Mechanism of Inhibition of Enveloped Virus Membrane
Fusion by the Antiviral Drug Arbidol
Elodie Teissier1, Giorgia Zandomeneghi2, Antoine Loquet1¤, Dimitri Lavillette3,4,5, Jean-Pierre
Lavergne1, Roland Montserret1, Franc ¸ois-Loı ¨c Cosset3,4,5, Anja Bo ¨ckmann1, Beat H. Meier2, Franc ¸ois
Penin1*, Eve-Isabelle Pe ´cheur1*
1Institut de Biologie et Chimie des Prote ´ines, UMR 5086, CNRS, Universite ´ de Lyon, IFR128 BioSciences Gerland-Lyon Sud, Lyon, France, 2Physical Chemistry, ETH-Zurich,
Zurich, Switzerland, 3Universite ´ de Lyon, UCB-Lyon1, IFR128, Lyon, France, 4INSERM, U758, Lyon, France, 5Ecole Normale Supe ´rieure de Lyon, Lyon, France
The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell
membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and
membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that
Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions
between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts
with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues
are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of
membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase
the strength of virus glycoprotein’s interactions with the membrane, due to a dual binding mode involving aromatic
residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational
changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like
interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.
Citation: Teissier E, Zandomeneghi G, Loquet A, Lavillette D, Lavergne J-P, et al. (2011) Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the
Antiviral Drug Arbidol. PLoS ONE 6(1): e15874. doi:10.1371/journal.pone.0015874
Editor: Annalisa Pastore, National Institute for Medical Research, Medical Research Council, London, United Kingdom
Received September 2, 2010; Accepted November 28, 2010; Published January 25, 2011
Copyright: ? 2011 Teissier 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: Funding was provided by ANRS to E-IP and FP (www.anrs.fr) and European Research Council (ERC-2008-AdG-233130-HEPCENT) to F-LC. 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: email@example.com (FP); firstname.lastname@example.org (E-IP)
¤ Current address: Department for NMR-Based Structural Biology, Max Planck Institute for Biophysical Chemistry, Go ¨ttingen, Germany
Distinct from specific antiviral compounds that target key viral
functions are a group of broad-spectrum medicinal drugs that were
originally designed for other treatments [1–3] or targeted toward a
number of viruses (; reviewed in ). The advantage of this
group of antivirals is that they have already met the pharmaco-
logical criteria for medicinal drugs and are already approved for
clinical use in some countries. Among these molecules, antiviral
agents targeting viral entry of enveloped viruses are of major
interest since they seize an early step in the viral life cycle, before
damages have occurred to cells (recently reviewed in [6,7]), and
since they can be incorporated into combinations of multiple drugs
with different targets. One of these compounds, arbidol [Arb; 1H-
indole-3-carboxylic acid, 6-bromo-4-[(dimethylamino)-methyl]-5-
hydroxy-1-methyl-2-[(phenylthio)methyl]-, ethyl ester, monohy-
drochloride; CAS Registry Number 131707-23-8 (Figure 1)], is
already licensed in Russia and China, and is described as an anti-
influenza drug with immunostimulant properties. Arb is in use for
several years as prophylaxis and treatment for influenza A and B
infections. It inhibits influenza virus-induced membrane fusion
and may have the capacity to induce serum interferon . Recent
studies extended its inhibitory activity to other human viruses such
as the respiratory syncytial virus, parainfluenza virus 3, rhinovirus
14, and hepatitis B virus (reviewed in [5,9]). We demonstrated that
it also inhibits hepatitis C virus (HCV) infection in vitro, and HCV
replication , HCV cell entry and membrane fusion using HCV
pseudoparticles (HCVpp) and HCV grown in cell culture (HCVcc)
[11,12]. Most recently, Ciliberto and coworkers demonstrated the
efficacy of Arb derivatives at inhibiting HCV entry and replication
into hepatoma cells in the low micromolar range . HCV
infection is a leading cause of liver diseases, including hepatocel-
lular carcinoma, and therapeutic options are still limited (for
recent reviews, see  and refs therein). There is thus an urgent
need to develop efficient and well tolerated drugs to combat this
Arb demonstrated a propensity to enter into hydrophobic
interactions with membranes, and with membrane-like environ-
ments such as detergent micelles . Here we further
characterize the mechanism of action of arbidol, and analyze at
the molecular and atomic level the interactions of Arb with
membranes, tryptophan-rich derivatives and peptides. We first
examined how Arb inhibits HCV entry and membrane fusion
using HCVpp of different genotypes, and found that Arb
inhibition was genotype-independent. By combining surface
plasmon resonance, fluorescence and NMR spectroscopy ap-
proaches, we showed that Arb directly interacts with the
phospholipid membrane interface, with an affinity in the
PLoS ONE | www.plosone.org1 January 2011 | Volume 6 | Issue 1 | e15874
micromolar range, comparable to the concentration inhibiting
HCVpp membrane fusion by 50% (IC50). Arb also displayed
micromolar affinity toward aromatic components of proteins such
as tryptophan and derivatives, and toward peptides containing
tryptophans and derived from HCV envelope glycoproteins.
Altogether our results demonstrate that Arb interacts with the
polar head of phospholipid membranes and protein motifs
enriched in aromatic residues, suggesting that the inhibitory
activity of Arb on HCV entry and fusion could involve both types
Materials and Methods
Phosphatidylcholine from egg yolk (PC, 99% pure), dimyr-
istoylphosphatidylcholine (DMPC, 99% pure), cholesterol (chol,
99% pure), lyso-phosphatidylcholine (lysoPC), dodecyl-phospho-
choline (DPC), Triton X-100, tryptophan octyl ester hydrochlo-
ride (TOE) and N-acetyl-L-tryptophanamide (NATA) were
purchased from Sigma. Octadecyl rhodamine B chloride (R18)
was from Molecular Probes. The peptides used were part of the
sequence of structural or non structural (NS) proteins of HCV and
of the bovine viral diarrheal virus (BVDV). The amphiphilic helix
of BVDV NS5A  and the transmembrane domain of HCV
NS4A  were obtained as described previously. The peptides
identified as important for HCV fusion  were purchased from
Clonstar Biotech (90% purity) or Sigma Genosys (70% purity),
respectively, and dissolved in DMSO before preparation of
lipid:peptide mixtures. Arbidol [Arb, 1H-indole-3-carboxylic acid,
nylthio)methyl]-, ethyl ester, monohydrochloride (Figure 1)] was a
kind gift from Stephen J. Polyak.
Arb was readily soluble in ethanol, and soluble in the mM range
in water. Ethanol stock solutions of Arb were diluted to a 1.88 mM
final concentration in milliQ water (the final stock solution
contained 10% ethanol). For SPR experiments, one mg of Arb was
resuspended in water, followed by centrifugation (160006g,
15 min, 4uC). Arb concentration in solution was measured at
280 nmin the supernatant
Liposomes and micelles preparation
Mixtures of lipids [DMPC; PC; PC:chol (70:30, M:M);
PC:chol:R18 (65:30:5, molar)], of lipid:peptide (20:1, M:M), of
lipid:TOE (20:1, M:M) or of detergent:TOE (800:1, M:M) were
prepared in chloroform:methanol mixtures. After solvent evapo-
ration, samples were resuspended in phosphate-buffered saline
(PBS, pH 7.4) or water, and underwent 5 freeze/thaw cycles
(liquid nitrogen and 37uC, respectively). Liposomes were prepared
by extrusion over a stack of Avestin polycarbonate filters (100 nm),
as described .
