Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.

Abstract

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177) core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177), as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.

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Structure and Functional Analysis of the RNA- and Viral
Phosphoprotein-Binding Domain of Respiratory
Syncytial Virus M2-1 Protein
Marie-Lise Blondot1, Virginie Dubosclard1, Jenna Fix1, Safa Lassoued2, Magali Aumont-Nicaise3,
Franc ¸ois Bontems2, Jean-Franc ¸ois Ele ´oue ¨t1*, Christina Sizun2
1Unite ´ de Virologie et Immunologie Mole ´culaires (UR892), INRA, Jouy-en-Josas, France, 2Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Gif-sur-Yvette,
France, 3IBBMC, Universite ´ Paris-XI, Orsay, France
Abstract
Respiratory syncytialvirus(RSV)protein M2-1 functionsas anessential transcriptionalcofactor of the viralRNA-dependent RNA
polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts
with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core
region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177core domain,
which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional
similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes
the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues
located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these
residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies,
which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA,
suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding
to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.
Citation: Blondot M-L, Dubosclard V, Fix J, Lassoued S, Aumont-Nicaise M, et al. (2012) Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-
Binding Domain of Respiratory Syncytial Virus M2-1 Protein. PLoS Pathog 8(5): e1002734. doi:10.1371/journal.ppat.1002734
Editor: Sean P. J. Whelan, Harvard Medical School, United States of America
Received October 27, 2011; Accepted April 20, 2012; Published May 31, 2012
Copyright: ? 2012 Blondot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: M.L.B. acknowledges a Ministe `re de l’Enseignement Supe ´rieur et de la Recherche grant and V.D. an ‘‘Ile-de-France 2007’’ postdoctoral fellowship. The
work was supported by the Agence Nationale de la Recherche (ANR 11 BSV8 024 02 grant) and the Centre National de la Recherche Scientifique (TGE RMN THC
FR3050). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jean-francois.eleouet@jouy.inra.fr
Introduction
Human respiratory syncytial virus (RSV), a pneumovirus of the
Paramyxoviridae family in the Mononegavirales order, is an important
respiratory pathogen and the major cause of bronchiolitis and
pneumonia in children [1]. Bovine RSV on the other hand
represents an important economic issue due to the high morbidity
and mortality of infected calves [2]. Whereas current efforts are
mainly focused on the development of safe RSV vaccines for
infants, the development of antiviral drugs specifically targeting
viral-specific functions such as the RSV RNA-dependent RNA
polymerase complex (RdRp) represents a promising alternative for
treatment.
Four of the 11 proteins (the nucleoprotein N, the phosphopro-
tein P, M2-1 and the large polymerase subunit L), encoded by the
RSV single-stranded negative-sense genomic RNA, are associated
with the viral genome to form the holonucleocapsid [3]. The
genomic RNA of RSV is maintained as a nuclease-resistant N-
RNA ribonucleoprotein complex, which acts as a template for the
RdRp that is responsible for both replication and transcription of
the genome. Whereas the highly processive replicase generates a
complete positive-sense RNA, which acts in turn as a template for
genomic RNA synthesis, the transcriptase produces ten different
subgenomic capped and polyadenylated mRNAs. Transcription
proceeds by a sequential stop-and re-start mechanism in which the
polymerase responds to cis-acting signals present in intergenic
regions [4]. Transcription is (re)initiated at a highly conserved 9–
10 nucleotide transcription promoter (gene start, GS) signal. Semi-
conserved 12–13 nucleotide gene ends (GE) signal for polyade-
nylation and release of the nascent mRNA [5]. The polymerase
has a propensity to dissociate from the N-RNA template, but
cannot reinitiate at a downstream gene in case of premature
termination [6], which leads to a decreasing transcription gradient
from the 39 to the 59 end of the genome.
For all known pneumoviruses, RdRp driven transcription
depends on M2-1. RSV M2-1 is a transcription antitermination
factor that is important for the efficient synthesis of full-length
mRNAs [7] as well as for the synthesis of polycistronic read-
through mRNAs [4,6,8,9]. The latter activity is thought to
facilitate polymerase access to promoter-distal regions of the
genome, and hence transcription of all genes [3]. It was shown that
the M2-1 protein reduced termination at all gene junctions, but
that the efficiency in the presence of M2-1 varied at the different
gene junctions [6,9]. However, mechanisms by which M2-1
prevents the polymerase from terminating transcription remain to
be clarified. There are at least three different scenarios. (i) M2-1
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could bind to the nascent mRNA transcript to facilitate
transcription elongation, perhaps by preventing the mRNA from
re-hybridizing to the template, or forming secondary structures
that might destabilize the transcription complex. This hypothesis is
sustained by the finding that RSV mRNA are co-precipitated with
M2-1 from RSV infected cells [10]. (ii) The polymerase
processivity enhancing effect of M2-1 could be due to an increase
of the affinity of the polymerase for the genomic RNA template in
a sequence non-specific manner. (iii) M2-1 could recognize GE
sequences either on the nascent mRNA or on the RNA template
and prevent the release of the polymerase complex from its
template, favoring transcription re-initiation at the downstream
GS sequences.
In RSV-infected cells, M2-1 co-localizes with the other RdRp
components in inclusion bodies (IBs) [11], which are regarded as
centers of RNA synthesis [12]. The basic 194-residue RSV M2-1
has been shown to be an RNA binding protein, but specificity of
M2-1 RNA binding has been debated. It was reported that M2-1
was able to bind to long RNAs without sequence specificity and
with an apparent Kdof 30 nM, and that it bound specifically to
short (80 nucleotides) but not long (700 nucleotides) RNAs
containing the positive-sense antigenomic leader sequence with an
apparent Kdof 90 nM [13]. Elsewhere it was demonstrated that
M2-1 interacts more specifically with viral mRNAs not containing
the leader sequence during infection [10]. In addition M2-1
interacts with P in vitro [14], competitively to RNA [15]. M2-1 is a
modular protein that contains four domains. M2-1 forms
tetramers in solution [15,16], and the oligomerization domain
was mapped to the region 33–62 [15]. The N-terminal region
(residues 1–30) contains a putative zinc binding domain with a
Cys3His motif, which is essential for the function of M2-1, but
whose exact role is still unknown [17,18,19]. The RNA and P
binding properties are related to the central part (or core domain)
of the molecule (residues 53–177) [13,15]. The C-terminal tail is
predicted to be unstructured.
Here we have addressed the molecular basis of the interaction
between RSV M2-1 and its partners. We have investigated the
structural aspects of P and RNA binding to the core domain of
M2-1 by NMR spectroscopy and report the solution structure of
RSV M2-158–177, which shows structural homology with the C-
terminal domain (CTD) of the Ebola virus (EBOV) VP30 protein.
In this a-helical domain we have identified residues that contribute
to two adjacent, partially overlapping contact surfaces, with P and
RNA respectively. We show that mutations of several of these
residues specifically disrupt the M2-1:P or M2-1:RNA interaction
in vitro and have a drastic effect on intracellular co-localization of
full-length M2-1 with P as well as on the function of M2-1 as a
transcription co-factor.
Results
Solution NMR structure of RSV M2-158–177
The boundaries of the protein fragment M2-158–177 were
chosen to focus on the binding regions of RNA and RSV
phosphoprotein determined previously, but also to exclude the
oligomerization domain and the disordered C-terminus, which are
not necessary for the interactions with RNA and P [15]. Line
widths of the solution NMR spectra were compatible with a
monomeric state, and M2-158–177 was amenable to structure
determination by NMR, in contrast to tetrameric full-length
M2-1. The resonance assignments were reported elsewhere [20].
