Cholesterol dependence of Newcastle Disease Virus entry
Juan José Martín, Javier Holguera, Lorena Sánchez-Felipe, Enrique Villar, Isabel Muñoz-Barroso ⁎
Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Edificio Departamental Lab. 108/112, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
a b s t r a c ta r t i c l ei n f o
Received 22 June 2011
Received in revised form 2 December 2011
Accepted 6 December 2011
Available online 13 December 2011
Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have
been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of
some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane
in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular choles-
terol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral
binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule ex-
pression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cho-
lesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-
resistant membrane microdomains (DRMs) at 4 °C and then shifted to detergent-soluble fractions at 37 °C.
These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.
© 2011 Elsevier B.V. All rights reserved.
Newcastle Disease Virus (NDV), a prototype of paramyxoviruses,
is an avian enveloped RNA-negative strand virus that causes respira-
tory disease in domestic fowls leading to huge economic losses in the
poultry industry. The envelope of NDV contains two associated glyco-
proteins that mediate viral entry: the hemagglutinin-neuraminidase
(HN) and fusion (F) proteins. HN is the receptor-binding protein
that recognizes and binds to sialoglycoconjugates at the cell surface
. The fusion protein is a metastable protein that undergoes a series
of irreversible conformational changes to trigger the fusion of the
viral and plasma membrane in a pH-independent manner . Never-
theless, we have previously reported an additional pathway of NDV
entry through caveolae-dependent endocytosis . For most para-
myxoviruses, including NDV, the triggering of F protein is HN-
dependent through its fusion promotion activity. The mechanism by
which HN protein activates F in a homotypic manner is not well un-
Lipid rafts are dynamic membrane microdomains preferentially
containing cholesterol as a major constituent, together with sphin-
golipids and specific associated proteins. Although their existence
is still controversial, the resistance of these components to cold de-
tergent extraction and mechanical disruption has been considered
proof of their existence (for a review, see ). Caveolae, a particular
membrane raft subset, are cholesterol- and glycosphingolipid-
enriched plasma membrane microdomains, identifiable as stable
invaginations of the plasma membrane, which are enriched in caveo-
lin, a membrane protein that is tightly bound to cholesterol .
Lipids rafts have been implicated in functions such as membrane sig-
naling and trafficking, signal transduction and the regulation of cell
Moreover, the involvement of lipid rafts and cell membrane cho-
lesterol has been demonstrated in different stages of the viral life
cycle, such as viral entry, assembly and budding (reviewed in [9–11]).
Some enveloped viruses have been shown to enter host cells in a
cholesterol-dependent manner, including poliovirus , polyomavi-
rus SV40 [13,14], coronavirus [15,16], arenavirus , togavirus
[18,19], poxvirus, such as vaccinia virus  and herpes virus
[21,22]. Lipid rafts have also been implicated in HIV entry [23,24],
and Ebola and Marburg filoviruses also require cholesterol in the plas-
ma cell membrane [25,26]. For some of these viruses, caveolae have
been proposed to be the portal of entry, including SV40 , picorna-
virus Echovirus 1 , the amphotropic murine leukemia virus
(A-MLV)  and the Ebola and Marburg filoviruses .
There are few reports describing the role of cell membrane choles-
terol in paramyxovirus entry, but with no evidence that lipid rafts
might be directly involved in it. It has been shown that the paramyxo-
virus canine distemper virus, genus Morbillivirus, does not require
cholesterol in the plasma membrane but does require it in the viral
envelope . The data concerning RSV are controversial. Thus,
some authors have reported the independence of RSV entry from cho-
lesterol , whereas in other works the entry of RSV through caveo-
lae in dendritic cells has been proposed . Moreover, using small
interfering RNA technology  showed that RSV use clathrin-
mediated endocytosis to productively infect cells. For NDV, it has
been shown that lipid rafts are sites of NDV assembly and release
Biochimica et Biophysica Acta 1818 (2012) 753–761
⁎ Corresponding author at: Departamento de Bioquímica y Biología Molecular, Uni-
versidad de Salamanca, Edificio Departamental Lab. 112, Plaza Doctores de la Reina
s/n, 37007 Salamanca, Spain. Tel.: +34 923 294465; fax: +34 923 294579.
