUACUACUUCUG-3′
5′-CGUACGUAAGCGCUUACGUACGUU-3′
5′-CGUACUCGAGAUAUCCUAGCUGGACUCUGAUU-3′
5′-UCAGAGUCCAGCUAGGAUAUCUCGAGUACGUU-3′
a
b
All oligos are RNA except for Dsi-1, Dsi-2, and Dsi-3, which are DNA.
Self-pairing regions are underlined.
siRNA formed two RNP complexes at high concentrations of the RHBV NS3 protein. However, this type
of binding pattern was not interpreted as a succession
of two binding events.17 In light of our result, RHBV
NS3 is likely also a size-independent dsRNA binding
protein that can associate with the 26-nt siRNA in two
copies. It would be surprising to find that RSV NS3
and RHBV NS3, which share 43% sequence identity
and 61% similarity, have different RNA binding
properties.
Gaining an understanding of the action mechanism of size-independent dsRBSs is complicated by
their dual ability to bind both siRNA and long
dsRNA. The latter activity could interfere with an
upstream step in the Dicer processing of long
dsRNA. The presence of cooperativity in a dsRBS
would strongly suggest that it naturally targets long
dsRNA, as this character is of no use for binding
short siRNA that only accommodates a single
protein molecule. The correlation between cooperativity and dsRNA binding targets has recently been
demonstrated in a comparison between two
dsRNA-binding proteins in the RNAi pathway.42,43
Most Dicer enzymes require a dsRNA-binding
protein as a partner, such as RDE-4 in C. elegans
and trans-activation response RNA-binding protein
(TRBP) in mammals. However, RDE-4 and TRBP
play different roles in siRNA processing. RDE-4 is
essential for Dicer to cleave long dsRNA into siRNA,
while TRBP functions downstream to load siRNA
into the RNA-induced silencing complex. Consistent
with their different roles, RDE-4 preferentially binds
long dsRNA with cooperativity, while TRBP binds
siRNA with high affinity and has no cooperativity in
binding long dsRNA.42,43
We have carried out the first quantitative cooperativity analysis of a size-independent dsRBS.
However, we find that NS3 lacks cooperativity.
One simple interpretation of this result is that NS3 is
not designed to target long dsRNA, and siRNA is
more likely the primary target of NS3. The high
affinity to siRNA (Kd = 2.4 nM) would allow NS3 to
sequester siRNA. Alternatively, the high intrinsic
affinity of NS3 might make cooperativity less critical
for NS3 to bind long dsRNA. Lack of cooperativity
means that it is difficult for NS3 to form a
continuous protein cluster on long dsRNA. This is
because, in this case, random association of the
ligand will create unoccupied gaps on the lattice that
are not large enough to accommodate a new
ligand.30,31 It is unknown whether or not other
size-independent dsRBSs bind cooperatively to long
dsRNA.11–13,15,18,28 Two dsRBSs, B2 and DCV-1A,
have been shown to bind long dsRNA with higher
affinities than siRNA, 11,16 suggesting that their
binding 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 likely
constitute the RNA-binding surface and make
contact with the phosphate group of RNA. These
mutants are defective in dsRNA binding and are
also impaired in the suppression of dsRNA-induced
GFP silencing in plants, indicating that dsRNA
binding of NS3 is responsible for its suppression
activity. In previous work, we have shown that a
triple-alanine mutation of residues K174, K175, and
R175 (NS3/3A) displays reduced activity in suppressing both local and systemic GFP silencing. We
have also shown that these three basic residues are
Recognition of dsRNA by RSV NS3
675
Fig. 9. The siRNA binding activity and silencing suppression activity of NS3 mutants. (a) Gel-shift titrations of a 21-nt
siRNA duplex, Si-1/Si-2, with NS3 mutants. The concentrations of NS3 proteins were 0.25, 2.5, 25, 250, and 2500 nM. The
binding buffer contained 25 mM Hepes-K (pH 7.5), 300 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NP-40, and 5% glycerol
in a 10-μl volume. The derived Kd values are indicated. (b) Silencing suppression activity of NS3 mutants. Leaves of the N.