M2-158–177contains a single globular domain spanning residues
G75-I171 and comprising six helices: a1 (G75-G85), a2 (K92-
E105), a3 (S108-D117), a4 (K124-K140), a5 (K143-R151) and a6
(D155-I171). The N-terminus (S58-L74), which corresponds to the
linker to the upstream oligomerization domain of M2-1, is
disordered. The a-helix bundle consists of a scaffold, formed by
a1, a2, a5 and a6, and an a3–a4 hairpin stacked on a6
(Figure 1A). M2-158–177 displays two oppositely charged faces
(Figure 1B). The positively charged face contains a large basic
cluster along a grove delimited by helices a2 (K92), a5 (K150,
R151) and a6 (K158, K159, K162, K169). Three smaller basic
clusters are found on a4 ( K124 and R126), on a3 (K112, K113
and R115), and between a4 and a5 (R139, K140 and K143) as
shown in Figure 1B. The putative overall tetrameric domain
organization of full-length M2-1 is schemed in Figure 1C.
RNA binds to the main basic cluster of M2-158–177
Incubation of M2-158–177with yeast RNA in a ,1:1 molar ratio
resulted in simultaneous shifting and broadening of several1H-15N
cross peaks in the
Figure S1). Treatment with RNAse A reversed these effects. This
experiment confirmed the RNA binding ability of M2-158–177in
vitro. The observed fast to intermediate exchange regime was an
indication for a weak interaction. To get rid of the broadening
contribution by transversal relaxation of large RNAs twice the
molecular weight of M2-158–177, we investigated the RNA:M2-
158–177 interaction by NMR by using short synthetic 10–15
nucleotide negative-sense (genomic) RNAs containing selected
transcriptional signals [3,21] as well as their complementary
positive-sense sequences. We tested the 39 polymerase entry site
(leader), the U-rich region upstream of the first GS signal, the GS,
and the F and SH gene ends (GE_F and GE_SH). Sequences are
detailed in Table 1. The oligonucleotides were designed to
minimize self-association or formation of secondary structure.
Only the leader-neg (59-CGCAUUUUUUCGCGU-39) and long
U-rich (59-CCCAUUUUUUUGGUU-39) sequences were pre-
dicted to form hairpins with a poly-U loop and a stem of two or
three G-C and A-U pairs with negative free energies. Calculations
were carried out on the mfold web server [22]. The absence of self-
association was assessed by1H NMR using 200 mM oligonucle-
otide solutions in water, except for leader-pos and GE_F-pos, for
1H-15N HSQC spectrum of M2-158–177(see
Author Summary
Premature termination of transcription by the RNA-
dependent RNA polymerase (RdRp) complex of respiratory
syncytial virus (RSV) is prevented by the M2-1 protein. This
transcription factor interacts with both RNA and viral
phosphoprotein P, the main RdRp cofactor, through a
specific ‘‘core’’ domain. Using NMR, we solved the 3D
structure of this domain and characterized the surface
residues involved in P- and RNA-binding. Based on these
data, we designed point mutations impairing binding to
either RNA or P. We studied the functional implications of
these mutations for transcription and co-localization of
full-length M2-1 in cytoplasmic inclusion bodies, where
viral transcription likely occurs. We found that the RNA-
and P-binding surfaces are in close proximity, accounting
for competitive binding in vitro. Our results suggest that
binding to both RNA and to P is necessary for transcrip-
tional antitermination by M2-1, but that the role of the
interaction with P is primarily to recruit M2-1 to the RdRp
complex. Finally we show that the M2-1 core domain is
homologous to the C-terminal domain of Ebola virus VP30
despite low sequence identity, solidifying the relationship
between these two proteins and transcriptional regulation
strategies shared by viruses belonging to the Filoviridae
family and the Pneumovirinae subfamily.
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which two broad imino proton signals were observed in the
guanosine and uridine regions respectively, corresponding to
partial formation of two G-C and U-A base pairs. Finally
formation of an RNA duplex with the two complementary strands
GE_F-pos and GE_F-neg was observed by NMR with sharp
signals corresponding to base-paired uridine imino protons.
Oligonucleotide binding was followed by chemical shift
perturbation experiments in1H-15N HSQC and1H-13C HSQC
experiments, as exemplified for the short polyA sequence in
Figure 2 (panels A to C). The chemical shift variation profile in the
1H-15N HSQC spectrum of short polyA is shown in Figure 2D.
Chemical shift perturbation profiles were similar for all RNAs and
consistent with those observed for yeast RNA. A complete set of
profiles for all oligonucleotides is given in Figure S2. For all RNAs
the largest chemical shift changes were observed for residues
belonging to the main basic cluster, i.e. K92-V97 (a2), L149-L152
(a5) and D155-K159 (a6), as illustrated in Figure 2E. These
residues contribute to forming a continuous positively charged
surface, which is consistent with an RNA binding surface
(Figure 2F). Chemical shift variations in1H-13C HSQC experi-
ments were observed for residues in the same region, solvent
exposed methyl side chains in the hinge between helices a5 and a6
as well as R151-Hdprotons being affected by RNA binding (see
Figure 2B and 2C).
Short A-rich RSV RNA sequences preferentially bind to
M2-158–177
The fast to moderately fast exchange regime in both1H-15N
and
1H-13C HSQCs allowed to extract apparent dissociation
Figure 1. Solution structure of the core domain of RSV M2-1. (A) Cartoon representation of the NMR structure of the a-helical domain of M2-
158–177(model of lowest energy). The color is ramped from blue to red from the N- to the C-terminus. (B) Electrostatic surface potential of M2-158–177
calculated with DELPHI [58] using PARSE parameters [59]. Two opposite faces of the protein are shown. The left-hand view is the same as for panel A.
Colors for charges are red to blue for potential energies 26 to +6 kBT. Basic residues belonging to the main cluster are labeled in black bold letters.
Basic residues belonging to the 3 minor clusters are indicated in blue, green and purple letters. (C) Schematic representation of the tetramer of full-
length M2-1.
doi:10.1371/journal.ppat.1002734.g001
Table 1. Apparent M2-158–177:oligonucleotide dissociation
constants determined from amide (1H and15N), methyl (1H
and13C) and R151-Hdchemical shift variations (at 14.1 T and
293 K).
Designation Oligonucleotide sequenceKd(mM)a
leader-neg39-UGCGCUUUUUUACGC-59
75615
leader-pos59-ACGCGAAAAAAU-39
2.561.5
long U-rich39-UUGGUUUUUUUACCC-59
125635
short U-rich39-GGUUUUUUUA-59.600
short A-rich59-CCAAAAAAAU-39
2266
GS-neg39-CCCCGUUUAU-59
110615
GE_SH-neg39-UCAAUUAAUUUUU-59
75615
GE_SH-pos59-AGUUAAUUAAAA-39
1365
GE_F-neg39-UCAAUAUAUUUU-59
85625
GE_F-pos59-AGUUAUAUAAAA-39
1165
GE_F-dsb
4.562.5
GE_F-DNA39-TCAATATATTTT-59
250670
UGA239-CGCGAAUUUUUUCGCG-59
60610
aKderrors were estimated from standard deviations of Kdvalues determined for
individual residues.
bDouble-stranded GE_F RNA was formed with equal amounts of the
complementary GE_F-neg and GE_F-pos strands.
doi:10.1371/journal.ppat.1002734.t001
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constants for a binding model with a 1:1 stoichiometry. The fitted
chemical shift variation curves for individual residues in a fast
exchange regime are given in Figure S3. The values of the
apparent dissociation constants (Kd) are recapitulated in Table 1.