Cell infection assays
Huh-7 cells  were maintained in DMEM containing 4.5 g/
L d-glucose and 4 mM L-glutamine (Invitrogen, Cergy-Pontoise,
France), supplemented with 100 U/ml penicillin, 100 mg/ml
streptomycin and 10% FCS (Lonza). Productions of pseudotyped
viruses were obtained by the transient transfection of 293T cells by
the calcium-phosphate method. For the genotype study, HCVpp
of genotypes 1a (H77; AF011752), 1b (Con1; AJ238799), 2a
(JFH1; AB047639), 2b (UKN2B 2.8, AY734983), 3a (UKN3A
1.28, AY734984), 4a (UKN4 21.16, AY734987), 5a (UKN5.14.4,
AY785283) and 6a (UKN6.5.340, AY736194) were produced as
described previously  from 293T cells co-transfected with a
murine leukemia virus (MLV) Gag-Pol packaging construct, an
MLV-based transfer vector encoding GFP as a marker protein,
and the E1–E2 expression constructs.
Supernatants were collected 48h post-transfection and filtered
on 0.45 mm. For genotypes 5a and 6a, pseudoparticles were
concentrated 100-fold after ultracentrifugation through a 20%
sucrose cushion at 75,0006g for 2h at 4uC. Pellets were
resuspended in the regular medium of Huh-7 cells. For infection
experiments, Huh-7 cells were seeded at 4000 cells/well in 96-well
plates. The following day, cells were infected in the presence of
increasing Arb concentrations for 6 h. Arbidol effect on viral
infectivity was evaluated by assaying GFP activity 72 hours after
infection using flow cytometry (FACScalibur). Pseudoparticles
harbouring at their surface the influenza hemagglutinin (HApp)
and the envelope glycoprotein of the RD114 feline oncovirus
(Rd114pp) were prepared as described in  and ,
Membrane fusion assays
Lipid mixing between pseudoparticles and PC:chol:R18lipo-
somes was monitored by fluorescent spectroscopy, as the
dequenching of R18. In brief, R18-labeled liposomes (1 ml,
12.5 mM final lipid concentration) were added to a 37uC-
thermostated cuvette containing pseudoparticles in PBS pH 7.4
[viral titers: H77 (1a) 5.10e5; W529A-HC 10e2; Con1 (1b) 4.10e4;
JFH1 (2a) 5.10e4; AY734983 (2b) 8.10e4; AY734984 (3a) 3.10e4;
Figure 1. Chemical structures of arbidol (A), N-acetyl trypto-
phanamide (NATA) (B), and tryptophan octyl ester (TOE) (C).
Note that numbering in panel A refers to proton numbers, as identified
in NMR (cf Figure 5 and Table 1).
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AY734987 (4a) 9.10e4; AY785283 (5a) 3.10e4; AY736194 (6a)
5.10e3; HA 8.10e8; Rd114 2.10e6], and incubated 2 min. Fusion
was initiated by acidification to pH 5 with HCl, and recorded on
an SLM Aminco 8000 spectrofluorimeter over a 10-min time
period, at excitation and emission wavelengths of 560 nm and
590 nm, respectively. Maximal R18dequenching was measured
after the addition of 0.1% Triton X-100 (final concentration) to
the cuvette to lyse liposomes. The same procedure was used to
follow pseudoparticle fusion in the presence of Arb; in this case,
after a 1-min incubation of pseudoparticles with liposomes, Arb
(11.3 mM final concentration) was added and incubated for 1 min,
and fusion initiated by acidification.
Indole emission fluorescence spectra of tryptophan derivatives
were recorded at excitation wavelength of 286 nm (spectral zone
of lowest absorption of Arb), under various conditions: NATA
(5 mM final) in PBS at pH 7.4 or 5.0; TOE at 5 mM in lyso-PC
micelles (TOE:lysoPC molar ratio 1:800), and in PC and PC:chol
liposomes (TOE:lipid molar ratio 1:20); peptides at 5 mM in
PC:chol liposomes (peptide:lipid molar ratio 1:20). Spectra were
obtained in the absence or presence of increasing concentrations of
Arb (0 to 100 mM). Samples were incubated 2 min at 37uC prior
to recording. Emission spectra were collected in the 300–400 nm
region (with 2 nm-increments), with blanks substracted, using a
black flat-bottom, low-binding 96-well microplate (Greiner Bio-
one). Measurements were recorded on a Tecan InfiniteH M1000
spectrofluorimeter. KDapp values were calculated from the
difference between the areas under the spectra in the absence or
presence of Arb (DA), at various Arb concentrations, by nonlinear
fitting using the equation DA=DA max C/(KD+C). Fluorescence
measurements were repeated three times to obtain averaged values
Preparation of giant unilamellar liposomes
GUVs were made by the electroformation method . The
flow chamber (Warner Instruments, Connecticut, USA) used for
vesicle preparation was equipped with two glass coverslips, each
coated with optically transparent and electrically conductive
indium tin oxide (ITO) (Philips, Eindhoven, NL). Mixtures of
lipids [PC:chol:R18 (65:30:5, molar ratio)] were prepared at
0.1 mM in chloroform. The lipid mixture (2 nmoles) was spread
into a thin and uniform film on the conductive face of ITO-coated
slide. After chloroform evaporation, the dried lipid film was
hydrated by adding water into the chamber (,400 ml) and an
alternative electrical field (10 Hz and 1.2V) was applied at room
temperature for 3 hours. GUVs in the absence or presence of
increasing amounts of Arb solubilized in water (0 to 40 nmoles),
were observed by epifluorescence microscopy.
Surface plasmon resonance (SPR)
Interaction of Arb with DMPC layers was investigated with a
BIAcore 3000H using a L1 sensor chip at 30uC. The sensor chip
surface was washed with a mixture of 50 mM NaOH and
isopropanol (6:4, v:v), at a flow rate of 20 ml/min for 1 min. The
running buffer was milliQ water. The influence of liposome
concentrations on the final SPR signal was tested; we assayed lipid
concentrations from 0.5 to 5 mM and measured the resulting
resonance units (RU). We obtained a well detectable, reproducible
and stable signal from 2 mM, and further increasing this
concentration did not improve the signal. We therefore chose
the 2 mM concentration for our experiments. DMPC liposomes
were resuspended in milliQ water and captured on sensor chip at
2 ml/min for 5 min. The flow rate was increased to 30 ml/min and
the liposome surface was then washed with 10 mM NaOH for
1 min. Liposomes immobilized on the chip surface gave approx. a
5000 RU signal. To calculate Arb affinity for lipids, its association
to and dissociation from DMPC layers were studied at different
Arb concentrations in water, from 0.5 to 10 mM, at a flow rate of
20 ml/min. After each binding cycle, the sensor surface was
regenerated to the original matrix by injecting 50 mM NaOH/
isopropanol (6:4, v:v). The sensor surface was then coated with a
fresh liposome suspension for the next binding cycle. KDvalues
were calculated from the equilibrium resonance signal (Req) as a
function of the analyte concentration. Reqvalues were estimated
by extrapolation to infinite time using plots of resonance signal as a
function of the reciprocal of time. Apparent KD were then
calculated by nonlinear fitting to the expression Req=RmaxC/
(KD+C), where Rmaxis the maximum binding capacity of the
surface and C is the analyte concentration, using the SigmaPlot
For the NMR studies, the bicellar system was prepared by
mixing 54 mg of 1,2-Dihexanoyl-sn-glycero-3-phosphocholine
(DHPC) and 40 mg of DMPC with 400 ml of D2O. The sample
with a lipid molar ratio [DMPC]/[DHPC]=0.49 was subjected to
3 cycles of vortexing (2 min), heating to 313 K (20 min), vortexing
(2 min) and cooling to 273 K (20 min). The clear lipid solution
was then added to 2 mg Arb in powder, and then subjected again
to the procedure of vortexing, heating, vortexing and cooling. The
final molar ratio [Arb]/([DMPC]+[DHPC]) was 1/48, with
[Arb]=9.4 mM. Another sample with similar lipid concentration
and higher Arb content (molar ratio 1/15) was also prepared. The
amount of free Arb in Arb:DMPC mixture was estimated after
rapid separation of lipids on ultrafiltration membrane (cutoff
5000 Da) and measure of Arb concentration in the ultrafiltrate at
280 nm. For Arb:DMPC molar ratio of 1:4 at neutral pH, free
Arb was found to be lower that 0.2%. We thus concluded that the
amount of free Arb in NMR samples was negligible.
