Biochem. J. (2012) 444, 405–415 (Printed in Great Britain)
405
doi:10.1042/BJ20112219
The disordered N-terminal region of dengue virus capsid protein contains
a lipid-droplet-binding motif
Ivo C. MARTINS*†, Francisco GOMES-NETO*‡, André F. FAUSTINO†, Filomena A. CARVALHO†, Fabiana A. CARNEIRO§,
Patricia T. BOZZA, Ronaldo MOHANA-BORGES¶, Miguel A. R. B. CASTANHO†, Fábio C. L. ALMEIDA*‡, Nuno C. SANTOS† and
Andrea T. DA POIAN*1
*Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil, †Instituto de Medicina Molecular, Faculdade de Medicina da
Universidade de Lisboa, Lisbon, 1649-028, Portugal, ‡Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro and National Institute of Structural
Biology and Bioimage, Rio de Janeiro, RJ, 21941-902, Brazil, §Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro, Duque de Caxias, RJ, 25245-390, Brazil, Instituto
Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, 21040-360, Brazil, and ¶Laboratório de Genomica Estrutural, Instituto de Biofı́sica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil
Dengue is the major arthropod-borne human viral disease, for
which no vaccine or specific treatment is available. We used
NMR, zeta potential measurements and atomic force microscopy
to study the structural features of the interaction between dengue
virus C (capsid) protein and LDs (lipid droplets), organelles
crucial for infectious particle formation. C protein-binding sites
to LD were mapped, revealing a new function for a conserved
segment in the N-terminal disordered region and indicating that
conformational selection is involved in recognition. The results
suggest that the positively charged N-terminal region of C protein
prompts the interaction with negatively charged LDs, after which
a conformational rearrangement enables the access of the central
hydrophobic patch to the LD surface. Taken together, the results
allowed the design of a peptide with inhibitory activity of C
protein–LD binding, paving the way for new drug development
approaches against dengue.
INTRODUCTION
encephalitis virus), JEV (Japanese encephalitis virus) and YFV
(yellow fever virus). Their genomic RNA is translated from
a single open reading frame, generating a polyprotein that is
cleaved by cellular and viral proteases into three structural
proteins [C (capsid), prM (pre-membrane) and E (envelope)]
and seven NS (non-structural) proteins (NS1, NS2A, NS2B,
NS3, NS4A, NS4B and NS5) [6]. Flavivirus capsid proteins
can be considered multifunctional proteins, since they bind to
viral RNA in nucleocapsid assembly [7], act as nucleic acid
chaperones [8] and interact with a variety of cellular proteins,
modulating transcription, cellular metabolism, apoptosis and
immune response [9–11]. The flexibility conferred by their
intrinsically disordered regions is expected to facilitate these
functions [5].
The present study focused on the structural features of
DENV C protein necessary for its interaction with LDs (lipid
droplets), endoplasmic reticulum-derived organelles crucial for
virus replication and infectious particle formation [12], whose
biogenesis is stimulated by infection [13]. DENV causes the most
important arthropod-borne human viral disease, with three billion
people at risk, 50–100 million infections and more than 20 000
deaths annually [14]. DENV infection can evolve to a severe
and life-threatening disease, for which no specific treatment is
currently available. Additionally, vaccine development against
DENV is still a challenge [14]. Therefore a detailed structural
understanding of the DENV life cycle is of utmost importance to
tackle this problem.
Viruses have evolved several strategies to manipulate cellular
functions during infection, allowing their efficient replication and
spread. This is possible even with very small viral genomes, as
viral proteins usually accumulate distinct activities and properties,
being remarkable examples of multifunctional proteins. It can
be hypothesized that the diversity of functions of viral proteins
could be achieved through a high structural flexibility, conferred
by the presence of intrinsically disordered regions, which are
interconverting dynamic structures that lack a defined folding, yet
are able to carry out specific functions [1,2]. Indeed, a comparative
structural analysis of viral and cellular proteins revealed that viral
proteins show a high occurrence of short disordered regions and
loosely packed cores, suggesting that they have evolved to be more
adapted to changes in the environment than to thermodynamic
stability [3]. An analysis of the functions carried out by the
intrinsically disordered regions of viral proteins showed that they
are usually involved in recognition, regulation, signalling and
interaction with nucleic acids and proteins, functions for which
structural flexibility is required [4].
The capsid (or core) proteins from viruses of the family
Flaviviridae are good examples in which the diversity of
functions can be attributed to their intrinsically disordered
nature [5]. Flaviviruses are positive-sense single-stranded RNA
viruses that comprise several human pathogens, including DENV
(dengue virus), WNV (West Nile virus), TBEV (tick-borne
Key words: atomic force microscopy (AFM), capsid protein,
conformational selection, dengue virus (DENV), intrinsically
disordered protein, lipid droplet, NMR.
Abbreviations used: AFM, atomic force microscopy; C, capsid; CSP, chemical shift perturbation; DENV, dengue virus; E, envelope; HCV, hepatitis C
virus; HSQC, heteronuclear single-quantum coherence; LD, lipid droplet; NCBI, National Center for Biotechnology Information; NOE, nuclear Overhauser
enhancement; NOESY, NOE spectroscopy; NS, non-structural; prM, pre-membrane; RMSD, root mean square deviation; TaxID, NCBI Taxonomy identifier;
TBEV, tick-borne encephalitis virus; TEE buffer, 25 mM Tris/HCl, pH 7.4, 1 mM EDTA and 1 mM EGTA; TPPI, time proportional phase incrementation; WNV,
West Nile virus; WNV-K, WNV strain Kunjin; YFV, yellow fever virus.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
406
I. C. Martins and others
DENV particles are composed of a lipid bilayer surrounding
a nucleocapsid, in which genomic RNA is located, complexed
with multiple copies of the DENV C protein [15]. This protein
forms symmetric homodimers in solution, with each monomer
containing four α-helices (α1–α4) connected by short loops
[16,17]. The segment containing the first 20 amino acid residues
in DENV C protein was found to be disordered in solution
[17], in agreement with the disorder prediction for the same
region of WNV and YFV C proteins [5]. The protein structure
and surface charge distribution suggest that capsid assembly
occurs via C protein interaction with lipid membranes through
a central hydrophobic region located in the α2/α2 interface,
which orientates the positively charged α4/α4 C-terminus area,
allowing its interaction with the viral RNA [17]. In agreement with
this proposal, mutation of two residues in the α2/α2 interface
disrupted LD targeting and abolished virus particle formation
[12].