They range from 2.5 mM to .600 mM and fall into two groups.
Except for a short U-rich RNA sequence, Kds for U-rich negative-
sense RNAs are in the 75–125 mM range. UGA2, an RSV-
unrelated stem-loop RNA comprising a U-stretch and five base
pairs, used as a control, binds with similar affinity (Kd=60 mM). A
second control was carried out with a short single stranded DNA
equivalent to the GE_F-neg RNA, showing that the same DNA
sequence (250 mM) binds with slightly less affinity than the RNA
sequence (85 mM). Apparent Kds for A-rich positive-sense RNA
sequences (leader-pos, GE_SH-pos, GE_F-pos and short A-rich)
are in the 2.5–22 mM range. A last experiment with the double-
stranded F gene end (GE_F-ds, 4.5 mM) shows that the affinity is
further increased as compared to the affinities of single-stranded
RNAs. Taken together, these results indicate that M2-158–177
binds to RNA with lower affinity than reported for full-length M2-
1 [10,13]. Importantly they show that the presence of A-rich
stretches increases binding affinity as compared to U-rich stretches
and that RNA base-pairing might also play a role.
As shown above, helices a2, a5 and a6 form a uniform RNA
binding surface. However, residues L74 and G75 in helix a1,
which are located on the opposite negatively charged side of the
protein, are also affected by RNA binding. For all RNAs, Kds
determined for residues in helix a1 were always the same as those
measured for residues of the main binding site. This strongly
suggests that perturbations in a1 on the one hand and in a2, a5
and a6 on the other hand are related to the same binding event.
Figure 2. Chemical shift perturbations of M2-158–177by RNA reveal a continuous RNA binding surface. Panels A, B and C show close-ups
of the superposition of HSQC spectra of15N13C-labeled M2-158–177(50 mM, 14.1 T, 293 K) in the presence of increasing amounts of the synthetic short
polyA RNA (59-CCAAAAAAAU-39). The spectra are shown in colors ranging from red to purple with 0/0.1/0.2/0.4/0.6/0.8/1.0/1.5/2.5/4.0/6.0 RNA
equivalents. (A)1H-15N HSQC spectrum, (B) methyl region of the1H-13C HSQC spectrum and (C) arginine side chain region of1H-13C HSQC. (D) Per
residue difference plot of weighted averaged chemical shifts Dd1H15N of15N-M2-158–177in the presence of short polyA determined with a 6:1
RNA:protein molar ratio. Bars are color coded from red to blue: lowest to highest value. The mean value and mean+1 standard deviation (sd) are
indicated with solid and dashed lines. Panels (E) and (F) show the mapping of chemical shift variations (Dd1H15N) on the M2-158–177structure, in
cartoon and surface representation respectively. Amide15N atoms of residues with Dd1H15N.mean+1sd for all tested RNA sequences (recapitulated
in Table 1) are indicated with red spheres. Side chains of R151 and K92, for which RNA induces13Cd-1Hd and13Ce-1He chemical shift variations, are in
stick representation. The surface formed by all these residues is colored in red. P153, for which no information is available from1H-15N HSQC data, is
shown in dark grey.
doi:10.1371/journal.ppat.1002734.g002
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Moreover as the hypothetical second contact surface with RNA
would be very distant from the main binding site, we exclude the
possibility of a second binding site at a1. However RNA binding to
a2 could be transmitted to a1 by slight alterations of helix-helix
packing.
The P binding region of M2-158–177is adjacent to the RNA
binding site
To compare RNA and P affinities for M2-158–177, we performed
isothermal titration calorimetry (ITC) experiments using this
truncated form of M2-1. Results showed that the phosphoprotein
P binds to M2-158–177with a stoichiometry of 1:1 and a Kdof
,3 mM (Figure S4). We investigated this interaction further by
NMR for atomic details. Perturbations induced by tetrameric full-
length P in1H-15N and1H-13C HSQC spectra of M2-158–177were
monitored. Due to the size of the complex and the unfavorable
exchange regime, addition of 0,5 molar equivalents of P was
accompanied by extensive overall line broadening in the1H-15N
HSQC spectra, except for the unstructured N- and C-termini (see
Figure S5A). At lower P concentrations, the majority of cross-
peaks remained detectable. Residues T130-L165 exhibited larger
line broadening, suggesting that P binds to the a4/a5/a6 region.
Transferred cross-saturation (TCS) experiments [23] were carried
out with2H15N-M2-158–177to more specifically identify residues
involved in P binding (Figure 3, panels A to D). Saturation of the
methyl protons of P resulted in reduction of M2-1 cross peak
intensities in the regions V127-S137 and L152-T164 (helices a4
Figure 3. Probing phosphoprotein P binding to M2-158–177by NMR perturbation experiments. (A, B, C and D) Results of transferred cross-
saturation (TCS) experiments carried out with 150 mM2H15N-M2-158–177in the presence of P (15 mM, 91% D2O, 14.1 T, 293 K). (A) Close-up of the
1H-15N HSQC spectra with methyl proton saturation of P (blue) and without (orange). (B) Per residue plot of reduced1H-15N HSQC cross-peak
intensities, calculated as a ratio between intensities with (Isat) and without (I0) methyl proton saturation and corrected by the reduced cross-peak
intensities measured in the absence of P. (C) and (D) Mapping of the TCS effect on the structure.15N atoms of residues with D(Isat/I0).mean+1sd are
shown as purple spheres. The surface formed by these residues is colored in purple. (E, F, G and H)1H-15N HSQC cross-peak intensity perturbation
experiments of15N13C-M2-158–177(50 mM) in the presence of P100–166(100 mM, 14.1 T, 298 K). (E) Close-up of the1H-15N HSQC spectra with P100–166
(blue) and without (red). (F) Per residue plot of the resulting cross-peak intensity reduction relative to the reference intensity. (G) and (H) Mapping of
the intensity variations on the structure.15N atoms are shown as spheres for residues with DI/I0.75% (blue) and .70% (cyan). The same color code is
used for the surface representation.
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and a6, and a5/a6 hinge). They form a nearly uniform surface on
M2-1 (Figure 3D). Since P residues 100–120 were reported to be
critical for M2-1 binding [14,24], we also monitored intensity
variations in1H-15N HSQC spectra in the presence of a truncated
form of P, P100–166, comprising this region as well as the
oligomerization domain of P [25,26]. The results are shown in
Figure 3 (panels E to H). Line broadening was enhanced for
helices a4 and a6 (Figures 3F and 3G), which is consistent with the
results obtained with full-length P. In summary, P binds to a region
proximal to the RNA anchoring surface. Helices a6 and a5 were
sensitive to both P and RNA binding in our NMR experiments,
and could thus contribute to both interaction sites, while a4 and
a2 appear to be involved specifically in P binding and RNA
binding, respectively.
Effects of surface mutations in the P and RNA binding
regions on M2-1 controlled transcription probed by a
minigenome
Based on these results, we designed single residue mutations of
the full-length M2-1 protein targeting the binding sites of P and
RNA. Solvent-exposed residues were first substituted by Ala. The
effect of these mutations on transcription antitermination by M2-
1, which was previously shown to be the same for RSV-specific
and heterologous sequences [6], was assessed using an RSV
dicistronic subgenomic replicon, pM/Luc. It contains the authen-
tic M/SH gene junction, and the Luc reporter gene downstream
of the gene start sequence present in this gene junction. The
expression of the Luc gene in this system is absolutely dependent
on the presence of a functional M2-1 [15]. The pM/Luc plasmid
was co-transfected in BHK-21 BSRT7/5 cells expressing T7 RNA
polymerase together with p-b-gal, pL, pP, PN, and pM2-1.