D2O from Cambridge Isotopes Lab (Cambridge, MA), and
Gd(DTPA-BMA) was a generous gift of Klaus Zangger. All
experiments were performed with freshly prepared samples.
1H NMR experiments were performed on a Bruker DMX 400
spectrometer operating at a proton frequency of 400 MHz.
Spectra were recorded with a 5 mm Triple Resonance Inverse
TXI probe equipped with z-gradient. The p/2 pulse was 10.3 ms,
the recycle delay was 3 s and solvent suppression with presatu-
ration was used. 1D
scans. These spectra were used for the assignment of the drug
signals together with 2D NOESY and
(data not shown). The assignment of the lipid resonances was
derived from the comparison with data in the literature . To
obtain Paramagnetic Relaxation Enhancements (PRE), a solution
of Gd(DTPA-BMA) in D2O (60 mM) was added to the Arb/
bicelle sample. Two sets of experiments were performed to
measure the proton T1 relaxation times on the Arb/bicelle
samples were performed. In the first one [Arb]/[Lipids]=1/15,
T=305 K and [Gd(DTPA-BMA)]=0, 2.0, 2.9, 4.6, 6.3, 7.9 and
9.7 mM. In the second one, [Arb]/[Lipids]=1/48, T=310 K
and [Gd(DTPA-BMA)]=0, 1, 2 mM. Proton T1 times were
measured by using inversion recovery experiments with an inter-
pulse delay ranging between 5 ms and 13 s. Each measurement
was repeated 3 times, adding 240 scans with a delay time between
scans of 15 s. All the spectra were processed using matNMR .
1H spectra were measured acquiring 120
1H/13C HSQC spectra
Arbidol Interacts with Membranes/Protein Motifs
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The1H frequency scale is given in terms of chemical shift relative
to the acetone signal used as an external reference (2.218 ppm).
Differential arbidol inhibition of cell entry and membrane
fusion of various HCVpp genotypes
We have previously shown that Arb could inhibit cell entry and
membrane fusion of HCVpp of genotypes 1a, 1b and 2a [10,12],
and HCVcc of genotype 2a . Here we sought to investigate the
effect of Arb on other major HCVpp genotypes as well. HCVpp
infectivity toward Huh-7 cells, objectifying HCVpp entry, was
assayed by counting cells positive for GFP (as the marker protein),
incubated with or without increasing Arb concentrations for 6h
(see Materials and Methods). A representative data set is shown in
Figure S1A, and inhibition obtained for the highest concentration
of Arb (11.3 mM) is presented in Figure 2A. The inhibitory effect
of Arb on HCVpp cell entry depends on HCVpp genotype.
Indeed, within biological intrinsic variability of HCVpp prepara-
tion and samples, three cases could be distinguished: entry of
HCVpp of genotypes 2a and 3a was inhibited by ca. 60%, while
1a, 1b and 2b exhibited a 40%-inhibition, but entry of genotypes
5a and 6a was weakly affected by the presence of Arb (Fig. 2A).
The influence of Arb on HCVpp-mediated lipid mixing was
assayed by fluorescence spectroscopy using fluorescent liposomes,
as previously described . Lipid mixing between HCVpp and
liposomes was only observed at low pH and optimal at pH 5.0
. In the presence of increasing Arb concentrations, lipid
mixing was inhibited in an Arb dose-dependent manner (Figure
S1B for HCVpp genotype 4a). In contrast to what was observed
for HCVpp infectivity, the effect of 11.3 mM Arb on HCVpp-
mediated membrane fusion (Figure 2B) was similar for all tested
genotypes, with about 50% inhibition of membrane fusion activity.
This indicates that membrane fusion inhibition by Arb is not
These data suggest that the differential inhibitory effect of Arb
on HCVpp infectivity of various genotypes is likely due to a
genotype-dependent modulation of HCV glycoproteins interaction
with the cellular proteins (e.g. HCV receptors) involved in HCV cell
entry. Conversely, Arb inhibition of HCVpp membrane fusion, as
assessed by a in vitro model system where the only proteins present
are the viral glycoproteins, could merely reflect the interaction of
Arb on lipids and/or on motifs present in HCV glycoproteins of
any genotype. To test these hypotheses, we further investigated
Arb interaction properties with lipids and protein fragments using
the approaches described in the following.
Arbidol interactions with lipid membranes
We previously showed that Arb could interact with liposomes
and membrane-like environments such as detergent micelles .
We further investigated this feature by studying the interactions of
Arb with giant unilamellar liposomes (GUV) by optical micros-
copy (Figure 3). GUV are pure lipid bilayers, intrinsically flexible
and unstable due to their very large size (in the range of tens of
mm) . Increasing Arb concentrations were added to the
chamber where GUV composed of PC:chol were electroformed
(see Methods section), with Arb-to-lipid molar ratios of 1:40, 1:20,
1:10, 1:1, 10:1 and 20:1. The GUV bilayer was unaffected by the
presence of Arb up to a 1:20 Arb-to-lipid ratio, with occasional
membrane flickerings (Fig. 3C and asterisk in Fig. 3E). At higher
ratios, membrane inhomogeneities and invaginations appeared
(Fig. 3F, asterisks in Fig. 3D), and a major overall membrane
reorganization was observed at a 20:1 Arb-to-lipid ratio (Fig. 3G).
Note that no lysis or membrane dislocation of GUV was observed
Figure 2. Arb inhibition of cell entry and membrane fusion of
HCVpp of various genotypes. A, HCV entry assays using HCVpp in
the absence or presence of 11.3 mM arbidol. Huh-7 cells were infected
by co-incubating HCVpp of indicated genotype with or without Arb for
6 h. Infectivity was evaluated after 72 h by counting the percentage of
GFP-positive cells, using a high-throughput flow cytometer (FACScali-
bur). The titer obtained in the absence of Arb was set to 100%, and the
resulting percentages of infection in the presence of Arb were
calculated. Results are the mean +/2 SEM of 5 separate experiments.
HApp are presented as control pseudoparticles sensitive to arbidol (cf
also ), and Rd114pp insensitive to arbidol (cf also ). *
mutant HCVpp W529A (cf ) are presented as a negative control of
entry, displaying very low infectivity. B, Membrane fusion between
HCVpp and R18-labeled liposomes was measured by recording the
kinetics of lipid mixing by fluorescence spectroscopy (excitation and
emission wavelengths were 560 and 590 nm, respectively), as described
in the Materials and Methods section. Values of the last 30 s of fusion
kinetics (final extent of fusion) were used to calculate the percentage of
fusion in the presence of Arb, relative to fusion kinetics without Arb
(100%). Results are the mean +/2 SEM of 4 separate experiments. HApp
and mutant HCVpp W529A were taken as controls. *2: no fusion was
observed for Rd114pp.