In the present study, using high-resolution NMR, zeta potential
measurements and AFM (atomic force microscopy)-based force
spectroscopy to map DENV C protein LD-binding sites, we
identified local structural changes involving specific conserved
amino acids residues located in the disordered N-terminal region,
in the central hydrophobic patch and in the loop between α1
and α2. On the basis of these results, it was possible to attribute
a new function to the N-terminal disordered region of DENV
C protein, reinforcing the concept of multiple functions for
intrinsically disordered regions in proteins. Furthermore, we
applied that information to develop an original and successful
peptide-based approach for the specific inhibition of the C
protein–LD interaction.
EXPERIMENTAL
Chemicals
The peptides pep14–23 (NMLKRARNRV) and pep5–26
(RKKTGRPSFNMLKRARNRVSTV) were purchased from JPT
Peptide Technologies. [15 N]NH4 Cl and 2 H2 O were purchased from
Cambridge Isotope Laboratories.
Recombinant DENV C protein expression and purification
The C protein of DENV serotype 2 New Guinea strain was
expressed and purified following established procedures [16,18],
except that M9 minimal medium supplemented with [15 N]NH4 Cl
was used for expression of 15 N-labelled C protein.
Production and purification of LDs
BHK (baby-hamster kidney)-21 cells were maintained in α-MEM
(minimum essential medium-α) supplemented with 10 % (v/v)
fetal bovine serum at 37 ◦ C in 5 % CO2 . At 80 % confluence, cells
were treated with 10 μM oleic acid for 24 h. LDs were isolated
by nitrogen cavitation (model 4639, Parr Instrument Company)
and purified by sucrose-gradient ultracentrifugation, as described
previously [18]. To ensure LD purity, only fractions negative for
lactate dehydrogenase activity were used. LDs samples were kept
at 4 ◦ C and used within 2 weeks.
NMR spectroscopic analysis
NMR experiments were performed at 300 K in Bruker Avance
III 800 MHz and 600 MHz instruments, equipped with tripleresonance (1 H,13 C,15 N) probes. C protein (200 μl) in 50 mM
c The Authors Journal compilation c 2012 Biochemical Society
phosphate buffer, pH 6.0, and 0.2 M NaCl, were added to
125 μl of LDs (∼ 4×105 /ml) in TEE buffer (25 mM Tris/HCl,
pH 7.4, 1 mM EDTA and 1 mM EGTA), or to TEE buffer
alone, to obtain a final concentration of uniformly labelled
15
N-DENV C protein between 250 and 275 μM. Chemical shifts
were referenced with respect to the H2 O signal at 4.77 p.p.m.
(pH 6.8, 25 ◦ C) relative to DSS (4,4-dimethyl-4-silapentane-1sulfonic acid). NMR spectra were processed and analysed using
NMRPipe [19] and NMRView [20]. Chemical shift assignments
were obtained by comparison with the deposited data for C protein
(Biomagnetic Resonance Data Bank 5973 [17]). Ambiguous
assignments were solved using three-dimensional 15 N-edited
NOESY [NOE (nuclear Overhauser enhancement) spectroscopy]HSQC (heteronuclear single-quantum coherence) and TOCSYHSQC by looking at αN (i,i + 1) and NN (i,i + 1) NOEs, and
85 % of the backbone amidic resonances were unambiguously
assigned. NOESY-HSQC (100 ms mixing time) and TOCSYHSQC (60 ms MLEV) were collected with and without LDs,
at 800.13 MHz, 1024×48×64 complex points. Residues Met1 ,
Asn2 , Thr11 , Leu35 and Arg55 were not assigned in the previously
determined NMR structure [17]. The assignments of the residues
Asp3 , Gln4 , Asn10 , Gly36 , Gln39 , Thr62 , Ala77 and Arg100 could
not be localized or confirmed. Quadrature detection in indirect
dimension was done using States-TPPI (time proportional
phase incrementation) (1 H) and echo–anti-echo (15 N). Gradient
selection HSQC spectra were acquired at 800.13 MHz
with 1024×128 complex points (echo–anti-echo). Peptide–LD
interaction experiments (using pep14–23 or pep5–26) were
performed at the same conditions as for C protein, except that the
final concentrations were 400 or 800 μM. Sequential backbone
resonance assignment was performed for free peptides using
(1 H,13 C)-gradient-selection-HSQC (natural abundance of 13 C),
NOESYs (100 and 200 ms mixing time) and TOCSY (60 ms
MLEV) in 10 % 2 H2 O, acquired at 800.13 MHz, with 4096×512
complex points for NOESY and TOCSY, and 1024×128 for
HSQC. Quadrature detection in indirect dimension was done
using States-TPPI (1 H) for NOESY and TOCSY and echo–antiecho (13 C) for the HSQC. Water suppression was achieved using
the 3-9-19 WATERGATE sequence for NOESY and TOCSY [21].
Alignment of the sequence of the C proteins from mosquito-born
flaviviruses
DENV C protein sequence from strain New Guinea was aligned
with other flavivirus C protein sequences using BLASTp [22]. The
search was performed against non-redundant protein sequences
and excluding the DENV group {TaxID [NCBI (National Center
for Biotechnology Information) Taxonomy identifier] 11052;
http://www.ncbi.nlm.nih.gov/Taxonomy/}. Only Flavivirus spp.
gave a score high enough to be considered for further analysis.
The best-scored sequence of each different virus strain was
selected. Virus taxonomical classification was accessed by the
NCBI Taxonomy browser and the International Committee
on Taxonomy of Viruses 2009 list (http://ictvonline.org/
virusTaxonomy.asp?version = 2009). Unclassified Flavivirus
spp. (TaxID 11051) were rejected. As a result, 16 polyprotein
sequences, each from an independent Flavivirus spp. strain
remained. In all of them, the C protein aligned sequence was in the
first ∼110 amino acid residues. The residues next to the NS2BNS3 protease cleavage site were excluded from further analysis
in all polyproteins, leaving the presumed C protein sequences,
which were aligned by multiple alignments on the Clustal W2
tool of the European Bioinformatics Institute web server [23]
against the DENV C protein sequence. In Supplementary Table
Dengue virus capsid protein binding to lipid droplets
S1 (at http://www.BiochemJ.org/bj/444/bj4440405add.htm), the
GenBank® accession numbers of each virus strain studied can be
retrieved.