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.
676
critical for nuclear localization of the GFP-NS3
fusion protein.32 Our new results suggest that the
three basic residues play an additional role in
dsRNA binding. In homologous RHBV NS3, the
equivalent residues have also been found to be
critical for siRNA binding and RNA silencing.44
NS3 exhibits only moderate structural specificity
for 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 possess
strict specificity for the dsRNA structure and cannot
bind ssRNA.9,12,18,25,26 B2 also distinguishes strongly
against dsDNA and RNA/DNA hybrid.12 In terms
of structural specificity, NS3 is similar to P21, which
also interacts with various nucleic acid structures.28
Further understanding of the size-independent and
unique recognition mode between NS3 and dsRNA
must await the determination of their complex
structure.
Materials and Methods
Plasmid construction
The RSV NS3 gene was PCR-amplified from plasmid
pBin438-35S-NS345 with the primers RSVNS3_NcoI_N1
(5′-TATCCATGGGCAACGTGTTCACATCGTC-3′, the
restriction site is underlined) and RSVNS3_C6H_
EcoRI_L211 (5′-CCGGAATTCTTAATGATGATGATGATGATGCAGCACAGCTGG-3′). The PCR product
was digested by NcoI and EcoRI restriction enzymes
and then purified and cloned into pET28a, resulting in the
pET28a-NS3 plasmid. Owing to the NcoI cloning, an extra
glycine residue was introduced after the starting Met
residue, but the residues were numbered as in the wildtype sequence (211 residues). In addition, six histidine
residues encoded in the reverse primer were added to the
C terminus of NS3 to facilitate affinity purification. Sitedirected mutations were generated on pET28a-NS3 with
QuikChange (Stratagene), using the appropriate primers.
The NS3 gene was also cloned into a modified pMal-c2x
plasmid (New England Biolabs), in which NS3 was
fused to the C terminus of MBP by a linker containing
a PreCission cleavage site, yielding the pMal-NS3
plasmid.
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-Dthiogalactopyranoside. Cells harvested from a 4-L culture
were 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 solution was then lysed using sonication. The cell lysate
was centrifuged at 30,000g for 60 min at 4 °C. The
supernatant was loaded onto a 5-ml HisTrap column and
then washed with buffer H300 and 50 mM imidazole in
H300. The bound protein was eluted with 500 mM
Recognition of dsRNA by RSV NS3
imidazole in H300. Fractions containing NS3 were pooled
and loaded directly onto a 5-ml heparin column (GE
Healthcare) equilibrated in buffer H100 [0.1 M KCl, 5%
glycerol, 20 mM Hepes-K (pH 7.6)]. The bound protein was
washed with buffer H300 and eluted at ∼0.6 M KCl in a
linear 0.3–1 M KCl gradient in buffer H [5% glycerol, 20 mM
Hepes-K (pH 7.6)]. The purified protein was supplemented
with 5 mM dithiothreitol, divided into aliquots, flash-frozen
in liquid nitrogen, and stored at −80 °C. The final yield was
about 3–4 mg per liter of culture. His-tagged NS3 was used
in biochemical assay by default.
MBP-NS3 was expressed in a similar way as His-NS3.
The clarified cell lysate was loaded into a maltose affinity
column and eluted with 20 mM maltose in buffer H300.
The protein was further purified by heparin chromatography. For the His-NS3/MBP-NS3 heterodimer, plasmids
pET28a-NS3 and pMal-NS3 were cotransformed into E.
coli Rosetta2 (DE3) which was cultured in medium
containing ampicillin and kanamycin. The heterodimer
was coexpressed and copurified through Ni–NTA affinity,
amylose affinity, and heparin chromatography. The
protein concentration of NS3, expressed in the monomeric
form, was determined by absorbance at 280 nm and a
molar extinction coefficient of 24,410 M− 1 cm− 1 for HisNS3 and 90,760 M− 1 cm− 1 for MBP-NS3.