E-mail address: firstname.lastname@example.org (I. Muñoz-Barroso).
0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
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We have previously shown that the caveolin phosphorylating fac-
tor PMA inhibits NDV fusion  and that cholesterol depletion exerts
an inhibitory effect on NDV interaction with the target cell, leading us
to propose that a fraction of NDV may penetrate the cell through
caveolae-mediated endocytosis . In the present paper, we extend
these studies with a view to clarifying the functional role of cholester-
ol in NDV entry. Depletion of cellular cholesterol by treatment with
methyl-β-cyclodextrin (MβCD) inhibited NDV binding, fusion and
infectivity. This inhibition was almost completely compensated by
replenishing cellular cholesterol levels, suggesting that the effect of
MβCD treatment on virus activities would be due to the removal
of cholesterol. Analysis of the distribution of viral envelope
glycoproteins in cholesterol-rich resistant membranes showed that
viral glycoproteins partially associate with lipid rafts during initial
virus adsorption and entry. Our data corroborate a role of cell mem-
brane cholesterol during the early stages of NDV infection. A clear un-
derstanding of the role of lipid membrane components in viral entry
should be useful in the future for designing antiviral agents and
2. Materials and methods
2.1. Cell lines and virus
East Lansing Line (ELL-0) avian fibroblasts and HeLa cells were
obtained from the American Type Culture Collection and maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with
L-GlutaMax(580 mg l−1, Invitrogen),
(100 U ml−1–100 μg ml−1) and 10% heat inactivated fetal bovine
serum (complete medium). The lentogenic “Clone 30” strain of NDV
was obtained from Intervet Laboratories (Salamanca, Spain). The
virus was grown and purified mainly as described previously .
2.2. Reagents and antibodies
Methyl-β-cyclodextrin (MβCD), cholesterol, lovastatin, Triton-X-
100, OptiPrep, FITC-MALI, monoclonal anti-β-tubulin antibody and
(MTT) were from Sigma-Aldrich; Octadecylrhodamine B chloride
(R18), Hoechst 33258 and Alexa fluor 488 donkey anti-mouse anti-
body were from Molecular Probes. Polyclonal anti-caveolin-1 (N20)
and anti-GAPDH antibodies were from Santa Cruz Biotechnology;
FITC-SNA was from Vector Laboratories; polyclonal anti-NDV, mono-
clonal anti-F (2A6) and HN (7B1) antibodies were generous gifts from
Dr. Adolfo García-Sastre (Emerging Pathogens Institute, Mount Sinai
School of Medicine, New York, USA).
2.3. Virus titration
Viral titres were calculated in plaque formation assays in Vero
cells, as described previously , or in monolayers of ELL-0 cells in
24-well plates infected with different dilutions of NDV for 24 h.
Cells were fixed for 30 min with PBS containing 2.5% paraformalde-
hyde, blocked in PBS containing 1% BSA for 1 h, and incubated for
1 h at room temperature with 5 μg ml−12A6 anti-F monoclonal anti-
body. Cells were then incubated with anti-mouse Alexa Fluor 488-
conjugated IgGs (dilution 1:400) for 45 min. After a wash with PBS,
cells were visualized under an inverted fluorescence microscope
(Olympus IX51) at 10× magnification. NDV infectivity was calculated
from the dilution of NDV that infected all cells of the monolayer and
was expressed as p.f.u. ml−1.