Structural comparison of DENV and WNV-K (WNV strain Kunjin) C
proteins
The UCSF Chimera software package [24] was used to
superimpose DENV C protein model number 21 (PDB
code 1R6R) [17], which is the energy-minimized averaged
structure of the 53 NMR models obtained, with WNV-K
(WNV strain Kunjin) C protein model number 2 (PDB
code 1SFK) [25], which presented the lowest RMSDs (root
mean square deviations) of the α-carbons of the amino
acids when superimposed with the selected DENV C protein
structure and analysed using the Swiss-PdbViewer software
package [26]. For superimposition analysis, only amino acids
43–96 of DENV C protein were used, since immediate
visual inspection showed that only the regions encompassing
α-helices 2, 3 and 4 could superimpose with the available WNV-K
C protein structure, with a final RMSD of 2.19 Å (1 Å = 0.1 nm).
Zeta potential analysis
Zeta potential (ζ ) measurements were performed using a
Malvern Zetasizer Nano ZS system, equipped with a helium–neon
laser (λ = 632.8 nm). LD interaction with C protein, pep14–23 or
pep5–26, in the buffer 20 mM Tris/HCl, pH 7.4, 100 mM KCl,
1 mM EDTA and 1 mM EGTA was analysed at 25 ◦ C (average
of 15 measurements, 100 runs each), by PALS (phase analysis
light scattering), using disposable zeta cells with platinum goldcoated electrodes. Values of viscosity and refractive index were
0.8872 cP and 1.330 respectively. The electrophoretic mobility
obtained was used for the zeta potential calculation through the
Smoluchowski equation [27]:
ζ =
4πηu
ε
(1)
where u is the electrophoretic mobility, η is the viscosity of
the solvent and ε is its dielectric constant. For the experiments
in which proteolysis of LDs was performed, LD samples were
previously incubated with 10 μM trypsin for 15 min at room
temperature (23◦ C), as described previously [18]. The reaction
was stopped by the addition of 1 mM PMSF to the mixture. After
5 min of incubation with PMSF, pep14–23 was added and the zeta
experiments were carried out.
AFM-based force spectroscopy measurements
A NanoWizard II atomic force microscope (JPK Instruments)
mounted on an Axiovert 200 inverted optical microscope (Carl
Zeiss), equipped with a 15 μm z-range linearized piezoelectric
scanner and an infrared laser, was used following standard
procedures [28], recently adapted to the probing of DENV C
protein–LD interaction [18]. Briefly, 50 μl of LD suspension were
allowed to deposit for 30 min at room temperature on a mica
surface. Non-adherent LDs were removed by five washes with
TEE buffer. Force spectroscopy measurements were performed
using OMCL TR-400-type silicon nitride tips (Olympus)
functionalized with C protein. Molecular recognition was
searched by pressing the DENV C protein-functionalized tips on
different points of the LDs in the presence of different pep14–23
concentrations. Force spectroscopy curves were analysed using
407
the JPK image processing program v.3. Each experiment was
performed at least three times, using different samples and
different functionalized tips, with approximately 5000 force–
distance curves collected, analysed and fitted to the WLC (wormlike chain) model [18]. Histograms of the (un)binding forces
of each dataset were constructed choosing the ideal bin size to
achieve the best fit (6 pN). Force rupture values below 10 pN were
considered to represent noise, artefacts or unspecific interactions.
From each histogram, the most likely single DENV C protein–
LD binding rupture force was determined fitting the distributions
of the rupture forces with the Gaussian model. The maximum
values of the Gaussian peaks represent a single-molecule-based
statistical measure of the strength of the molecular bond.
RESULTS
The N-terminal region of the DENV C protein interacts with LDs
The interaction between the DENV C protein and LDs was
studied by NMR, taking advantage of the previously determined
structure of the protein in solution [17]. The comparison of the
(15 N,1 H)-HSQC spectra of DENV C protein in the absence and
in the presence of LDs revealed specific local interactions that
could be mapped by CSPs (chemical shift perturbations) and
changes in peak intensity for specific residues, with maintenance
of the overall protein structure (Supplementary Figure S1 at
http://www.BiochemJ.org/bj/444/bj4440405add.htm, and spectra
sections shown in Figures 1a–1d).
Chemical shifts are highly sensitive to small changes in the
chemical environment, which may occur as a result of the direct
participation of the atom in the interaction, or by an indirect effect
of the binding (e.g. conformational changes induced by the
molecular recognition) [29]. Analysis of the (15 N,1 H)-HSQC
spectra revealed that the larger CSPs occurred for residues located
in the N-terminal region (Arg5 , Lys6 , Arg9 , Arg18 , Arg22 , Ser24 and
Thr25 ), in the L1–2 loop (Gly40 ) and in the α2–α2 dimer interface
(Ala49 , Val51 , Phe53 and Leu54 ) (Figure 1e). In contrast, residues
located elsewhere were almost not affected by the presence of
LDs, as exemplified for residues within helices α1 (Gln28 ), α3
(Arg68 ), α4 (Asn79 and Val80 ) or in the C-terminus (Arg98 ).
Peak intensity (I) change upon binding is also a good reporter
for measuring protein recognition [30]. The intensities ratios
for LD-bound to free C protein residues (I LD /I 0 ) are shown in
Figure 1(f). For most of the residues, I LD /I 0 was slightly less than
1, as expected for non-interacting regions of a protein complex.
Remarkably, a significant increase in I LD /I 0 was observed for
several residues in the N-terminal region (Arg5 , Lys7 , Ala8 , Arg9 ,
Asn14 , Leu16 , Arg18 and Arg22 ), for Leu38 , located in the L1–2
loop, and for Ala52 and Phe53 , located in the α2–α2 central
hydrophobic patch (Figure 1f). In addition, Ile59 , located in
the second loop (L2–3), and Glu87 , located in α4, were also
affected by the interaction. The increase in intensity may be
explained by the conformational selection as the mechanism for
molecular recognition. This mechanism of interaction presumes
that the protein in the free state contains regions in conformational
exchange. Residues in conformational exchange show low peak
intensities due to the line-broadening caused by the exchange
contribution to transverse relaxation rate. Binding stabilizes one
conformation, thus leading to an increase in peak intensity. The
results showed that a significant increase in I LD /I 0 was observed
for regions that are prone to be in conformational exchange, such
as the N-terminal region and the α2–α2 dimer interface.