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for any condition, even at the highest ratio (data not shown). These
results reveal that only very high concentrations of Arb with
respect to lipids could significantly perturb the lipid organization
of these bilayers. This also indicates that the direct interaction of
Arb to lipid bilayers at the concentrations used to inhibit HCVpp
infectivity and membrane fusion (panel E) do not perturb lipid
In addition, HCVpp pre-incubated at neutral or acidic pH with
Arb, even at very high concentrations (100 mM), displayed similar
morphology (visualized by transmission electron microscopy) as
those observed in the absence of the drug (data not shown). Indeed
we counted over 160 HCVpp for each condition, and no
difference in HCVpp morphology could be observed between
the parameters assessed. This indicates that Arb inhibition of
HCVpp fusion is not due to viral particle disruption/damage.
Surface plasmon resonance
To gain insight into the molecular details of the interaction of
Arb with lipid membranes, we next investigated the lipid binding
properties of Arb by using surface plasmon resonance (SPR,
BiacoreH technology). We used a Biacore’s L1 sensor chip to
capture DMPC liposomes. This sensor chip displays lipophilic
groups attached on the surface of a carboxymethylated dextran
layer, and was shown to provide a quick and reproducible method
for the preparation of bilayer-mimetic systems . We first tested
whether arbidol per se could bind or not to the chip. Arb at
11.3 mM (the highest concentration relevant in the biological
context) was injected onto the chip devoid of liposomes. This led to
approx. 60 resonance units (RU, see Methods section). DMPC
liposomes (2 mM) captured onto the sensor chip reached about
5000 RU, and a further ,600 RU was seen when Arb was pulsed
onto the liposome-coated chip. The binding of arbidol alone on
the L1 chip remains therefore negligible.
Measures of Arb/DMPC association and dissociation were
performed with various Arb concentrations ranging from 0.5 to
11.3 mM. After passage over the surface of the sensor chip, Arb
bound to immobilized DMPC in a concentration-dependent
manner (Figure 4). Arb initial binding was fast, but then slowed
down without reaching saturation equilibrium (from 0 to 240 s).
After stopping the Arb flow onto the sensor chip (from 240 s),
bound Arb was rapidly but incompletely dissociated from DMPC
membranes. Indeed, for all Arb concentrations tested, about 50%
of Arb remained bound to DMPC. This demonstrates that Arb is
capable of interacting with lipid membranes, in a stable association
between Arb and DMPC. However the behaviour of Arb binding
to membranes rendered difficult the fitting of a kinetic model to
the data, and hence the determination of reliable on- and off-rates.
Indeed using global fitting, binding curves could not be fitted
properly with the different models included in the BIAevalution
3.0 software (1:1 Langmuir binding, bivalent analyte, heteroge-
Figure 3. Arb interacts with lipid bilayers of giant unilamellar liposomes. GUV composed of PC:chol:R18(2 nmol) were electroformed in
water and observed by optical epi-fluorescence microscopy (A). Various concentrations of Arb in water were added to GUV, for final Arb-to-lipid
molar ratios of: B, 1:40; C, 1:20; D, 1:10; E, 1:1; F, 10:1 and G. 20:1. Asterisks indicate small invaginations (panel D) or occasional GUV flickering (panel E).
Bar, 25 mm.
Figure 4. Binding of Arb to immobilized DMPC membranes. Arb
in water at concentrations of 0.5, 1, 2, 4, 6, 8 and 11.3 mM was injected
over immobilized DMPC membranes (ca. 5000 resonance units) for
4 min at a flow rate of 20 ml/min, followed by water. Blank curves
without Arb were substracted from those obtained with Arb. Inset,
representative set of data of non-linear regression fits to the equilibrium
resonance signal (Req), obtained by extrapolation to infinite time (see
Materials and Methods), vs Arb concentration, used to obtain apparent
equilibrium dissociation constant (KD) as well as the maximum binding
capacities (Rmax). Kinetics were reproduced 4 times. Dotted curves
represent the sensorgram and solid curves the non-linear fit. RU,
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neous ligand, heterogeneous analyte, conformational change), with
or without mass transport. Furthermore, because equilibrium was
not reached during the association phase, the direct use of
Scatchard analysis to calculate the apparent equilibrium dissoci-
ation constant was not allowed. Instead, the apparent equilibrium
dissociation constant KD was calculated from the equilibrium
resonance signal (Req) as a function of analyte concentration, Req
values being estimated by extrapolation to infinite time using plots
of resonance signal as a function of the reciprocal of time [26,27].
Apparent KD was then calculated by nonlinear fitting to the
expression Req=RmaxC/(KD+C), where Rmaxis the maximum
binding capacity of the surface and C is the analyte concentration,
using SigmaPlot software. This calculation, performed on 4
separate experiments, gave an apparent KDof 6.860.4 mM (see
inset to Figure 4 for a representative experiment). This dissociation
constant is in the same order as the IC50 of HCVpp fusion
(11.3 mM). This result indicates that the inhibitory effect of Arb on
HCVpp membrane fusion is at least in part deriving from Arb
association to lipid membranes.
NMR spectroscopy was used to characterize the Arb insertion
in a model membrane system. The1H NMR spectrum of Arb
recorded at 305 K in deuterated water is shown in Figure 5A
(black spectrum), and the assignment of the proton signals deduced
from 2D spectra analysis (data not shown) are reported in Table 1.
In order to study Arb in a membrane-mimetic environment, we
used isotropic phospholipid bicelles consisting of a mixture of
DMPC/DHPC in water. Long-chain phospholipid molecules of
DMPC self-assemble into planar bilayers, while the short-chain
Figure 5. NMR of Arb into lipid bicelles. A,1H NMR spectrum of Arb in D2O (in black) and in DMPC/DHPC bicelles (in red) with [Arb]/[lipids]=
1/15 and T=305 K. B, influence of the concentration of the paramagnetic agent Gd(DTPA-BMA) on the proton relaxation times for Arb in the bicelle
system. C, paramagnetic relaxation enhancements (PRE) measured on Arb (marked by dotted vertical lines) and on the phospholipid protons (marked
by histogram bars). The phospholipid is used as a yardstick to roughly estimate the arbidol proton positions inside the membrane. Red circles
indicate the yardstick marker closest to a given arbidol PRE value. Error bars indicate the standard deviation derived from the calculation of PRE. Error
bars for Arbidol are comparable. D, sketch of the positioning of Arb in a DMPC membrane system. The Arb molecule was produced by generating an
extended structure, and regularized by 1000 cycles of a Powell type minimization using XPLOR-NIH . The positioning in the membrane system
was done manually by taking into account the relative proton depth measured by the PRE (panel C).
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org6 January 2011 | Volume 6 | Issue 1 | e15874
molecules of DHPC segregate to edge regions of high curvature
. Bicelles with [DMPC]/[DHPC] molar ratio ,1 form fast
and isotropically tumbling aggregates, amenable to solution NMR
studies. Still, isotropic bicelle systems are used as a phospholipid
bilayer mimetic, since DMPC has been shown to form a flat
bilayered surface [29–31]. The1H NMR spectrum of Arb in this
bicellar phase is shown in Figure 5A (red spectrum). In this system,
the spectral crowding due to the presence of phospholipid
resonances allowed only the observation of protons denoted 1, 2,
3, 4, 5 and 6 of the arbidol molecule (see Fig. 1A) where only 2
signals can be distinguished for the three protons 2, 3, 4.
Additional1H resonances could be resolved from 2D1H NOESY
corresponding chemical shifts are shown in Table 1 together with
the assignment of the proton lines for Arb in water. Arbidol
interaction with lipids induce chemical-shift changes in the Arb
resonances when compared to that observed in water.