Size-exclusion chromatography
The apparent molecular masses of NS3 and its RNA
complex were analyzed with a Superdex 200 10/30 GL
column (GE Healthcare). The column was equilibrated at
4 °C in a running buffer containing 300 mM KCl, 5%
glycerol, and 20 mM Hepes-K (pH 7.6). The calibration
curve was based on the following standard proteins:
lysozyme (14 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and bovine serum albumin (67 kDa). The
logarithm of molecular mass of the standards was fit to a
linear function of elution volume using OriginPro 8. The
initial NS3 sample had a concentration of 62 μM (1.5 mg/
ml) and was diluted to 25 and 5 μM with the running
buffer. The NS3–RNA complex was formed by mixing
3 nmol NS3 and 2 nmol SD-16B RNA. Each sample was
loaded 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 reactions
containing 25 mM Hepes-K (pH 7.6) and 300 mM KCl for
30 min at room temperature. The reactions were quenched
by adding 1 μl of 1 M Tris–HCl (pH 7.5) and resolved in a
4–20% 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 containing
100 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.01% NP-40, and
25 mM Hepes-K (pH 7.6) and incubated on ice. Samples of
appropriate volume were frozen in liquid nitrogen at 0.5,
1, 2, 4, 8, and 22 h. The 0.1 and 1 nM samples were
concentrated to ∼ 10 nM, whereas the 100 and 1000 nM
677
Recognition of dsRNA by RSV NS3
samples were diluted to 10 nM. Twenty microliters of
these 10 nM samples was mixed with 32P-labeled 21-nt
siRNA (Si-1/Si-2). After incubation on ice for 20 min, the
reactions were resolved in a native gel and visualized by
autoradiography.
The macroscopic binding constant Ka (the inverse of
Kd) is related to the microscopic intrinsic binding
constant Kint by37,40:
Ka = 1 = Kd = ðM − c + 1ÞKint
ð2Þ
where M is the lattice length and c is the interaction site
size of NS3, which was fixed at 9 for NS3.
RNA preparation
Short RNAs were chemically synthesized by Dharmacon
or Takara. DNA oligos were purchased from Invitrogen. The
two strands of 100-bp dsRNA were prepared by in vitro
transcription following standard protocols. The transcription
template for the sense strand RNA was amplified by PCR
with the primers S100-T7F (5′-CGCGTAATACGACTCACTATAGGGCATGGATATTCTCATCATTAGTTTG-3′) and
S100-R (5′-CGGCTCCGGCTACTGC-3′) using the P25 gene
as template.46 The template for the antisense strand RNA
was prepared with the primers AS100-F (5′-ATGGATATTCTCATCATTAGTTTG-3′) and AS100-T7R (5′CGCGTAATACGACTCACTATAGGGCCGGCTCCGGCTACTGC-3′). Transcribed RNAs were dephosphorylated
prior to 5′-labeling.
Electrophoretic mobility shift assay
Oligonucleotides were 5′-end labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-32P]ATP
(Furui Biotech, Beijing) in a 20- μl reaction at 37°C for
40 min and purified through a MicroSpin G-25 column (GE
Healthcare). In EMSA, ∼ 0.1 nM of labeled oligos was
mixed with various amounts of NS3 in a 10-μl reaction
containing 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 important
for RNA binding. In the binding reactions for NS3 mutants,
the binding buffer contained additionally 300 mM KCl and
5% glycerol to increase the solubility of some mutants. The
binding reactions were incubated on ice for 20 min and
resolved in a 5% native polyacrylamide gel run in 1× Tris–
glycine buffer (pH 8.3) at 4 °C or room temperature. The
gels were dried and autoradiographed using a Typhoon
PhosphorImager (GE Healthcare). The amounts of free and
bound RNA molecules were measured by integrating the
volumes of the corresponding bands with ImageQuant
(Molecular Dynamics). The fraction of each RNA species
was calculated in comparison with the total amount of
RNA in that lane.
Analysis of one-NS3 binding data
For oligos that accommodated only one NS3 dimer, the
fractions of bound RNA, Θ1, were fitted to the function:
Q 1 = L = ðL + K d Þ
ð1Þ
where Kd is the apparent macroscopic dissociation
constant and L is the concentration of free NS3 molecules
(as monomer). Since the concentration of labeled RNA
was ∼ 0.1 nM in our experimental conditions, the amount
of bound ligand was negligible and the free ligand
concentration L could be well approximated by the
known total concentration of ligand LT.