Interestingly, the three major C protein regions found to be
affected by LD interactions are in the same side of the protein,
probably facing the LD upon binding (Figure 1g).
c The Authors Journal compilation c 2012 Biochemical Society
408
Figure 1
I. C. Martins and others
DENV C protein residues involved in the interaction with LDs are in the disordered N-terminal region and in α-helix 2
(a–d) Sections of the HSQC spectra of 15 N-labelled DENV C protein in the presence (red) and absence (black) of LDs, highlighting Arg5 , Arg18 and Arg22 , which belong to the disordered N-terminal
region; Gly40 , located in the L1–2 loop; Phe53 and Leu54 , located in α2; and Gly28 of α1, Arg68 of α3, and Asn79 , Val80 and Arg98 of α4. (e) CSP and (f) peak intensity ratios (I LD /I 0 ) of bound/free
protein as a function of protein residue. Bars are coloured to indicate the residues showing CSP values higher than 0.05 in (e), and I LD /I 0 values higher than 3 in (f): green, residues within the
N-terminal region for which no structure is available; dark blue, residues within the disordered N-terminal region for which a structure is available; cyan, the residue in the L1–2 loop; and orange,
residues in α2. (g) Ribbon structure of DENV C protein (PDB code 1R6R) in two alternative views, with the residues that showed significant CSP highlighted with the same colour code used in
(e) and (f). Note that the segment containing residues 1–20 is missing, since its structure could not be determined by NMR [17]. Residues Met1 , Asn2 , Thr11 , Leu35 and Arg55 were not assigned
in the previously determined NMR structure [17]. The assignments of Asp3 , Gln4 , Asn10 , Gly36 , Gln39 , Thr62 , Ala77 and Arg100 could not be localized or confirmed. Pro12 , Pro43 , Pro60 and Pro61 are
not represented in the graphs.
c The Authors Journal compilation c 2012 Biochemical Society
Dengue virus capsid protein binding to lipid droplets
Figure 2
Analysis of the backbone dynamics of DENV C protein
(a) Peak intensity derived from the (15 N,1 H)-HSQC spectrum of DENV C protein in the free
state. (b) Order parameter of the N–H bond (S 2 ) predicted from the random coil index obtained
from chemical shift values deposited in the Biomagnetic Resonance Bank (BMRB) [32]. (c)
Chemical shift index (CSI) predicted by the program TALOS + [33] as a function of residue
number. A value of − 1 indicates the presence of an α-helix. Filled rectangles indicate the
four α-helices displayed in the PDB structure 1R6R. Note that there is a small helix segment
predicted by the chemical shift index − 1 in the L1–2 loop. Residues Met1 , Asn2 , Thr11 , Leu35
and Arg55 were not assigned in the previously determined NMR structure [17]. The assignments
of Asp3 , Gln4 , Asn10 , Gly36 , Gln39 , Thr62 , Ala77 and Arg100 could not be localized or confirmed.
Pro12 , Pro43 , Pro60 and Pro61 are not represented in (a).
DENV C protein displays intrinsically disordered behaviour for its
N-terminal region
The central hydrophobic patch at α-helix 2 has previously been
assigned to participate in C protein interaction with membranes
[17,31] and LDs [12]. In the present study we show that loop
L1–2 and the previously unstudied N-terminal region also play
a role in C protein binding to LDs. Thus, to get information
regarding the conformational flexibility of these regions, two
parameters for each residue in the protein sequence were analysed
and compared: the peak intensities in the (15 N,1 H)-HSQC of the
free protein (Figure 2a) and the prediction of the order parameter
(S2 ) of the N–H bond based on the random coil index obtained
from the deposited chemical shift values [32] (Figure 2b). Most of
the residues in the N-terminal region presented lower intensities
and lower S2 values when compared with the residues in the
globular region. Additionally, within the N-terminal region, values
of peak intensities inversely correlate with S2 , as clearly observed
for residues Ala8 , Met15 and Glu19 , which display the highest
intensities and the lowest S2 values within this region. This
behaviour is expected, since regions of low S2 , and probably
high thermal flexibility, generally show sharper resonance lines.
A remarkable observation was the presence of high order (S2
∼0.8) for Pro12 and Phe13 within this region, which also display
low peak intensities. This segment is a hydrophobic triad (Thr11 Pro12 -Phe13 ) in which Thr11 was not assigned in the previous
determined structure [17]. For Asn10 , only 13 C and the amidic
1
H and 15 N could be found, and Pro12 was found only in the
trans-conformation. The lack of those assignments could be a
consequence of conformational exchange in the milli- to microsecond timescale, leading to line-broadening and, consequently,
to the decrease in peak intensity. Conformational exchange results
from an equilibrium among two or more discrete conformational
states, indicating some degree of conformational order in the
flexible N-terminal region, possibly due to the hydrophobic
character of the triad and the presence of a proline. This is a typical
behaviour of intrinsically disordered proteins, which display
flexibility, but yet some degree of order [1,2]. Taken together, these
409
observations agree with the proposed conformational selection
mechanism for C protein binding to LDs suggested by the
increased peak intensity ratio (Figure 1f).
Both S2 values and peak intensities agreed on the presence of
higher ordering in the C protein globular region, which extends
from Val23 to the C-terminus. All secondary structure elements
(residues contained in α1–α4) presented an S2 value greater than
0.8. On the other hand, a decrease in the order parameter on the
L1–2 loop could be observed, indicating thermal motion within
this long loop that extends from Arg32 to Pro43 . The most-reduced
order parameters were observed for the segments extending
from Arg32 to Leu35 (RFSL) and from Gln39 to Pro43 (QGRGP)
(Figure 2b). The reduction in S2 was followed by a significant
increase in peak intensity for residues Ser34 and Arg41 (Figure 2a).
TALOS+ prediction of secondary structure [33] suggests the
presence of one turn of a helix within these segments, comprising
residues Gly36 , Met37 and Leu38 (Figure 2c). Analysis of the DENV
C protein structure in solution (PDB code 1R6R) corroborates the
presence of the one-turn helix involving these residues. Indeed, for
this segment, an S2 value greater than 0.8 was found (Figure 2b).
Additionally, reduced order parameters, but not as pronounced as
for the L1–2 loop, were observed for the L2–3 and L3–4 loops
(Figure 2b). It is important to mention that the assignments of
Leu35 and Arg55 were missing in the previously determined C
protein structure [17], possibly due to conformational exchange,
which is interesting since Arg55 is within the central hydrophobic
patch that is also involved in the binding to LDs.