In order to investigate the immersion depth of Arb in the
membrane, we monitored the proton longitudinal relaxation rate
of Arb protons upon the addition of the soluble paramagnetic
agent gadolinium-diethylenetriamine pentaacetic acid-bismethyla-
mide Gd(DTPA-BMA) . This paramagnetic contrast agent
stays soluble in the water surrounding the membrane and induces
a paramagnetic relaxation enhancement (PRE) on the spin of the
atoms close to the surface of the membrane. Recently, PRE effects
due to Gd(DTPA-BMA) were used to probe the immersion depth
and orientation of an anti-microbial peptide [33,34]. Here we
measured the proton T1 relaxation times of both Arb and
phospholipid protons to probe the immersion depth of Arb in the
membrane, using the PRE values of the phospholipids as an
approximated yardstick. A titration of Arb with Gd(DTPA-BMA)
was performed at increasing concentrations of the paramagnetic
agent, from 2.0 to 9.7 mM. At each step of the titration, the
proton T1 relaxation times for the protons 1, 2, 3, 4, 5 and 6 of
Arb, and for the protons alpha, beta, gamma, G1, G2, G3, C2, C3
and omega14 of the phospholipids, were measured. The plot in
Figure 5B shows the relaxation times as a function of the
Gd(DTPA-BMA) concentration. This titration leads to a curve for
each proton whose slope corresponds to the PRE values. As shown
in Fig. 5C, PRE measured on the Arb-bicelle system range from
0.90 sec21to 0.03 sec21for the phospholipid protons and from
0.90 sec21to 0.20 sec21for Arb protons. The PRE observed for
each spin can be described as an overall relaxation enhancement
, due to all the paramagnetic agents in solution. For a planar
1H/13C HSQC spectra (Figure S2) and the
membrane surrounded by a buffer containing a non-interacting
paramagnetic probe, the total PRE of a nucleus with immersion
depth d is given by the equation [33,34]: PRE=z/d3, where d is
the immersion depth of a specific nucleus within the membrane
plus the radius of the magnetic probe, and where the constant z is a
combination of various parameters, among them a correlation
time, itself a combination of the electron relaxation time, the
lifetime of the intermolecular adduct bicelle-Gd(DTPA-BMA), and
the rotational correlation time. In order to determine the
immersion depth of Arb, instead of determining z, we used the
phospholipids as a yardstick by comparing their PRE with the one
The PREs of the resolved signals of Arb and phospholipids are
reported and compared in Figure 5C. This procedure is based on
two assumptions: (i) the amount of free Arb in solution in the
presence of lipids is negligible (see NMR sample preparation in
Experimental Procedure section), and it does therefore not affect
significantly the PRE of Arb in the membrane ; ii) the constant z is
the same for lipids and Arb.
Figure 5C shows that methyl protons 6 of Arb are at the lipid/
water interface as the gamma-proton of the hydrophilic head-
group of the lipids. Protons 5 and 1 of Arb are at the level of
respectively protons alpha and G1 of the phospholipids. The
aromatic protons 2 and 3 are the most buried protons of Arb, close
to the beginning of the hydrophobic chain (protons C2).
The validity of assumption (ii) is supported by the NOE cross-
peaks detected between proton 1 of Arb and the glycerol moiety of
phospholipids. In addition, the maximum PRE measured for Arb
(protons 6) and phospholipids (c protons) are about the same,
suggesting that the corresponding molecule regions are the most
exposed to water. This estimation of the immersion depth of Arb
protons relative to the phospholipids protons enables us to propose
a model for the positioning of Arb in the membrane, shown in
These NMR data clearly demonstrate that Arb interacts at the
membrane interface, mainly at the level of phospholipid polar
head. This result supports the assumption that the Arb inhibitory
effect on HCVpp membrane fusion is dependent, at least in part,
from this interaction.
Arbidol interaction with tryptophan derivatives and
A second possibility regarding Arb activity is that Arb might
interact with key motifs present in viral proteins, thereby impeding
their structural reorganization at the onset of fusion and thus
leading to fusion inhibition.
A first set of experiments was designed to investigate whether
the order of addition of fusing partners would affect Arb-induced
fusion inhibition. For this purpose, we measured fusion after pre-
incubation of HCVpp or liposomes or both in the absence or
presence of 11.3 mM Arb. As shown in Table 2, when Arb was
pre-incubated with both partners before fusion was initiated by
lowering the pH, fusion inhibition was ca. 50%. In contrast, only
ca. 10% fusion inhibition was observed when Arb was pre-
incubated with either HCVpp or liposomes. The greater inhibitory
effect of Arb when it has simultaneously access to both viral and
target membranes suggests that Arb could also act by interacting
with selective residues of the HCV glycoprotein sequences.
This assumption was tested by studying the interaction behaviour
of Arb with tryptophan (Trp) derivatives, as tryptophan is a
constituent of proteins often found in regions located close to
membrane interfaces, such as stem regions in several viral fusion
proteins (e.g. HIV gp41 ). We also reasoned that Arb, being an
indole derivative, might interact with tryptophan and tyrosine
Table 1. NMR assignment of1H chemical shift of Arb in water
and in the presence of [DMPC]/[DHPC] bicelles at 305 K.
Arb protonsArb in water (ppm)Arb in bicelles (ppm)
02/04/10 7.18–7.34 7.25–7.33
5 4.63 4.93
9 4.52 4.68
nd, protons 7 and 8 could not be resolved from the lipid resonances (see Figure
S2). Protons 9 and 10 can be resolved only in the1H/13C HSQC spectrum.
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org7January 2011 | Volume 6 | Issue 1 | e15874
residues through aromatic ring stacking. For this purpose, we tested
the effect of increasing concentrations of Arb on the fluorescence of
N-acetyl tryptophanamide (NATA) as a water-soluble Trp
derivative, and tryptophan octyl-ester (TOE) as a membranotropic
molecule (Figure 1B–C). The fluorescence of NATA and TOE was
recorded between 300 and 400 nm, using an excitation wavelength
of 286 nm, which corresponds to an absorption minimum of Arb
. Results are presented in Table 3 and Figure 6. The apparent
affinity of Arb for indole of Trp derivatives was calculated as the
difference (DA) between spectral areas (AUC) in the absence and
presence of Arb (i.e. DA=AUCno Arb–AUCArb), for each Arb
concentration at pH 7.4 or 5.0. Apparent KDvalues were then
calculated from the plots DA=f([Arb]), by non-linear fitting.
Apparent KDof Arb for NATA in solution was found in the range
of ca. 60 mM at both pH (Table 3). Similarly, the KDof Arb for
TOE in lyso-PC micelles was in the 50 mM range at both pH. This
indicates that Arb is able to interact with indole rings, but with a
line (DPC) micelles in the presence of TOE further confirmed this
interaction (TOE/Arb/DPC molar ratios: 1:0.5:50; 1:1:50; 1:2:50
and 1:5:50). Indeed, chemical shift variations of aromatic protons
from Arb and TOE were observed when comparing the NMR
spectra of Arb/DPC, TOE/DPC, and Arb/TOE/DPC samples
(data not shown).
In contrast, when Arb was added to TOE associated to
liposomes (1:20, TOE/lipid molar ratio), a marked increase in
affinity was observed (Table 3, compare TOE/micelles and TOE/
liposomes), reaching KDvalues in the 10 mM range. Note that
TOE fluorescence could not be measured in DPC micelles, due to
a great intrinsic fluorescence of the DPC used for our experiments.
Interestingly, these KD values are comparable to Arb IC50
inhibition of HCVpp fusion (see above and Discussion section).
Indole fluorescence decreased when Arb concentration increased,
with virtually no measurable fluorescence for 100 mM Arb
(Figure 6). This further confirms that Arb interacts with indole
rings, but with a higher affinity when indole is incorporated into
lipid membranes. This affinity was higher for PC than for PC:chol
liposomes (Table 3), and at neutral than at acidic pH (Table 3 and
Figure 6). At acidic pH, Arb is most likely protonated in the lipid
Table 2. Influence of the order of addition of fusing partners on HCVpp 1a fusion inhibition by Arb.