Luciferase activities were determined and normalized based on b-
galactosidase expression [5,8,15,27,28]. Except for L148A and
N163A, most of the Ala substitutions had only little effect on
transcription, as assessed by Luc expression (Figure 4A). As the P-
and RNA-binding surface identified by NMR is highly positively
charged, residues were substituted by Asp to emphasize the
electrostatic effect of mutations. When substituted by Asp, K92 (in
helix a2), a residue involved in RNA but not in P binding
according to the NMR results, and R126 and T130 (in helix a4),
two residues involved in P but not in RNA binding, appeared to be
critical for transcription. A similar effect on transcription was
observed for four other residues (K150, R151, T160 and N163, in
helices a5 and a6), that belong to the dual RNA/P binding
surface. In contrast, Ala and Asp mutants of K158, which is also
Figure 4. Effect of M2-1 mutations on RSV transcription and association with the nucleocapsid. (A) Analysis of RSV specific M2-1-
controlled transcription with WT and M2-1 substitution mutants. BSRT7/5 cells were transfected with RSV pP, pN, pL, and pM2-1 plasmids and an RSV
specific minigenome containing the firefly luciferase reporter gene, together with p-b-Gal constitutively expressing b-galactosidase. Luciferase
activity, measured 24 h after transfection, was normalized by b-galactosidase activity, and the luciferase activity gained with WT M2-1 set to 100%.
The mean value and confidence intervals (error bars) result from 3 separate experiments performed in duplicate. A control was run without M2-1. (B)
Expression of M2-1 mutant proteins in BSRT7 cells. Cells were co-transfected with plasmids encoding M2-1 mutants and N. Cell extracts were
analyzed by Western blotting with rabbit polyclonal antibodies against M2-1 and N. Expression levels of M2-1 were normalized against N expression
and compared to tubulin. (C, D) Colocalization studies of M2-1 with N-P complexes. Plasmids encoding N, P, and M2-1 mutants were transfected into
BSRT7/5 cells. Immunofluorescence analysis was performed on cells fixed 24 h after transfection, by using rabbit polyclonal anti-N (1:100) or anti-P
(1:500) and Alexa Fluor 594 goat anti-rabbit (1:1000) antibodies, and mouse monoclonal anti-M2-1 (1:40 dilution) and Alexa Fluor 488 goat anti-
mouse (1:1000) antibodies. Horizontal bars correspond to 10 mm.
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located in the RNA/P binding region, still displayed more than
80% activity as compared to the WT (Figure 4A). As a control, we
further verified by NMR that the deactivating mutations did not
disrupt the protein fold (Figure S6).
Interaction of M2-1 surface mutants with P-N complexes
in cellulo
Co-expression of P and N proteins (in the absence of other viral
proteins) in cells induces the formation of cytoplasmic inclusion
bodies containing P-N complexes, as observed in RSV infected
cells [11]. When co-expressed with P and N, WT M2-1 also
localizes preferentially in these IBs as seen in Figure 4D [11]. We
analyzed the intracellular localization of the M2-1 mutants in cells
co-transfected with expression vectors for P, N and M2-1 by
fluorescence microscopy (Figure 4C and 4D). Contrary to WT
M2-1, the mutants R126D, T130D, L148A, T160D and N163D
were excluded from the IBs and spread all throughout the
cytoplasm. In contrast K92D, K150D and R151D retained their
localization to cytoplasmic IBs. These mutants were expressed at
comparable levels, as determined by Western blotting (Figure 4B),
and were correctly folded when purified from E. coli and analyzed
by NMR (Figure S6).
In vitro binding of M2-1 mutants to P and RNA
To verify that the residues identified using NMR and the
minigenome assay are critical for RNA- and/or P-binding, the in
vitro RNA and P binding capacities of eight M2-1 mutants selected
by the Luc assay were investigated. As M2-1 did not migrate in
native agarose gel, it was not possible to obtain electrophoretic
mobility shift assays (EMSA) with the GST-free forms. We thus
used M2-1 fused to GST for the in vitro binding assays with RNA
and P. For RNA binding assays, we used either full-length
(tetrameric) or truncated 58–177 (monomeric) forms of M2-1 fused
to GST. GST-M2-158–177, incubated with tRNA, was analyzed by
EMSA. Formation of GST-M2-158–177:RNA complexes was only
impaired by the K92D, K150D and R151D mutations, which did
not prevent M2-1 association with IBs (Figure 5A and 5B). The
effect of single mutations of the full-length form on in vitro RNA-
binding was smaller than with truncated M2-158–177, probably
owing to the higher avidity of tetramers for RNA compared to
monomers, and the Lys/Arg repetitions that limit the effect of
single substitutions (data not shown). In vitro M2-1:P interactions
were assessed by GST pull-down assays. As shown in Figure 5C
and 5D, a$50% decrease in P binding was observed for the
mutants R126D, T130D, L148A, T160D and N163D, which were
excluded from IBs, but could still bind RNA. The two separate
binding surfaces for RNA and P, determined from these 8
residues, are illustrated in Figure 5E. Together with the cellular
localization experiments, these results show that there is a strong
correlation between the reduced capacity of M2-1 to pull-down P
in vitro and its exclusion from IBs. Conversely, since residues
specifically involved in RNA binding appear not to be crucial for
recruitment of M2-1 to IBs, the M2-1:RNA interaction is not
required for this process.
Discussion
Similarity between pneumovirus M2-1 and filovirus VP30
transcription co-factors
In contrast to other members of the Mononegavirales order for
which transcription is driven by only three proteins (N/NP, P and
L), pneumoviruses and filoviruses encode a fourth transcription co-
factor (M2-1 and VP30 respectively). Parallels have been drawn
between VP30 and M2-1 on the basis of their functions and
domain organization [17,29]. Both were shown to be necessary for
efficient transcription in reconstituted minigenome systems [7,30].
In addition, although VP30 and M2-1 were shown to be
dispensable for RNA replication [30,31,32], both are required to
rescuerecombinant EBOVor Marburg
[33,34,35]. M2-1 and VP30 contain an N-terminal Cys3His1
motif that does not bind RNA directly, but that is essential for
VP30 RNA binding [15,17,36,37]. In the case of M2-1 this motif
is indispensable for function, but its exact role is still unknown
[17,18,19]. Like M2-1, VP30 also contains an oligomerization
domain downstream of the Cys3His1motif, which is necessary for
its function during transcription [15,36]. But, despite functional
similarities, the sequence identity between the two proteins is very
low and it was not possible to know that they were evolutionary
related.
Here we show that the globular domains M2-158–177 and
EBOV VP30CTD[38] are structurally homologous and that they
display the same a-helix bundle fold. The structures were aligned
on the DALI server [39] which yielded a Z-score of 5.7 and an
rmsd of 3.9 A˚for 92 aligned residues and 9% sequence identity
(see Figure 6). The core helices a1, a2, a5 and a6 align well, with a
slight shift of a1M2-1with respect to a1VP30. The a3–a4 hairpins
are skewed relative to each other. Moreover M2-158–177 and
VP30CTDcontain several identical hydrophobic residues in helices
a5 and a6 (indicated in Figure 6), which are involved in stabilizing
inter-helical contacts and appear to be semi-conserved among
pneumovirus/metapneumovirus M2-1 and filovirus VP30 protein
sequences (Figure S7). Although they mainly contribute to inter-
helix packing, it is noteworthy that one of them (L148) is also
critical for M2-1 transcription antitermination. VP30CTDcontains
an additional 7th C-terminal helix, which has no counterpart in
M2-1. It stabilizes the crystallographic VP30CTD dimer by
interaction with a1. Since full-length M2-1 forms tetramers with
the oligomerization domain just upstream of the core domain, its
monomeric state in isolation suggests that the core domains may at
best be loosely associated with each other in the tetramer, in the
absence of partner molecules, as schemed in Figure 1C.