C protein residues involved in the interaction with LDs are located
within conserved regions
Sequence comparison of C proteins from different flaviviruses
revealed that most of the residues implicated in the DENV
C protein–LD interaction are conserved in evolutionary terms
(Figure 3a). Most interestingly, Asn14 , Met15 , Leu16 , Arg18 , Asn21
and Arg22 , located in the disordered N-terminal region, are part of
an NML + R conserved motif (with + representing a positively
charged residue, mostly lysine). Moreover, Leu38 and Gly40 ,
contained in the L1–2 loop, are also highly conserved. Phe53
is highly conserved as well, being part of the α2–α2 central
hydrophobic patch (Val51 , Ala52 , Phe53 and Leu54 ), which, in
turn, is part of an hAFL/F conserved sequence, where h is a
hydrophobic residue.
Comparison of the two available structures of C proteins
from Flavivirus spp., DENV [17] and WNV-K [25], reinforces
our findings (Figure 3b). Structure superimposition clearly
showed two regions in the protein structure: a globular
conserved core (from residues 44 to the C-terminus), and
a plastic region with two alternative ways of facing the
conserved globular core. The plastic region is composed
of the disordered N-terminal region (residues 1–26) and α1. The
residues affected by the LD interaction are located within both
regions. The loop connecting these two regions (L1–2) works as
a flexible hinge, enabling the plasticity of C proteins. Since CSP
was found within this loop, it is plausible to believe that it enables
the accommodation of DENV C protein in the LD interface.
A peptide corresponding to the conserved N-terminal motif of C
protein binds to LDs
To further evaluate the structural features involved in the
interaction between the disordered N-terminal region and LDs,
two peptides were designed, corresponding to residues 5–26
(pep5–26), and to residues 14–23 (pep14–23). In both peptides,
Glu19 was replaced with an alanine residue, in order to make their
c The Authors Journal compilation c 2012 Biochemical Society
410
Figure 3
I. C. Martins and others
C protein residues involved in the LD interaction are conserved among Flavivirus spp.
(a) Alignment of the DENV C protein sequence with C protein sequences from other flaviviruses, highlighting fully conserved segments (red) and partially conserved residues (black). The conserved
segments and the DENV C protein sequence are depicted, with the residues shown to interact with LDs underlined and coloured according to the colour code used in Figure 1(b). Both the N-terminal
NML + R motif and the α2 hAFL/F motif are conserved and contain the residues indicated by the NMR studies to interact with LDs. (b) Comparison of C protein structures of DENV (grey) and
WNV-K (red). Two regions can be depicted: a conserved fold (from residues 44 to the C-terminus), and a particularly flexible region with alternative folds, containing α1 and the disordered section
(residues 1–26). DENV C protein residues that showed a significant CSP are highlighted in blue, cyan and orange (following the colour code used in Figure 1b).
sequences more similar to that of C proteins of all other DENV
strains, as well as of all other flaviviruses, in which a neutral amino
acid is present at that position. pep5–26 includes all N-terminal
residues affected by the interaction with LDs, starting at residue 5
and ending just before the beginning of α1, at residue 26. pep14–
23 is a shorter version of pep5–26 that excludes the N-terminal
residues and the hydrophobic segment that showed a high order
parameter. pep14–23 includes the conserved motif NML + R,
beginning in the first residue of the conserved motif (Asn14 ) and
extending to the last residue affected by LD interaction, just before
the beginning of α1.
c The Authors Journal compilation c 2012 Biochemical Society
To quantify peptide binding to LDs, zeta potential
measurements were performed. This parameter is dependent
on the charge of a given scattering particle in solution [27].
Upon the addition of DENV C protein to an LD suspension,
there was a concentration-dependent increase in the zeta potential
values, from the initial negative value characteristic of LDs
(approximately − 20 mV) to a limit positive value, as expected
for the binding of the positively charged C protein (Figure 4).
pep14–23 (net charge + 4) was also able to bind to LDs, leading
to an increase in the zeta potential with a magnitude similar to
that observed when LDs interact with DENV C protein. In
Dengue virus capsid protein binding to lipid droplets
411
Figure 4 Zeta potential analysis of DENV C protein and pep14–23
interaction with LDs
Zeta potential measurements of the titration of LD samples (filled symbols) or LD samples after
limited proteolysis (empty symbol) were performed for DENV C protein (squares), pep14–23
(circles), or pep5–26 (triangles). Results are means +
− S.E.M. for three independent samples
for each experimental point, with 15 zeta potential measurements per sample.
contrast, pep5–26 (net charge + 8) did not change the measured
zeta potential values, indicating an absence of significant binding
to LDs. In a previous study, we found that C protein binds to
a protein component in the surface of LDs [18]. To evaluate
whether binding of pep14–23 to LDs was also dependent on a
surface protein, LD limited proteolysis was performed before zeta
potential measurements. As previously observed for C protein, LD
trypsinization eliminated the ability of pep14–23 to interact with
LDs (Figure 4).
NML + is the LD-binding motif of pep14–23
To map the interaction of pep14–23 with LDs, free pep14–23
was firstly assigned using a sequential assignment strategy [34],
with a combination of TOCSY and NOESY spectra. A (1 H,13 C)HSQC spectrum was also used to solve ambiguities and to
assign the 13 C resonances. It was possible to unambiguously
and fully assign Met2 , Leu3 , Lys4 , Ala6 and Val10 . The amide
resonances of Met2 and the N-terminal Asn1 were not found in
the spectrum, probably due to line broadening. Both Asn1 and
Asn8 were assigned, although they could not be unambiguously
differentiated. All arginine residues were superimposed in the
spectrum, showing degenerated chemical shift for the amide and
side-chain resonances. The only exception was for the (1 H,13 C)chemical shift, for which two sets of peaks could be differentiated.