ComponentTime (min)Component Time (min) Inhibition of fusion with Arba(%)
Liposomes1 HCVpp1 13611
aArb (11.3 mM) was pre-incubated sequentially with either HCVpp or R18-labeled liposomes or both for the indicated times at 37uC before initiating lipid mixing by
decreasing pH to 5.0 as described in the legend to Figure 2. The extent of inhibition of fusion by Arb was calculated relatively to the fusion observed in the absence of
Arb and normalized to 100%. Results are the mean 6 SEM of 3 separate experiments.
Figure 6. Indole emission fluorescence spectrum of TOE into
PC:chol liposomes. PC:chol (70:30 molar ratio) liposomes containing
TOE (5 mM final, lipid-to-TOE ratio 20:1) were equilibrated to 37uC in PBS
at pH 7.4 or 5.0, in the absence (bold line) or presence (standard lines)
of increasing concentrations of Arb (2, 5, 10, 25 and 100 mM). Indole
fluorescence was measured between 300 and 400 nm, with excitation
at 286 nm. The apparent affinity of Arb toward TOE was calculated from
the plot of the difference DA between spectral areas (AUC) of TOE
without or with Arb (DA=AUCno Arb2AUCwith Arb) as a function of Arb
concentration (see inset for a range of Arb concentrations between 0
and 30 mM) (see KDvalues reported in Table 3).
Table 3. Apparent dissociation constants between Arb and
the indole ring of the tryptophan derivatives NATA and TOE.
Tryptophan derivatives and media pH 7.4pH 5.0
NATA in solution6461055610
TOE in lyso-PC micellesb
TOE in PC liposomesc
TOE in PC:chol liposomesc
aFor experimental details, see legend to Figure 6.
bTOE-to lyso-PC molar ratio was 1:800.
cTOE-to-lipid molar ratio was 1:20.
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org8 January 2011 | Volume 6 | Issue 1 | e15874
environment , as is probably TOE as well. Arb affinity for
TOE under these conditions might then be lower than that of
uncharged Arb at neutral pH, because of repulsive electrostatic
A third set of experiments was designed to assess the behaviour
of Arb in the presence of aromatic residues into protein sequences,
more specifically toward Trp present in peptides. For this purpose,
we used synthetic membrane-binding peptides of known structure
and containing only one tryptophan residue, expected to be
localized at the membrane interface: the transmembrane helix of
HCV NS4A protein  and the N-terminal amphipathic helix of
BVDV NS5A protein interacting in-plane of the membrane
interface . Intrinsic tryptophan fluorescence of both peptides
was monitored in the presence of increasing concentrations of Arb;
this is illustrated in Figure 7 for HCV NS4A peptide inserted into
PC:chol liposomes (and in Figure S3 for BVDV NS5A, 1:20
peptide-to-lipid molar ratio). The Arb dose-dependent quenching
of tryptophan fluorescence at both neutral and acidic pH clearly
indicates Arb interaction with both peptides (insets in Figure 7 and
Fig. S3). For the NS4A peptide at both pH, a red shift of the
spectral maximum, proportional to Arb concentration, accompa-
nied the fluorescence quenching; this effect was more pronounced
at acidic pH (6 nm at pH 7.4 for 5 mM Arb, and 12 nm at pH 5.0
for 100 mM Arb). This red shift suggests that Arb, when
interacting with NS4A peptide, relocates its tryptophan residue
to a more shallow zone of the membrane, where the Trp
environment would be more hydrophilic. The measure of the
apparent affinity of Arb for these peptides inserted into PC:chol
liposomes was performed as described above. Interestingly Arb
displayed an apparent KDtoward peptide Trp between 3.3 and
5.6 mM (Table 4), twice lower than that observed for TOE in
PC:chol liposomes. Since both peptides contain one or two
tyrosine residues (HCV NS4A and BVDV NS5A, respectively) in
addition to the Trp, interaction of Arb molecules with these
aromatic residues might account for a higher affinity of Arb for
peptides than for a small molecule such as TOE.
Since Arb is an inhibitor of HCV membrane fusion, we
reasoned that it might interact with the regions of E1 and E2
described as important for HCV fusion [11,17]. These peptides
were described as membranotropic on model membranes  and
contain aromatic residues. We therefore analyzed the effect of
increasing concentrations of Arb on the fluorescence quenching of
two peptide sequences derived from HCV E2 (positions 415–432
and 606–625, see aa sequences in Table 5), and inserted into
PC:chol liposomes (1:20 peptide-to-lipid molar ratio). Note that we
also tested a third peptide located at position 270–283 of E1,
containing only one Tyr; but its fluorescence quantum yield was
too low to monitor any interpretable fluorescence signal (data not
shown). We then calculated the KDvalues as described above. As
shown in Table 5, E2 415–432 contains only one Trp, whereas E2
606–625 contains one Trp and 3 Tyr. The apparent affinity was in
the 15 mM range at pH 7.4 for both peptides, reminiscent to Arb
IC50 of HCVpp fusion. This indicates that Arb is able to interact
with the aromatic residues of both peptides in the membrane, and
lends further support to our hypothesis that Arb could interact
with key residues/motifs in viral fusion proteins, which would
constitute a possible (partial) explanation to its inhibition of
HCVpp fusion. Strikingly this affinity decreased at acidic pH for
both peptides, and even drastically to 70 mM for E2 606–625.
Interestingly, this relatively high KDvalue is reminiscent of that
observed for Arb interaction with NATA in solution (Table 3).
This suggests that the interaction between the E2 peptide and the
membrane would be weak at acidic pH, and that most of the
peptide could be in solution. Moreover an histidine residue,
located in the immediate vicinity of Trp in the sequence of both
peptides, is expected to be charged at pH 5.0. Since Arb is also
protonated at that pH value, this could create repulsive forces
affecting the interaction between Trp and Arb. Moreover, as
protonation of the histidine cycle is expected to decrease the free
energy of partition from lipids to water, the peptide could be
released from the membrane at acidic pH, possibly in relation with
peptide conformational change(s). This behavior in not in favor
with their direct role as fusion peptides of HCV, a virus dependent
on low pH for its membrane fusion activity. However, due to their
membranotropism , and since our and other studies showed
their involvement in HCV membrane fusion [17,38], it is possible
that the conformational changes they might undergo at low pH
would lead to a proper relocation of the actual fusion peptide/loop
toward the target membrane  (and see Discussion section).
This study aimed at further investigating the molecular
mechanism of action by which arbidol (Arb) inhibits virus cell
entry and membrane fusion, using HCVpp as a model of an
We showed that Arb displayed a dual binding capacity, on lipid
membranes interface on one hand and on the aromatic residue
Figure 7. Trp emission fluorescence spectrum of an HCV model
peptide into PC:chol liposomes. PC:chol (70:30 molar ratio)
liposomes containing HCV NS4A peptide (KKGGSTWVLVGGVLAA-
LAAYCLSTGSGGKK, 5 mM final, lipid-to-peptide ratio 20:1) were equili-
brated to 37uC in PBS at pH 7.4 or 5.0, in the absence (bold line) or
presence (standard lines) of increasing concentrations of Arb (2, 5, 10, 25
and 100 mM). Trp fluorescence was measured between 300 and 400 nm,
with excitation at 286 nm. The apparent affinity of Arb toward Trp was
calculated from the plot of the difference DA between areas under the
curve (AUC) of peptide without or with Arb (DA=AUCno Arb2AUCwith Arb)
as a function of Arb concentration (see inset for a range of Arb
concentrationsbetween0 and30 mM)(see KDvaluesreportedinTable4).