The overall structural match rationalizes the relationship
between M2-1 and VP30. Importantly both proteins associate
with the nucleocapsid by means of their globular core domains
[10,14,38,40]. Whereas the VP30CTD:nucleocapsid interaction is
mediated by the EBOV nucleoprotein but not by RNA [41], the
M2-1:nucleocapsid interaction has been proposed to be mediated
by RNA [10] and by P [14]. RNA binding to VP30CTDhas not
been observed [38], in contrast to what we report for M2-158–177.
As no direct M2-1:N complex was evidenced in vitro [10,14], the
correlation found between M2-1 localization to cytoplasmic IBs,
containing N and P, and the capacity of M2-1 to bind P in vitro
indicates that binding of M2-1 to P drives recruitment of M2-1 to
the holonucleocapsid.
virusand RSV
M2-158–177binds to mRNA transcripts rather than to
genomic RNA
M2-1 was first described as an antitermination factor, prevent-
ing cessation of chain elongation and release of the nascent mRNA
[7]. Later M2-1 was reported to inhibit transcription termination
at the GE signals to produce polycistronic readthrough mRNAs
[6,8,9], depending on GE sequences. The semi-conserved 12–13
nucleotide GEs fall into three groups with respect to this property:
the first (NS1/NS2, NS2/N, M2/L, and L/trailer) contains
sequences inefficient for transcription termination and is insensi-
tive to M2-1; the second (N/P, P/M, M/SH, SH/G, G/F) is very
efficient in transcription termination but not sensitive to M2-1;
finally (F/M2) is highly sensitive to M2-1 [9]. M2-1 did not direct
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readthrough at the leader-NS1 junction [6]. Thus, it was suggested
that M2-1 would not only prevent inappropriate intragenic
termination, but also allow the polymerase to access to promot-
er-distal regions of the genome and to transcribe downstream
genes [9]. Cuesta et al. reported that renatured M2-1 bound
preferentially to a short positive-sense leader RNA in vitro [13],
while Cartee and Wertz co-immunoprecipitated RSV mRNA with
M2-1 from infected cells treated with Actinomycin D [10]. These
observations suggested that M2-1 could modulate transcription
termination by recognizing specific viral RNA sequences, either on
transcribed mRNAs or on the genomic RNA template. Contrary
to transcribed viral mRNA, genomic RNA is encapsidated.
However if the model of RNA synthesis by the vesicular stomatitis
virus, a prototype of nonsegmented negative-strand RNA viruses,
could be transposed to RSV, genomic RNA would become
accessible as the nucleocapsid is being locally disassembled by the
transcription complex [42,43]. By comparing the affinities of M2-
158–177for short 10–15 nucleotide RSV RNA sequences, we found
Figure 5. Effect of mutations affecting M2-1-controlled transcription on M2-158–177:RNA and M2-1:P complex formation in vitro. (A
and B) Electrophoretic mobility shift assay (EMSA) of M2-1:RNA complex formation. Eluted GST-M2-158–177(WT and mutants selected using the
minigenome assay, 100 mM final concentration) were incubated with yeast tRNA (,50 mM final concentration) for 1 h at room temperature. (A)
Complexes were resolved by agarose gel electrophoresis stained with ethidium bromide. (B) Proteins were revealed by amido black staining. M2-1
mutations are indicated above each lane. (C and D) GST pull-down of purified P by GST-M2-1 (WT and the same mutants as in A and B). (C) GST-M2-1
or GST were incubated alone (2) or in the presence of P (+), washed, and analyzed by SDS-PAGE. P was also run alone (lane P). (D) Coomassie blue-
stained gels were scanned and M2-1:P binding was quantified using ImageJ software and corrected for nonspecific binding to GST. Errors were
estimated to 65%. (E) The surfaces formed by the 8 residues, for which mutants were analyzed, are indicated on the M2-1 structure according to their
binding partner: in red (RNA binding) and blue (P binding).
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that M2-158–177 bound preferentially to positive-sense RNA
sequences such as the 12 first bases of the positive-sense leader
(Kd=2.5 mM), positive-sense GEs (11 and 13 mM) and an A-rich
sequence located on the positive-sense leader (22 mM). Affinities of
the negative-sense signal sequences were systematically lower by
one order of magnitude or more than those of their complemen-
tary sequences. But there was no difference between the SH and F
gene ends, which are respectively insensitive and highly sensitive to
M2-1. The 39 and 59 extremities of the leader and the GS
displayed a similar behavior to that of GEs of same polarity,
indicating that M2-158–177does not discriminate between tran-
scription signals of same polarity. Sequence specificity of RSV
transcription antitermination thus appears not to be linked to the
M2-1 core domain. These results are also consistent with the
observation that M2-1 is not required for initiation of RNA
transcription [7]. Our results show that M2-158–177 displays a
preference for purine-rich and especially A-rich RNAs found in
positive-sense RSV RNAs over pyrimidine-rich sequences con-
taining U-stretches found in negative-sense RNAs. Thus, they
reinforce the hypothesis that M2-1 binds preferentially to positive
sense RNA transcripts rather than to the template, in agreement
with previous reports [10,13]. An additional finding is that M2-1
binds to the double-stranded F gene end with similar or better
affinity (4.5 mM) than to the positive-sense sequence alone
(11 mM). This implies that M2-1 could bind to the nascent
mRNA transcript, either still bound to the template or released
from it. In both cases, in order to facilitate transcription
elongation, M2-1 could prevent formation of mRNA secondary
structures that might destabilize the transcription complex, in
analogy to the function of N protein that binds to the nascent
RNA during replication.
Functional implications of the interaction of M2-1 with P
Using in vitro experiments, we have previously shown that RNA
and P bind to M2-158–177in a competitive way [15]. Here we have
characterized two distinct binding surfaces for P and RNA by
NMR (Figure 5E). Their edges are partially overlapping. The P
binding epitope, which is buried between helix a4 and the hinge
between a5 and a6 (Figure 3D and 3H), might be occluded by
RNA binding in the vicinity, and conversely RNA binding might
be hampered by bound P due to steric hindrance. Both binding
sites are located on the positively charged face of M2-158–177,
underlining the importance of electrostatic interactions for
association with RNA and with P. This hypothesis is also in
agreement with the predicted negatively charged surface in
computer models of the M2-1 binding region of the P tetramer,
spanning residues 100–120 of P [44]. Redundancy of positive
charges in the P and RNA binding region certainly accounts for
the weak effect on Luc expression observed for Ala mutants of
residues in this region in the minigenome assay, as compared to
Asp substitution, which introduced opposite charges. Electrostatic
interactions could be further emphasized in the M2-1 tetramer if
the core domains were arranged to provide an extended binding
surface consisting of two adjacent positively charged clusters. In
addition to charge effects, specific hydrophobic interactions may
also contribute, as suggested for the interaction between P and
M2-1 in [14]. This could apply to RNA as well. Although the
range of RNA affinities for M2-158–177(micromolar to millimolar)
is an indicator for non-specific binding mediated by the RNA
phosphate backbone, corroborated by binding of a DNA sequence
equivalent to GE_F-neg, the difference between A-rich and U-rich
RNA affinities indicates that the nature of the bases also comes
into play.