The 80 % assignment of the (1 H,13 C)-HSQC spectrum enabled
the measurement of the CSP for each residue after addition
of LDs to the peptide sample. Met2 and Leu3 Hα/Cα crosspeak showed the largest CSP, along with one of the asparagine
residue, which has been attributed to Asn1 due to the proximity
in the sequence to Met2 and Leu3 (Figure 5). It should be
noted that Hα/Cα cross-peaks for the arginine residue did not
change with the addition of LDs. Smaller changes in CSP for
Lys4 , Ala6 and Val10 were also observed. It is important to
remark that all chemical shift changes were double-checked in
the HSQC and TOCSY spectra. Figure 5(b) shows a significant
perturbation in the Hβ/Cβ cross-peak for the same asparagine
residue, attributed as Asn1 . CSP was not observed for any other
side-chain resonance. The Hα chemical shift changes promoted
by the presence of LDs were quantified, showing that the highest
changes occurred for the resonances of Asn1 , Met2 and Leu3
(Figure 5c). The chemical shift of the basic arginine residues did
not change significantly, showing that the positive charges of the
Figure 5
pep14–23 interacts with LDs via specific conserved residues
(a) and (b) Sections of the (13 C,1 H)-HSQC spectra of pep14–23, in the presence (black)
and absence (grey) of LDs, showing the Hα/Cα paired resonances and side-chain Hβ/Cβ
paired resonances. Asterisks (*) indicate that Asn1 and Asn8 were assigned but could not be
unambiguously differentiated. (c) CSP changes for each pep14–23 residue as a result of the
interaction with LDs.
N-terminal residues and Lys4 were dominant for the interaction.
To complement the findings, the interaction of pep5–26 with LDs
was evaluated employing the same methodology. As previously
observed using zeta potential, no interaction of this peptide
with LDs was detected by NMR (Supplementary Figure S2 at
http://www.BiochemJ.org/bj/444/bj4440405add.htm).
pep14–23 inhibits the binding of DENV C protein to LDs
Since pep14–23 was shown to specifically interact with LDs,
a relevant question is whether the peptide is able to compete
with C protein for its LD-binding site. In order to tackle
this issue, the effects of pep14–23 on the strength of the C
protein–LD interaction was evaluated by AFM-based force
spectroscopy, following procedures established previously
[18,28]. Tapping on LDs with DENV C protein-derivatized
AFM tips allowed measurement of the frequency of C protein–
LD-binding events (by the subsequent unbinding), as well as
measurement of the force necessary to break the bond between a
single C protein dimer and an LD. The (un)binding frequencies
and forces for C protein–LD interaction were determined in the
absence and in the presence of different concentrations of pep14–
23 (Figures 6a–6e). It was found that C protein binding to LD
was inhibited by the addition of the peptide, in a concentrationdependent manner (Figure 6f). The force necessary to break the
c The Authors Journal compilation c 2012 Biochemical Society
412
Figure 6
I. C. Martins and others
DENV C protein binding to LDs is inhibited by pep14–23
Force-rupture histograms for the C protein-functionalized AFM tips interacting with LDs in the absence of pep14–23 (a), or in the presence of 4 μM (b), 20 μM (c) 40 μM (d) or 100 μM (e)
pep14–23. Each histogram was constructed with the results of approximately 5000 single rupture-force measurements, performed with an applied force of 0.2 nN, pulling speed of 2 μm/s and
loading rate of 4 nN/s. (f) Percentage of (un)binding events (circles) and force adhesion values of the first fitted peak (squares) of DENV C protein-functionalized AFM tips binding to LDs at each
peptide concentration tested. Results are means +
− S.E.M.
binding decreases from 33 pN, in the absence of the peptide, to
19 pN in the presence of 100 μM pep14–23. Most striking was the
abrupt decrease in the number of (un)binding events, from 54 %
in the absence of peptide to only 23 % at the highest peptide
concentration tested.
DISCUSSION
DENV C protein is known to interact with LDs and this
association was shown to be mandatory for virus particle
formation [12]. Taking into account the biological relevance
of that interaction, we investigated the determinant structural
features of C protein for the binding to LDs. Local
structural changes involving specific amino acid residues could
be mapped in the disordered N-terminal region, in the central
hydrophobic patch and in the L1–2 loop.
The finding that four consecutive residues in helix α2 (residues
51–54) are affected by LD interaction agrees with the suggestion
that the interaction with membranes would occur through the
central hydrophobic patch formed by the α2–α2 dimer helical
interface [17,31]. Additionally, mutational analysis showing that
Leu50 and Leu54 are essential for targeting C protein to LDs [12]
also supports the NMR data of the present study. Finally, it is clear
that the residues within the hydrophobic α2–α2 core involved in
LD interaction are conserved among flaviviruses, both in terms
of sequence (are part of an hAFL/F conserved sequence) and
structural alignment, reinforcing their importance for C protein
functions.
c The Authors Journal compilation c 2012 Biochemical Society
Furthermore, our findings expanded the previous understanding
of DENV C protein interaction with LDs by pointing to a
role of specific residues in the disordered N-terminal region
and in the L1–2 loop that have been overlooked so far. Our
hypothesis is that loop residues do not participate directly in
the interaction, but provide structural flexibility, as observed for
other viral proteins [35,36]. Most intriguing is the involvement
of the N-terminal residues located in the intrinsically disordered
region, which had not been studied before. The intrinsically
disordered nature of this region points to its involvement
in molecular recognition events, such as the novel function
of LD binding described in the present paper. Intrinsically
disordered proteins or regions undergo equilibrium among several
conformational states [1,2]. Thus, the pre-existent conformational
states allow recognition of different targets. Binding to each of
the targets, in turn, leads to conformational selection [37–39].
Indeed, conformational selection as a recognition mechanism
in C protein–LD interaction is supported by the increase in
peak intensity ratios. Conformational selection may offer an
evolutionary advantage for multifunctional proteins, such as the
C proteins of flaviviruses. In fact, despite their low sequence
similarity, these proteins share similar activities attributed to their
intrinsically disordered regions, such as RNA binding [40,41] or
RNA chaperoning [8]. In the case of DENV C protein, a nuclear
localization signal has already been identified in the disordered
N-terminal region, but mutations in this region had partial effects
on nuclear migration [42]. Interestingly, antibodies produced
against DENV C protein presented specific reactivity to the
Dengue virus capsid protein binding to lipid droplets
disordered N-terminal region, suggesting that it corresponds
to the predominant immunogenic segment [43]. The high
immunoreactivity of C protein N-terminal region may be related
to the intrinsically disordered nature of this segment, enabling its
interaction with multiple targets, including LDs, as shown in the
present study.
As the interaction with LD did not significantly change the
globular core of C protein structure, we hypothesized that LD
binding may alter the relative orientation of the N-terminal region
and α1 with respect to the globular core. This hypothesis was
supported by the dynamics of the C protein in the absence of
LDs, which clearly identified flexible segments corresponding to
the N-terminal region and the L1–2 loop, contrasting with a rigid
core, extending from α2 to the C-terminus. The comparison of the
C protein structures of DENV [17] and WNV-K [25] also revealed
that the segment from the N-terminus to residue 43 behaves as
a plastic region, with alternate folds, corroborating the analysis
of DENV C protein dynamics and reinforcing our hypothesis.