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org9 January 2011 | Volume 6 | Issue 1 | e15874
tryptophan of proteins on the other hand. It therefore appears
plausible that the observed inhibitory effect of Arb on viral entry
and membrane fusion might result from a combined effect of
binding of Arb on membranes and on (fusion) proteins.
From a physico-chemical point of view, Arb displayed tropism
for membranes or membrane-like environments such as detergent
micelles, particularly prominent at low pH . By combining
several biochemical approaches, we show here that Arb has the
propensity to bind to and incorporate into lipid bilayers, with
calculated apparent affinities in a similar range as the IC50 value
for fusion, i.e. ca. 10 mM. Our NMR studies of Arb interaction
with DMPC leads to a model where Arb binds at the membrane
interface and establishes contacts mainly with the polar heads of
phospholipids (Fig. 5D).
Altogether these data suggest that at least part of Arb inhibitory
activity could be explained by its membranotropism. This physico-
chemical property has been further emphasized in a recent work
by Villalain , using Fourier-transform infrared spectroscopy.
Arb interaction with phospholipids would disturb membrane
fluidity crucial to the fusion process, thereby rendering the lipid
bilayer less prone to fusion. Such a model is consistent with the
behavior of other indole derivatives, that were shown to exhibit a
preference for membrane interfaces [41,42], due to the flat rigid
structure of these molecules and to their aromaticity, which allows
them to establish cation-p interactions with the positively charged
quaternary ammonium lipid headgroups [41,43]. At low pH, the
optimal pH for HCV fusion, these interactions would be favored
due to the protonation of the amino groups. As was described for
other substituted indoles , it is possible that protonation of the
carbon bearing the ester group of Arb could displace this group
out of the indole plane, and place it in a better position to bond
with neighboring molecules. This could in turn lead to a better
membrane association. Arb might therefore have the propensity to
intercalate into lipids of the viral and target membranes while
adopting a consistent orientation by filling the gaps between lipid
molecules. The interfacial region of the lipid bilayer provides a
suitable environment for a wide range of chemical groups, as long
as they possess a large enough hydrophobic moiety and a group
capable of forming hydrogen bonds with the lipid carbonyl groups.
Several compounds with antiviral pharmacological properties
belong to this category, in particular adamantanes active against
influenza A viruses [45,46] and against some HCV clones but not
all [47,48], the natural triterpene glycyrrhizin efficient in the
treatment of chronic viral hepatites  and the flavonolignan
molecules composing silymarin, an herbal extract with potent anti-
HCV activities [1–3,50,51].
In a previous study, we noticed that Arb inhibition of cell entry
concerned HCVpp and pseudoparticles bearing the influenza
hemagglutinin (HApp), but not pseudoparticles bearing the
envelope glycoprotein of a feline oncogenic retrovirus (Rd114pp)
. These data suggest that Arb might display selectivity for the
recognition of key motifs inside envelope proteins. This hypothesis
was tested by assessing the influence of Arb on the fluorescence
properties of aromatic compounds derived from tryptophan (Trp)
and of peptides containing Trp. Trp is a component of proteins
with interfacial properties [41,42], often located at the lipid/water
interface and grouped into so-called tryptophan-rich motifs crucial
to protein/membrane association , and found in the envelope
(fusion) proteins of the SARS coronavirus or HIV-1 [36,53,54].
Trp is also enriched at protein/protein binding interfaces of the
small envelope protein of the hepatitis B virus  and of
membrane proteins in general . We demonstrated here that
Arb was able to alter/quench the fluorescence properties of small
Trp derivatives in solution (NATA), in detergent micelles and in
liposomes (TOE, ), in a dose-dependent manner. This occurred
most likely through stacking of the aromatic rings of both molecules
which is often involved in stabilization of inter-cations. Interestingly
the apparent affinity of the Arb/Trp derivative interaction was in
the order: lipid bilayers.micelles.solution, indicating that Arb
binding strength for Trp could increase in membrane environments
where both molecules accomodate and get packed. Indeed Arb
apparent affinity for TOE in liposomal membranes was in the
10 mM range, a value comparable to the IC50 of fusion. Arb affinity
was even greater for membrane peptides containing Trp and
tyrosine (Tyr) residues (ca. 4 mM). Due to its indole group, it is
conceivable that Arb might display selectivity not only for indole
rings (Trp) but more generally for aromatic groups, as the phenol
ring of Tyr. A greater number of Arb molecules could therefore
interact with aromatic residues in peptide sequences, leading to
some cooperativity in the quenching effect and to an overall larger
Table 4. Apparent dissociation constants between Arb and Trp residues of model membrane-binding peptides inserted into
Membrane peptidesainserted in PC:chol liposomesb
pH 7.4pH 5.0
HCV NS4A[1–22]* transmembrane peptide KKGGSTWVLVGGVLAALAAYCLSTGSGGKK3.360.6 5.660.3
BVDV NS5A[1–28] membrane anchor peptide SGNYVLDLIYSLHKQINRGLKKIVLGWA 3.360.4 4.160.4
aThe NMR structures of synthetic peptides HCV NS4A[1–22]* and BVDV NS5A[1–28] peptides have been reported in references  and , respectively. The
solubilization tags KKGG and GGKK at the N- and C-terminal ends, are indicated in italic. Aromatic residues Trp and Tyr are indicated in bold, His is underlined.
bPeptide-to-lipid molar ratio was 1:20.
cKDvalues were calculated as described in legend to Figure 7.
Table 5. Apparent dissociation constants between Arb and
Trp residues of synthetic peptides involved in fusion.
Membrane peptidesainserted in PC:chol
pH 7.4pH 5.0
E2[415–432] NTNGSWHINSTALNCNES 1564 2867
E2[606–625] RCMVDYPYRLWHYPCTINYT 1363 70610
aPeptides from HCV strain H (genotype 1a, accession number AF009606; ).
Aromatic residues Trp and Tyr are indicated in bold, His is underlined.
bPeptide-to-lipid molar ratio was 1:20.
cKDvalues were calculated as described in legend to Figure 7.
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org10 January 2011 | Volume 6 | Issue 1 | e15874
Although HCV entry inhibition by Arb was found genotype-
dependent, HCV membrane fusion was inhibited by Arb in a
genotype-independent manner. HCV entry and fusion are early
steps in the life cycle of the virus [58,59]. HCV first interacts
through its envelope glycoproteins with a set of coreceptors at the
plasma membrane level (recently reviewed in [60,61]) and
eventually becomes endocytosed [62–64]. Due to a combined
action of acidification in the endosome and particular lipids like
cholesterol and sphingomyelin [11,18], viral fusion occurs over a
broad spectrum of pH’s ranging from 6.3 to 5.0 [11,18,65]. HCV
binding to the hepatocyte membrane followed by endocytosis
therefore requires several cellular proteins, and most likely involves
several levels of interactions (interactions between viral proteins,
between cellular and viral proteins, between viral/cellular proteins
and lipids). These features might explain the differential effect
exerted by Arb on entry of various HCV genotypes: indeed subtle
differences in protein sequences could translate into modified
interactions with several partners and/or at several levels.
Conversely some common principles of action apply to all fusion
reactions, viral fusion and cellular fusion processes alike .
Indeed all fusion processes involve two partners: lipids and the
fusion protein(s). This might account for the similar inhibitory
effect of Arb on HCV fusion observed for all genotypes. This is in
line with the observations that Arb displayed potent antiviral
activity against some antigenic serotypes of influenza viruses, but
not against all .