The large M2-158–177:RNA binding surface determined by
NMR coincides with the main positively charged cluster of M2-
158–177 (Figure 1B and 2F), which is well conserved among
Pneumovirinae (Figure S7). We confirmed by mutagenesis that the
three basic residues K92, K150 and R151, included in this
epitope, are crucial for in vitro RNA binding to M2-158–177and for
transcription enhancement in vivo by full-length M2-1. However
they did not prevent association with cytoplasmic IBs or P binding.
In contrast, mutants R126D, T130D, L148A, T160D and N163D,
for which M2-1-controlled transcription was impaired and which
still bound RNA in vitro, had lost their ability to bind P in vitro and
did not co-localize with N-P complexes in cytoplasmic IBs. These
residues are not well conserved in Pneumovirinae M2-1 proteins
(Figure S7A), but this would be consistent with the sequence
variability of the M2-1 binding region of P(100–120), which has
co-evolved with M2-1. Altogether the NMR and mutagenesis
results provide a rationale for the competitive binding previously
observed between P and RNA [15]. They highlight the role of P
for the recruitment of M2-1 to cytoplasmic IBs that contain N and
P and where viral RNA synthesis takes place, by analogy to
Rhabdoviridae [12].
Possible cooperative effects in full-length tetrameric M2-1,
involving the N-terminal Cys3His motif and interactions between
core domains, may induce an increased affinity for both P and
RNA as well as sequence specific recognition of RNA, with respect
to the core domain of M2-1. However the overall lower affinities of
RNA for M2-158–177, as compared to P, suggest that M2-1 binds P
Figure 6. Structural alignment of RSV M2-158–177and EBOV VP30CTD. Cartoon representations of aligned RSV M2-158–177(residues L74 to
T172) and EBOV VP30CTD(pdb 2I8B, [38]). Disordered N- and C-termini are not shown. Structural alignment with M2-158–177was generated by the Dali
server [39] (Z-score=5.7; rmsd=3.9 A˚; 92 aligned residues; 9% sequence identity). VP30CTDhelix a7, which has no counterpart in M2-158–177, is not
represented. Identical hydrophobic residues in helices a5 and a6 are represented with sticks for M2-158–177and VP30CTD.
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preferentially in the absence of viral RNA so that M2-1 would be
recruited as an RNA-free M2-1:P complex to the IBs. This
hypothesis is in agreement with our fluorescence microscopy
observations, since WT M2-1 and mutants that do not bind RNA
are found likewise in IBs containing N and P (Figure 4C and 4D).
Indeed, higher affinity to RNA would result in sequestration of
M2-1 in the cytoplasm, where cellular RNA is highly accessible,
and M2-1 would not be recruited to the IBs. The relatively low
Kds are in the same range as those measured for the Measles virus
P-N interaction [45], this intrinsically weak association being
probably required for movement of the viral polymerase during
RNA transcription.
In summary, our results indicate that not only P:M2-1, but also
RNA:M2-1 interactions are required for efficient transcription
activation by M2-1. Association with P is strictly required for
recruitment to the viral RNA synthesis site. As suggested by the
proximity of the binding sites, it is likely that the P:M2-1
interaction is displaced in favor of other interactions, RNA:M2-1
interactions in particular, in the context of the holonucleocapsid.
The higher affinity of M2-158–177for the 59 end of the positive-
sense Leader RNA (2.5 mM), which is in the same range as the
affinity for P (3 mM), suggests that M2-1 could be loaded onto the
polymerase during transcription initiation. It can further be
speculated that an L:M2-1 interaction also takes place when the
P:M2-1 interaction breaks down, thus altering the sensitivity of the
polymerase to transcription termination signals. A similar L:VP30
interaction was recently reported for EBOV [46]. This hypothesis
is currently under investigation for RSV.
Materials and Methods
Protein expression and purification
Recombinant P, P100–166, M2-1 and M2-158–177 (WT and
mutants) as well as15N-,13C- and/or2H-labeled M2-158–177were
expressed in E. coli BL21(DE3) strain. Full-length M2-1 and M2-
158–177were produced as GST fusion proteins as described in [15]
and [25] with a thrombin cleavage site. M2-1 amino acid
substitution mutants were obtained with the Quickchange site-
directed mutagenesis kit (Stratagene) by using pGEX-M2-1,
pGEX-M2-158–177and pM2-1 as templates. Isotopically labeled
proteins for NMR were produced in minimal M9 medium
supplemented with 1 g/L15NH4Cl and 4 g/L13C- or unlabeled
glucose (Cortecnet). Protocols are detailed as Supplemental
Materials and Methods (Text S1).
Minigenome assay
BSRT7/5 cells stably expressing T7 RNA polymerase [47] were
maintained in EMEM supplemented with 10% FCS/L-glutamine/
penicillin-streptomycin solution. The cells were grown in an
incubator at 37uC under 5% CO2. pN, pP, pM2-1 and pL
plasmids coding for HRSV (strain Long) N, P, M2-1 and L
proteins respectively, under the control of the T7 promoter, have
been described previously [15,48]. An encephalomyocarditis virus
internal ribosome entry site (IRES) sequence was placed between
the T7 promoter and the inserted ORF to enhance protein
expression in BSR/T7-5 cells, as previously described [48,49].
The pM/Luc subgenomic replicon was derived from the pM/SH
subgenomic replicon [8] and has been described previously [15]. It
contains two transcription units, the second encoding the firefly
luciferase (Luc) gene under the control of the M/SH intragenic
sequence. The expression of the Luc gene in this system is
absolutely dependent on the presence of M2-1 [15]. BSRT7/5
cells were transfected with pN, pP, pL, pM2-1, p-b-Gal coding for
beta-galactosidase under the control of the Rous sarcoma virus
promoter (Promega) and pM/Luc, where luciferase expression is
controlled by the RSV M/SH intergenic region [15]. Luciferase
activity was determined in triplicate 24 h post-transfection as
previously described [15]. Cells were lysed in luciferase lysis buffer
(30 mM Tris, pH 7.9, 10 mM MgCl2, 1 mM dithiothreitol
[DTT], 1% [vol/vol] Triton X-100, and 15% [vol/vol] glycerol),
and luciferase activities were evaluated twice for each cell lysate
with an Anthos Lucy 3 luminometer (Bio Advance).
Transfections and indirect immunofluorescence analysis
BSRT7/5 cells were transfected with pP (0.4 mg), pN (0.4 mg)
and pM2-1 (0.2 mg) containing either WT or mutant M2-1 by
using Lipofectamine2000 (Invitrogen). Samples were fixed after
24 h in 4% paraformaldehyde, and permeabilized in PBS
containing 0.1% Triton X-100 and 3% BSA. Each coverslip was
incubated with primary antibodies: anti-N (1:100 dilution) and
anti-P (1:500 dilution) rabbit polyclonal sera, and 37M2 and 22K4
anti-M2-1 monoclonal antibodies (1:40 dilution) [50]. These
samples were incubated for 1 h at room temperature, washed,
and then incubated for an additional hour with Alexa Fluor 488
goat anti-mouse and Alexa Fluor 594 goat anti-rabbit (1:1000) IgG
(Invitrogen). Cells were observed with a Nikon TE200 inverted
microscope equipped with a Photometrics CoolSNAP ES2
camera. Images were processed using MetaVue software (Molec-
ular Devices).