Furthermore, the fact that the same N-terminal residues that
interact with LDs are contained in a conserved flavivirus C protein
segment (NML + R, residues 14–18) highlights the importance
of these residues for the C protein functions. Interestingly,
NS5A from HCV (hepatitis C virus, also a member of the
Flaviviridae), a protein that is found associated with surface of
LDs in infected cells [44], contains a relatively similar sequence
(Y299 MLPKRR304 ), further supporting the role of the NML + R
sequence as a LD-binding motif.
Taking into account the findings described above, we
hypothesized that the disordered region, rich in positively charged
residues, containing the NML + motif, might prompt an initial
interaction with the negatively charged LDs, after which a
conformational rearrangement facilitated by the conformational
freedom provided by the flexible L1–2 loop enables the access
of LDs to the later specific interactions with the central
α2–α2 hydrophobic dimer interface. Indeed, the increase in
peak intensity observed for α2-helix residues Ala52 and Phe53
corroborates the stabilization of binding through this region. The
results of the present study suggest that the α2–α2 interface is in
conformation exchange in the free state, possibly interconverting
between an open and a closed state. Indeed, Arg55 is missing
in the previous assignment of DENV C protein structure [17],
and residues in helices α1 and α2 showed consistently lower
peak intensities than those of α3 and α4, possibly indicating
line-broadening due to conformational exchange. This would
facilitate the DENV C protein interaction with LDs and may
have special importance in the case of WNV-K, in which α1
partially blocks the access to the α2–α2 hydrophobic core [25].
In this model, the disordered N-terminal residues would be the
key to initiate the interaction with LDs. These data may explain
some intriguing results reported for WNV and TBEV C proteins,
in which deletions in large parts of its inner core lead to stillviable viruses [45,46]. In those studies, viruses containing large
deletions in the genome that resulted in the complete removal of
C protein helices α2 and α3 generated viable viral progeny. In
those shortened C protein versions, the disordered N-terminus,
helix α1, part of the L1–2 loop and part of helix α4 remained
intact and seemed to be sufficient for viral replication to proceed.
Following this line of reasoning, it can be speculated that
peptides mimicking the interacting region of the C protein,
especially if the NML + R motif is included, might be targeted
to the LD–C protein binding site. Thus we rationally designed
two peptides based on this region, and tested their ability to
interact with LDs by zeta potential measurements. The advantage
of employing zeta potential analysis is that it allows monitoring
the changes in the surface charge density of particles, such as LDs,
413
as a result of an interaction with small ligands, without any further
modification of the ligand [27]. The results showed that pep14–23
is able to interact with LDs to a similar extent as the full protein.
The extensive zeta potential variation (approximately 34 mV),
changing from negative to positive values, fits well with a model
of interaction in which the positively charged α4 is exposed,
conferring a positive charge to the outer surface of the C protein–
LD complex, and supporting the previously described conclusions
based on NMR data [17]. Additionally, the interaction between
pep14–23 and LDs was dependent on a protein component in
the surface of LD, exactly as previously found for C protein–
LD interaction [18]. The detailed analysis of the Hα chemical
shift changes in the (1 H,13 C)-HSQC spectra of pep14–23 in the
presence and in the absence of LDs revealed that the higher
changes occurred exactly for the conserved NML + motif, fitting
adequately with the NMR analysis of the full-length protein. As
pep14–23 is rather small and highly basic, with the five positive
charges distributed along positions 1, 4, 5, 7 and 9, one can
hypothesize that electrostatic interactions contribute to a first
stage of the peptide binding to the negatively charged surface
of the LDs. Then, the NML + R residues may become more
tightly bound, due to the contribution of hydrophobic interactions,
which may also occur in the full-length protein and, in this way,
anchor the C protein to the LDs, allowing further interaction of the
hydrophobic α2–α2 core. On the other hand, both zeta potential
and NMR data showed that pep5–26 does not interact with LDs.
This result is similar to those found for other peptide–ligand
interactions, in which shorter sequences were more effective in
eliciting a response than longer versions [47]. The longer size
of this peptide may render the NML + R sequence inaccessible
to LD binding. The inaccessibility of the NML + R sequence
in pep5–26 may result from presence of the hydrophobic triad
(Thr11 -Pro12 -Phe13 ) that displays high order parameter, which may
impose some conformational constraint. Remarkably, pep14–23
comprises only the low order parameter segment. This binding
behaviour is another evidence of the intrinsically disordered
nature of the C protein N-terminal region.
Taken together, the results of the present study point to
the ability of using pep14–23 to inhibit C protein binding
to LDs. Indeed, the inhibitor potential of the peptide was
demonstrated by single-molecule AFM-based force spectroscopy
data. The peptide blocked C protein binding to LDs in a
concentration-dependent manner, with a marked decrease in
the probability of the occurrence of the binding to one fifth
of the initial value at the highest peptide concentration tested.
It is important to bear in mind that the histograms (obtained
by thousands of measurements for each peptide concentration)
clearly show that even the small fraction of binding events
occurring at high pep14–23 concentrations corresponds mostly
to low (un)binding forces, typical of unspecific interactions. To
conclude, we propose that sequences encompassing or within
pep14–23 actively contribute to the C protein–LD interaction.
Moreover, therapeutic approaches using peptides and peptide
analogues were effective against other viruses, such as HIV
[48,49] and HCV [50], a strategy that may be followed also for
DENV and similar flaviviruses.
In summary, the results of the present study revealed that the
disordered N-terminal region of DENV C protein is crucial for
its interaction with LDs, and that a peptide designed on the basis
of a conserved segment within this region was able to inhibit
this interaction, which is a key event of the replication of DENV
(and possibly of other flaviviruses). Therefore the novel advances
reported in the present paper pave the way to drug development
approaches, in which peptide inhibitory efficiency may be further
improved through lead optimization strategies.
c The Authors Journal compilation c 2012 Biochemical Society
414
I. C. Martins and others
AUTHOR CONTRIBUTION
Ivo Martins, Fabio Almeida, Nuno Santos and Andrea Da Poian conceived and designed
the experiments. Ivo Martins, Andre Faustino, Filomena Carvalho and Fabiana Carneiro
performed the experiments. Ivo Martins, Francisco Gomes-Neto, André Faustino, Filomena
Carvalho, Fabio Almeida, Nuno Santos and Andrea Da Poian analysed the data. Ronaldo
Mohana-Borges, Patrı́cia Bozza, Fabio Almeida, Nuno Santos and Andrea Da Poian
contributed reagents/materials/analysis tools. Ivo Martins, Miguel Castanho, Fabio
Almeida, Nuno Santos and Andrea Da Poian wrote the paper.