Previously we noticed that Arb inhibition of primary infection of
Huh-7.5.1 cells with HCV (clone JFH-1) was efficient only when
cells were preincubated with Arb 24 or 48h before infection ;
in addition, inhibition of HCVpp and HApp cell entry was most
efficient when Arb was pre-incubated with both viral and cell
membranes . Here, using our in vitro fusion assay, we observed
that Arb inhibition of HCVpp fusion was maximal when both viral
and target membranes were incubated with Arb, before fusion was
initiated. This suggests that a certain level of membrane
impregnation and/or saturation with Arb must be achieved to
efficiently inhibit viral infection. Membranes might therefore act as
‘‘concentrators’’ of arbidol, and high concentrations of the
molecule might be locally achieved. This could explain why
Arb, exhibiting an apparent (medium to low) affinity for
membranes in the mM range, exerts a relevant antiviral activity
without noticeable membrane damages. Along these lines, in spite
of its marked membranotropism, Arb displays only low toxicity
[9,10]. Arb exhibited a comparable micromolar apparent affinity
for aromatic residues present in membrane peptides in a
membrane environment. Altogether, these observations lead us
to propose a mechanistic model of the way Arb would inhibit
HCV entry and fusion. Through its membranotropism, Arb is able
to freely interact with viral and target membranes, and could
locally get highly concentrated. Arb is also able to interact with
aromatic residues within viral proteins involved in membrane
interactions and membrane destabilization necessary for fusion.
Through this dual binding capacity, Arb could then locally
impede the structural rearrangements required for the fusion
protein to adopt its fusion conformation. The fact that Arb is
active in the mM range suggests that Arb would act by reducing the
overall speed of the fusion reaction rather than by blocking a
specific protein conformation. This could therefore explain the
broad antiviral spectrum of Arb, and the genotype independence
of its inhibitory effect on HCV fusion, since HCV envelope
proteins contain well-conserved aromatic residues in all genotypes.
Mechanistically, the key point is the relative accessibility of these
residues to Arb at the membrane interface. A cooperative effect
between Arb and several aromatic residues might therefore occur.
Also the local environment of these aromatic aa is important, since
the presence of residues such as histidines (His) in their vicinity
could modify their accessibility with respect to pH. Interestingly
enough, in the sequence of both HCV E2 peptides studied here
(Table 5) and shown to be involved in HCV fusion , His is
contiguous to Trp, and in the 606–625 peptide, His is surrounded
by three tyrosines. The concept of His as a critical pH sensor at a
key intramolecular domain interface in a viral fusion protein has
recently emerged [67,68]. Indeed, the protonation of a sole His in
the E protein of the tick-borne encephalitis flavivirus (TBEV)
triggers large-scale conformational changes leading to viral fusion.
Concerning HCV, Rey and coworkers recently proposed a model
of the 3D arrangement of the E2 ectodomain . In this model,
the fusion loop/peptide would lie within the poorly structured
domain II, and the E2 606–625 peptide would be found in the
globally unstructured domain III, where a critical His residue is
disposed at the interface with domain I. The putative fusion loop
contains a phenylalanine and a tyrosine . At low pH, the
optimal pH for HCV membrane fusion, key histidine(s) could
become protonated. This could result in conformational rear-
rangements and, in the context of Arb fusion inhibition, aromatic
residues might consequently become more or less accessible to Arb
molecules present in their vicinity. We noted that the apparent
affinity of Arb for HCV peptides was weaker at pH 5.0 than at
pH 7.4. At low pH, Arb is also protonated, and this protonated
form could exhibit a greater preference for the interfacial region of
the lipid bilayer than the deprotonated form, as demonstrated for
adamantanes . Combined with the notion that key aromatic
and His residues would also display interfacial (re)localization at
low pH, this would in turn explain the higher efficiency of Arb at
inhibiting fusion at acidic pH .
In conclusion our data reveal that Arb directly interacts with the
lipid membrane-water interface, and is able to bind to aromatic
residues present in HCV glycoproteins, in their membrane-
associated form. Through a subtle binding interplay between Arb,
lipids, viral and cellular proteins, Arb might efficiently block HCV
entry and membrane fusion interacting with the main actors of the
early steps of viral entry. Most interestingly, Arb inhibition of these
processes demonstrated an affinity in the mM range, although the
membranotropic properties of Arb suggest that it could become
locally more concentrated in membranes. Together, these findings
suggest that Arb could increase the strength of viral glycoprotein’s
interactions with the membrane due to a dual binding mode,
involving aromatic residues and phospholipids. The resulting
complexation would inhibit the expected viral glycoprotein
conformational changes required during the membrane fusion
The antiviral mechanism of Arb therefore opens promising
perspectives for the development of small membranotropic low
affinity molecules, that would become locally concentrated in
membranes and would mainly act on the kinetics of the
conformational rearrangements of the viral fusion protein.
in a dose-dependent manner. A, Infectivity. Results are the
mean 6 SEM of 5 separate experiments. Black, no Arb; blue,
1.9 mM; green, 5.6 mM and red, 11.3 mM Arb, respectively. B,
Membrane fusion between HCVpp of genotype 4 (4.11.21) and
R18-labeled liposomes. The lipid mixing kinetic was followed by
fluorescence spectroscopy using excitation and emission at 560
and 590 nm, respectively. Fluorescent liposomes (12.5 mM final
lipid concentration) were added to 40 ml of HCVpp in PBS pH 7.4
Arb inhibits infectivity and membrane fusion
Arbidol Interacts with Membranes/Protein Motifs
PLoS ONE | www.plosone.org11 January 2011 | Volume 6 | Issue 1 | e15874
at 37uC, in the absence or presence of the indicated concentrations
of Arb. After a 2 min-equilibration, lipid mixing was initiated by
decreasing the pH to 5.0 with diluted HCl, and R18 dequenching
was recorded. Maximal fluorescence was obtained after addition of
0.1% final Triton X-100. Average value of the last 30 s of fusion
(i.e. final extent of fusion) was used to calculate the percentage of
fusion in the presence of Arb, relative to 100% fusion without Arb
(Figure 2). Black, no Arb; blue, 1.9 mM green, 5.6 mM and red,
11.3 mM Arb, respectively.
HSQC spectrum; [Arb]/[lipid] ratio was 1/15 and temperature
305 K. B, extract of 1H NOESY spectrum; [Arb]/[lipid] ratio was
1/10, temperature 290 K and mixing time 200 ms.
NMR of Arbidol into lipid bicelles. A, 1H/13C
BVDV model peptide into PC:chol liposomes. BVDV
NS5A peptide (SGNYVLDLIYSLHKQINRGLKKIVLGWA,
5 mM final) reconstituted into PC:chol liposomes (70:30 molar
ratio, peptide-to-lipid ratio 1:20) were equilibrated to 37uC in PBS
at pH 7.4 or 5.0 in the absence (thick line) or presence (thin lines)
Trp emission fluorescence spectrum of a
of increasing concentrations of Arb (2, 5, 10, 25 and 100 mM from
top to bottom). Trp emission fluorescence was measured between
300 and 400 nm, with excitation at 286 nm. The apparent affinity
of Arb toward Trp was calculated from the plot of the difference
DA between areas under the curve (AUC) of peptide without Arb
(DA=AUCno Arb2AUCwith Arb) as a function of Arb concentra-
This work was presented at the 16thInternational Symposium on Hepatitis
C and related viruses, (Nice, France, October 3–7, 2009).
The authors gratefully acknowledge Sylvie Ricard-Blum for SPR
analysis expertise. Fluorescence experiments were performed on the
platform ‘‘Production et Analyse des Prote ´ines’’ of the IFR 128 BioSciences
Conceived and designed the experiments: E-IP FP BHM AB. Performed
the experiments: ET J-PL DL RM AL GZ. Analyzed the data: ET GZ AL
J-PL BHM AB FP E-IP. Contributed reagents/materials/analysis tools: F-
LC. Wrote the paper: ET AL FP E-IP.
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