Analysis of in vitro RNA and P binding by M2-1 and M2-1
mutants
GST-M2-158–177 fusion proteins (WT or mutants, final
concentration 100 mM) were eluted by GSH and incubated with
yeast tRNA (Sigma, final concentration 50 mM) in a final volume
of 10 ml. Complexes were resolved by 1.5% agarose gel
electrophoresis in 16 Tris-Glycine buffer at 4uC, stained with
ethidium bromide and amido black. GST pull-down of purified
recombinant P by full-length GST-M2-1 fusion proteins (WT and
mutants) was performed by incubating 10 ml aliquots of a 50%
slurry of Glutathione-Sepharose 4B beads (GE Healthcare)
containing ,25 mM GST-M2-1 in PBS with a 3-fold molar
excess of P for 1 h at 20uC under agitation. Beads were washed
extensively with PBS, boiled in 25 ml Laemmli buffer and analyzed
by SDS-PAGE and Coomassie blue staining. Bands were
quantified using ImageJ software. Affinity between P and M2-1
was determined by isothermal titration calorimetry.
Isothermal titration calorimetry assays
Raw ITC data were processed and quantitative analysis of the
P-M2-158–177interaction was performed on a MicroCal ITC200
microcalorimeter (Microcal, Northampton, MA). Samples were
dialyzed against 16PBS for 15 h. The experiments were carried
out at 20uC. The P concentration in the microcalorimeter cell
(1.4 mL) was of 33 mM. In total, 20 injections of 2 mL of M2-158–
177solution (concentration 335 mM) were carried out at 180 s
intervals, with stirring at 1000 rpm. The experimental data were
fitted to a theoretical titration curve with software supplied by
MicroCal (ORIGIN). This software generates titration curves
based on the relationship between the heat generated by each
injection and DH (enthalpy change in kcal/mol), Kd(dissociation
constant), n (number of binding sites), the total protein concen-
tration and free and total ligand concentrations.
RNA oligonucleotide synthesis
10–15 nucleotide RNAs corresponding to viral RNA sequences
of negative and positive polarity were synthesized on a Pharmacia
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Gene Assembler Plus using phenoxyacetyl b-RNA phosphorami-
dites and Universal Support resin (Glen Research) with a protocol
adapted for RNA from DNA solid-phase synthesis [51]. The
products were HPLC-purified (Beckman) by anionic chromatogra-
phy on a DEAE-Sepharose column (Pharmacia) in 10 mM
phosphate buffer pH 6.8 and eluated with a linear gradient from
0.55 to 1 M NaCl in 80 min. The oligonucleotides were extensively
dialyzed in water. If necessary the pH was adjusted to 7 with 0.5 M
NaOH and the concentration determined using the A260 value.
Aliquots were then lyophilized and stored at 220uC. The
oligonucleotide sequences are given in Table 1. Folding properties
of RNAs were estimated by using the mfold web server [22].
NMR spectroscopy
NMR measurements were carried out at 293 K (or 298 K) on
Bruker Avance 600, 800 and 950 MHz spectrometers equipped
with triple resonance cryoprobes. All samples were in 50 mM
sodium phosphate buffer pH 6.8, 150 mM NaCl, 1 mM DTT
and 7% D2O. Resonance assignment was reported elsewhere [52].
15N-NOESY-HSQC(800 MHz)
(950 MHz) experiments, simultaneously edited for aliphatic and
aromatic13C, with 80 and 100 ms mixing times, were recorded
using 120 mM
samples respectively. Data were processed with Topspin 2.1
or NMRPipe [53]. Spectra were analyzed with Sparky [54].
Residual dipolar couplings (RDCs) were collected on15N-labeled
M2-158–177in two alignment media. The first sample contained
120 mM15N-M2-158–177in a stretched 6% acrylamide/bisacryla-
mide (37.5:1) gel (deuterium splitting D2H=3 Hz). The second
sample contained 200 mM
C12E5 phase with r=0.96 molar ratio (D2H=26,7 Hz).
RDCs were extracted from spin-state selective InPhase-AntiPhase
1H-15N HSQC experiments acquired in interleaved fashion.
and
13C-NOESY-HSQC
15N- M2-158–177 and 100 mM
13C-M2-158–177
15N-M2-158–177 in a 5% hexanol/
1DNH
NMR structure calculation
M2-158–177structures were calculated with CYANA 2.1 [55],
using distance restraints obtained from
HSQC spectra and backbone torsion angles generated with
TALOS. They were refined in Xplor-NIH [56] using
residual dipolar couplings measured in a stretched polyacrylamide
gel and a hexanol/C12E5 phase. Structure statistics are summa-
rized in Table S1. More details are provided in Text S1. Graphic
representations were performed with PyMOL [57].
15N- and
13C-NOESY-
1DHN
NMR M2-158–177chemical shift and cross-peak intensity
variation experiments with RNA and phosphoprotein
Chemical shift and cross-peak intensity perturbations experi-
ments were carried out with yeast RNA (Roche) and short
synthesized oligonucleotides (see Table 1). 0.25–20 equivalents of
lyophilized RNA were added stepwise to 50–60 mM15N-,13C- or
15N13C-labeled M2-158–177.1H-15N and1H-13C HSQC spectra
were recorded at 293 K at each step, at magnetic fields of 14.1 T
and/or 22.3 T.
Interaction with phosphoprotein was probed by cross-peak
intensity perturbations in
ments, with full-length P (P1–241) and truncated P100–166. Samples
with P1–241were prepared by mixing concentrated solutions of
P1–241 (30 mM) and
the same phosphate buffer to a final concentration of 40 mM
M2-158–177and P:M2-1 molar ratios of 0.25:1:and 0.5:1. Samples
with P100–166wereprepared by mixing solutions of P100–166(600 mM)
and15N13C-labeled M2-158–177(140 mM) to a final concentration of
45 mM M2-158–177and a P:M2-1 molar ratio of 2:1.
1H-15N and
1H-13C HSQC experi-
15N13C-labeled M2-158–177 (275 mM) in
Titration experiments with RNA and dissociation
constant determination
Spectra were typically recorded at 14.1 T and 293 K. For each
RNA sequence, addition of RNA was followed by1H-15N HSQC
experiments of15N-M2-158–177. Weighted averaged chemical shift
differences were calculated from
according to following equation, where the 1/10 scaling factor for
15N chemical shifts corresponds to the ratio of gyromagnetic ratios
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
The apparent dissociation constant Kdwas obtained by fitting
1H or15N chemical shift differences at each titration point with
two parameters in a binding model with 1:1 stoichiometry and
with a user-defined function in Origin 7 software as follows:
1H and
15N chemical shifts
of15N and1H: Dd~
d1H{dfree
1H
??2z d15N{dfree
15N
??2=100
r
.
d{dfree~2 ? B ? (Azx{
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
(Azx)2{4x
q
),
where x~RNA
B~ dbound{dfree
ð
½?tot= M2{158{177
Þ.
½?tot, A~1zKd= M2{158{177
½?tot,
Transferred cross-saturation experiments
Samples were prepared with a 30 mM solution of P1–241
centrifuged at 15 krpm in a TLA55 rotor (Beckman) for 15 min
and exchanged into D2O buffer with biospin columns according to
the manufacturer’s instructions (Biorad). Transferred cross-satu-
ration experiments [23] were conducted with 150 mM
labeled M2-158–177in 91% D2O in the presence of 15 mM (10%)
unlabeled P1–241protein. Broadband proton saturation (2 s) was
achieved with a 1.8 kHz Wurst pulse centered at 0.8 ppm. Spectra
were recorded in interleaved fashion with and without saturation,
with 1.5 s recycling delay, at 14.1 T and 293 K. Intensity ratios
were determined based on experiments with and without
saturation. Control experiments were carried out without P to
account for spin diffusion and effects of residual aliphatic protons
in M2-158–177.
2H15N
Data deposition
Atomic coordinates and structural constraints have been
deposited in the Protein Data Bank (PDB accession code 2L9J).