Analysis of two-NS3 binding data
We consider the case where the lattice containing M site
is capable of binding two n-site ligands, but is not long
enough to bind three ligands (i.e., 2n ≤ M b 3n). According
to the exact combinatorial expression,37,39 the fraction of
RNA bound to i ligands, Θi (i = 0, 1, 2), is given by:
Q0 = 1 = Z
ð3Þ
Q1 = ðM − n + 1ÞKint L = Z
ð4Þ
Q2 = ½0:5ðM − 2n + 1ÞðM − 2nÞðKint LÞ2
+ ðM − 2n + 1ÞðKint LÞ2 x = Z
ð5Þ
where Kint is the intrinsic microscopic binding constant
between the ligand and a single binding site, ω is the
cooperativity factor between adjacent interacting ligands,
L is the concentration of free ligand, and Z is the
normalization factor:
Z = 1 + ðM − n + 1ÞKint L
+ ½0:5ðM − 2n + 1ÞðM − 2nÞðKint LÞ2
+ ðM − 2n + 1ÞðKint LÞ2 N
ð6Þ
The parameters Kint and ω were determined by
nonlinear global fit to Eqs. 3–6 of experimentally
measured values Θi (i = 0,1,2) for the 21-, 22-, and 30-bp
dsRNAs with overhangs. L was approximated by LT. The
lattice length M was corrected as M0 + 2b + v, where M0 is
the length of the dsRNA duplex region, 2b is the correction
factor for the ligand border effect, and v is the correction
factor for the dsRNA overhang. The lattice length M and
site size n were fixed, but were subjected to a grid search
by varying the values of n and v. The site size n was tested
at values of 13, 14, and 15, which resulted in border
corrections (2b) of 4, 6, and 8, respectively. We found that
when n = 13, 14, and 15, the overhang correction factor v
was necessarily 1, 2, and 3, respectively. This ensured that
M ≥ 2n and that cooperativity had a nonnegative value.
The fitting residuals reached a minimum when n = 13 and
v = 1. The nonlinear fit was carried out in MATLAB 6.1
using home-written scripts.
RNA silencing suppression activity in plants
The RSV NS3 wild-type and 11 mutant genes were PCRamplified from the corresponding pET28a-NS3 plasmid
with the primers NS3_BamHI-F (5′-GGATCCATGAACGTGTTCACATCGTCT-3′) and NS3_SalI-R (5′GTCGACCTACAGCACAGCTGGAGA-3′) and then
cloned into vector pGEM-T (Promega). The construct
integrity was confirmed by sequencing. The NS3 genes
were digested with BamHI and SalI and inserted into the
678
binary vector pBin438 between the 35S promoter and
nopaline synthase terminator.32 Plasmids expressing 35SGFP, an inverted repeat sequence of GFP (35S-dsGFP),
and the Cymbidium ringspot virus P19 gene (35S-P19) were
described previously.32 All constructs were electroporated
into A. tumefaciens strain C58C1 with a Gene Pulser II
system (Bio-Rad). For co-infiltration assays, A. tumefaciens
containing various plasmids were grown individually to
an OD600 of 0.6–0.8. The cultures were pelleted and
resuspended in an infiltration medium containing 20 mM
MgCl2, 10 mM MES (pH 5.6), and 100 μM acetosyringone
to 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 tissues
of 4-week-old N. benthamiana plants by using 1-ml
syringes. The leaves were photographed under UV
illumination (UV Products) at 3 dpi using a Canon 400D
digital camera with a 58-mm yellow filter.
Recognition of dsRNA by RSV NS3
11.
12.
13.
14.
15.
Acknowledgements
We are grateful to Xinxing Yang for help in
MATLAB programming. K. Ye was supported by
the Chinese Ministry of Science and Technology
through the 863 and 973 projects and the Beijing
Municipal Government. X. Zhou was supported by
the National Natural Science Foundation of China
(grant 30870110).
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