ACKNOWLEDGEMENTS
We thank Teresa Freitas (IMM, FMUL, Lisbon, Portugal) for technical assistance and
Marco Domingues (IMM) for help with the zeta potential measurements.
FUNDING
This work was supported by the European Union 7th Framework Programme PEOPLE
IRSES (International Research Staff Exchange Scheme) project MEMPEPACROSS, the
National Institute of Science and Technology in Dengue (INCTD, Brazil), the Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq, Brazil), the Fundação
Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ,
Brazil), the Fundação para a Ciência e a Tecnologia (FCT)–Ministério da Educação e
Ciência (MEC) (Portugal) [grant number PTDC/QUI-BIQ/112929/2009], the Fundação
Calouste Gulbenkian (Portugal), and FCT–Fundação Coordenação de Aperfeiçoamento
do Pessoal de Nı́vel Superior (CAPES) Portugal–Brazil joint co-operation projects. I.C.M.
is the recipient of consecutive postdoctoral funding from a Marie Curie International
Outgoing Fellowship [grant number MC-IOF237373] and FCT postdoctoral fellowships
[grant numbers SFRH/BPD/46324/2008 and SFRH/BPD/74287/2010].
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Received 22 December 2011/6 March 2012; accepted 19 March 2012
Published as BJ Immediate Publication 19 March 2012, doi:10.1042/BJ20112219
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 444, 405–415 (Printed in Great Britain)
doi:10.1042/BJ20112219
SUPPLEMENTARY ONLINE DATA
The disordered N-terminal region of dengue virus capsid protein contains
a lipid-droplet-binding motif
Ivo C. MARTINS*†, Francisco GOMES-NETO*‡, André F. FAUSTINO†, Filomena A. CARVALHO†, Fabiana A. CARNEIRO§,
Patricia T. BOZZA, Ronaldo MOHANA-BORGES¶, Miguel A. R. B. CASTANHO†, Fábio C. L. ALMEIDA*‡, Nuno C. SANTOS† and
Andrea T. DA POIAN*1
*Instituto de Bioquı́mica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil, †Instituto de Medicina Molecular, Faculdade de Medicina da
Universidade de Lisboa, Lisbon, 1649-028, Portugal, ‡Centro Nacional de Ressonância Magnética Nuclear, Universidade Federal do Rio de Janeiro and National Institute of Structural
Biology and Bioimage, Rio de Janeiro, RJ, 21941-902, Brazil, §Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro, Duque de Caxias, RJ, 25245-390, Brazil, Instituto
Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, 21040-360, Brazil, and ¶Laboratório de Genomica Estrutural, Instituto de Biofı́sica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil
Figure S1 (13 C,1 H)-HSQC spectra of DENV C protein in the presence (red)
and absence (black) of LDs, showing that the overall protein structure is
maintained
Figure S2 (13 C,1 H)-HSQC spectra of pep5–26 in the presence (red) and
absence (black) of LDs
(a) Cα region. (b) Cβ region. (c) Methyl region of the spectra. Hα/Cα paired resonances in
three different regions of the (13 C,1 H)-HSQC spectra do not show significant chemical shift
changes, reinforcing the previous conclusion of the absence of pep5–26–LD binding.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
I. C. Martins and others
Table S1
Virus strains and sequences used to construct the alignment with different Flavivirus spp. capsid proteins in Figure 3(a) in the main paper
N.A., not applicable (query sequence).
Virus name
Abbreviation
Strain
NCBI accession number
Total score
Query coverage
Taxonomy (Group, species )
Dengue virus serotype 2
West Nile virus
WNV serotype Kunjin
Japanese encephalitis virus
St. Louis encephalitis virus
Murray Valley encephalitis virus
Kokobera virus
Kedougou virus
Zika virus
Spondweni virus
Aroa virus
Iguape virus
Usutu virus
Alfuy virus
Bagaza virus
Ilheus virus
Rocio virus
DENV-2
WNV
WNV-K
JEV
SLEV
MVEV
KOKV
KEDV
ZIKV
SPOV
AROAV
IGUV
USUV
ALFV
BAGV
ILHV
ROCV
PR-159S1
K6453
MRM62C
YL
GML 903797
MVE-1-51
AusMRM 32
DakAR D1470
MR766
SM-6 V1
BEAN 4073
SPAn 71686
SAAR-1776
MRM3929
DakAR B209
Original
SPM 34675
1R6R_A
ADM88862.1
AAP78941.1
AAL93186.1
ABN11822.1
NP_051124.1
YP_001040007
ABI54477.1
ABI54475.1
ABI54480.1
YP_001040004
AAV34154.1
AAS59401.1
AAX82481.1
YP_002790883
YP_001040006
AAV34158.1
N.A.
76.6
75.5
75.5
79.3
73.2
85.9
85.5
84.0
87.8
83.6
80.1
79.7
77.0
76.6
65.9
64.7
N.A.
91
91
93
93
83
91
96
98
99
95
87
91
93
85
94
86
Dengue virus group, Dengue virus
Japanese enc. virus group, West Nile virus
Japanese enc. virus group, West Nile virus
Japanese enc. virus group, Japanese enc. virus
Japanese enc. virus group, St. Louis enc. virus
Japanese enc. virus group, Murray Valley enc. virus
Kokobera virus group, Kokobera virus
Dengue virus group, Kedougou virus
Spondweni virus group, Zika virus
Spondweni virus group, Zika virus
Aroa virus group, Aroa virus
Aroa virus group, Aroa virus
Japanese enc. virus group, Usutu virus
Japanese enc. virus group, Murray Valley enc. virus
Ntaya virus group, Bagaza virus
Ntaya virus group, Ilheus virus
Ntaya virus group, Ilheus virus
Received 22 December 2011/6 March 2012; accepted 19 March 2012
Published as BJ Immediate Publication 19 March 2012, doi:10.1042/BJ20112219
c The Authors Journal compilation c 2012 Biochemical Society