Accession numbers
Swiss-Prot
UniProt
P04545.1
P28887.1
Supporting Information
Figure S1
followed by
done at 298K and a field of 14.1 T. The reference spectrum of
15N-M2-158–177 (50 mM) is shown in black, the spectrum after
addition of 4 mg/mL yeast RNA (,1:1 molar ratio) in red and
after treatment by RNAse A in green.
(EPS)
Binding of yeast RNA to
1H-15N HSQC spectra. Measurements were
15N-M2-158–177
Figure S2
for15N-M2-158–177in the presence of RNA.1H-15N HSQC
spectra were recorded at 14.1 T and 293 K with 50 or 60 mM
protein. Per-residue plots of combined1H/15N chemical shifts in
absolute values are represented for (a) Yeast RNA (Roche) (4 mg/
mL), for (b) an RSV unrelated hairpin UGA2 hairpin (20:1
RNA:protein molar ratio), for single-stranded RSV genomic
negative-sense (neg) and positive-sense (pos) RNA sequences, (c)
neg leader (6:1),(d) neg gene start (2.5:1), (e) neg SHgeneend (2.5:1),
1H-15N chemical shift perturbations measured
Structure and Functional Analysis of RSV M2-1
PLoS Pathogens | www.plospathogens.org 11May 2012 | Volume 8 | Issue 5 | e1002734

Page 12

(f) neg F gene end (2.5:1), (g) neg long U-rich (30:1), (h) neg short U-
rich (7.5:1), (i) pos short A-rich (6:1), (j) pos leader (2:1), (k) pos SH
gene end (6:1), (l) pos F gene end (6:1) and for (m) double-stranded F
geneend (6:1) and(n)a DNAequivalent toneggeneendF (4:1).The
exact sequences are given in Table 1. Mean and mean+1sd values
are indicated by solid and dashed lines respectively.
(EPS)
Figure S3
constants by titration experiments with
experiments of
differences of backbone amides or Q93 side chain (denoted H, N,
He21, He22, and Ne2 respectively) are displayed versus the ratio of
RNA concentration relative to protein for chosen residues showing
the largest chemical shift amplitudes. Measurements were done at
14.1 T and 293 K. Protein concentration was 50 or 60 mM.15N
chemical shifts were scaled with a factor 1/10 to be of comparable
magnitude to1H. Plots are shown for following RNA sequences,
‘‘pos’’ and ‘‘neg’’ denoting RSV genomic positive-sense and
negative-sense sequences : (a) UGA2 hairpin, (b) neg leader, (c)
neg gene start, (d) neg SH gene end, (e) neg F gene end, (f) neg long
U-rich, (g) pos SH gene end, (h) pos short A-rich, (i) pos F gene end
and(j)double-strandedFgeneend.Thedatawerefittedtoa binding
model with 1:1 stoichiometry in Origin software. The mean values
of apparent Kdand the statistical error are indicated for each RNA.
(EPS)
Determination of apparent RNA dissociations
1H-15N HSQC
15N chemical shift
15N-M2-158–177.
1H and
Figure S4
158–177. (A) Raw binding data obtained for 20 automatic
injections of M2-158–177(2 ml for each injection, 335 mM protein
concentration) into a cell containing P (200 mL initial volume;
33 mM initial protein concentration). Proteins were suspended in
16PBS. (B) Integrated titration curve obtained from the raw data
in panel A after baseline subtraction. The solid squares represent
the experimental data, while the solid line corresponds to the
standard multiple independent binding-site model that was fitted
to the data. The corresponding average Kdvalue is 3 mM with a
stoichiometry of 1:1.
(TIF)
ITC binding isotherms for P binding to M2-
Figure S5
inthepresenceofphosphoprotein.(A)Superpositionof1H-15N
HSQC spectra of15N13C-labeled M2-158–177without (black) and
with 0.5 equivalent of full-length RSV phosphoprotein P (blue) shows
overall line broadening on addition of P. (B) Arginine13Cd-1Hdand
lysine13Ce-1Heregion of1H-13C HSQC spectra of15N13C-labeled
M2-158–177in the presence of 0.25 (red) and 0.5 (blue) equivalents of
full-length P (22.3 T, 298 K) overlaid on the reference spectrum
(black). Broadening is observed for K92 and K158.
(EPS)
IntensityvariationsofM2-158–177HSQCspectra
Figure S6
single mutants K92D, R126D, T130D, L148A, K150D,
R151D and T160D and N163D. K92D, K150D and R151D
strongly reduced in vitro RNA binding to M2-1 whereas R126D,
T130D, L148A, T160D and N163D were excluded from
cytoplasmic inclusion bodies containing RSV phosphoprotein. All
Changes in1H-15N HSQC spectra of M2-158–177
resulted in a nearlytotal loss of transcription in the Luc minigenome
assay. (A)1H-15N HSQC spectra of15N-labeled M2-158–177single
mutants (125 mM, 18.8 T, 293 K) have a similar pattern to that of
wild type and show that the mutant proteins are well folded. (B)
Chemical shift difference plots relative to WT M2-158–177indicate
that chemical shift changes occur only for residues close to the
mutation in the sequence, as expected if no conformational
rearrangement takes place. In the case of K92D (in helix a2), the
spatially close region around K150 (in helix a5) is also affected, and
vice versa. A15N NOESY-HSQC experiment with 80 ms mixing
time was recorded withK150D to verify the presence of HN,i-HN,i+1
correlations, characteristic of a-helices.
(EPS)
Figure S7
teins. (A) Alignment of primary sequences of M2-1 proteins of
human Respiratory Syncytial Virus (hRSV, strain Long, UniProt
accession numberP28887.1),
ATue51908, NC_001989.1), Pneumonia virus of mouse (PVM,
YP_173333), human Metapneumovirus (HMPV, AAM12941.1),
and avian Metapneumovirus (AMPV, YP_443841). Strictly iden-
tical residues are highlighted in red. Residues with more than 70%
similarity are boxed in blue. Secondary structures extracted from
the M2-158–177structure are indicated above each bloc. Single
mutated M2-158–177residues which severely impair RSV transcrip-
tion are indicated by a red star if the residues are strictly conserved
and a blue star if they diverge in more than 2 sequences. (B) The
primary sequences of M2-1 proteins were manually aligned with
VP30 protein sequences of Zaire Ebola virus (EBOV_Z,UNP
Q05323), Reston EBOV (EBOV_R, NP_690585.1) and Lake
Victoria Marburg virus (MARV_LV, YP_001531157.1). Strictly
identical residues are highlighted in red and located in the Cys3His
putative zinc finger and in the oligomerisation domains. Residues
with more than 70% similarity are boxed in blue. (C) Primary
sequences of VP30 proteins were aligned separately from M2-1 and
secondary structures extracted for EBOV_Z VP30 CTD (pdb
2I8B). Alignments were edited with ESPript 2.2 [60].
(EPS)
Sequence alignment of M2-1 and VP30 pro-
bovineRSV(bRSV, strain
Text S1
(DOC)
Supplemental Materials and Methods.
Table S1
(DOC)
NMR structure statistics of RSV M2-158–177.
Acknowledgments
We are grateful to Jose ´ A. Melero and Robert P. Yeo for providing anti-P
and anti-M2-1 monoclonal antibodies, to Gail Wertz for providing the
pM/SH minigenome, and to Ce ´line Urien for confocal microscopy. We
thank Marie Galloux and Felix Rey for helpful discussions and critical
reading of the manuscript.
Author Contributions
Conceived and designed the experiments: CS JFE. Performed the
experiments: CS FB JF MAN MLB SL VD. Analyzed the data: CS FB
JFE MLB. Wrote the paper: CS JFE.
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