2.4. Fluorescence-activated cell sorting (FACS) analysis
Monolayers of ELL-0 cells in 60 mm plates were infected with 5
mois of NDV for 24 h at 37 °C. Infected cells were removed from the
plates by scraping, pelleted by centrifugation and incubated for 1 h
at room temperature in the presence of both 5 μg ml−1mAb anti-F
and mAb anti-HN. After centrifugation, cells were resuspended in
PBS and incubated with anti-mouse Alexa Fluor 488-conjugated IgG
(dilution 1:100) for 45 min in the dark at room temperature, followed
by fixation in FACS lysing solution (Becton-Dickinson) for 10 min.
Cells were then pelleted by centrifugation and resuspended in a suit-
able volume of PBS for analysis in a FACScalibur flow cytometer
(Becton Dickinson). At least 5×104cells were analyzed for each sam-
ple. Mean fluorescence values and the % of cells with fluorescence
higher than the background were combined to quantify viral protein
expression and were normalized to the control values. Data were
analyzed with the WinMDI 2.9 software.
2.5. Cholesterol depletion and replenishment experiments
2.5.1. Cholesterol depletion
ELL-0 cells seeded on 35 mm plates were left either untreated or
treated with increasing concentrations of MβCD and 4 μg ml−1lova-
statin in OptiMEM (Gibco) for 1 h at 37 °C. Under these conditions,
cell viability was not significantly affected, as determined with the
trypan blue exclusion method. The viability of untreated or MβCD-
treated cells was found to be higher than 90% as analyzed by trypan
blue exclusion. Then, the medium was removed and the cells were
washed 3 times with OptiMEM to remove MβCD. Following this, the
cells were processed as required for each experiment in the presence
of 4 μg ml−1lovastatin a competitive inhibitor of the rate limiting en-
zyme HMG-CoA reductase in the cholesterol synthesis pathway, pre-
venting the synthesis of endogenous cholesterol .
2.5.2. Replenishment of cholesterol
The replenishment of cellular cholesterol was accomplished as de-
scribed previously [12,38]. Briefly, after depletion of cellular choles-
terol as described above, ELL-0 cells were treated either with
medium alone or with cholesterol-MβCD-containing medium, with
soluble cholesterol at final concentrations ranging from 50 to
200 μM for 1 h at 37 °C. Then, cells were washed six times with Opti-
MEM to remove MβCD.
2.5.3. Determination of cholesterol levels
Cellular cholesterol was extracted according to [12,20]. Briefly,
ELL-0 cells were lysed by three cycles of freeze-thawing followed by
ultrasonication and the cholesterol was extracted from the cell lysates
by the addition of chloroform/methanol 1:1. The bottom layer (chlo-
roform) was collected and evaporated under vacuum. The residual
cholesterol was dissolved in ethanol and assayed with a fluorimetric
Amplex Red cholesterol assay kit (Molecular Probes) according to
the manufacturer's protocol. Data were referred to the cholesterol
levels of the same number of untreated control cells, considered as
2.6. Virus-binding assays
Plated control, cholesterol-depleted and ELL-0 cells replenished
with cholesterol as described above were washed three times with
ice-cold OptiMEM, and incubated with 5 mois of NDV for 1 h at 4 °C.
Then, the cells were washed with ice-cold PBS to remove unbound
virus and were collected by scraping, performing FACS analysis as
2.7. NDV–cell fusion assays
Purified NDV was labeled with the fluorescent probe octadecylr-
hodamine (R18) essentially as described previously . Control,
cholesterol-depleted and cholesterol-replenished ELL-0 cells plated
in 35-mm plates were incubated with 3 μg of R18-NDV per plate in
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
OptiMEM for 1 h at 37 °C. Then, the plates were washed 3 times with
PBS and the transfer of the rhodamine probe to cells was observed
under an Olympus IX51 inverted fluorescence microscope. R18 trans-
fer was considered to be represented by bright, full cell-shaped
events (Supplementary Fig. S1). Nuclei were stained with Hoechst
33258 (10 μg ml−1). The percentage of fusion was calculated as the
number of positive red-stained cells in 6 random fields with respect
to the total number of cells in these areas of the well.
2.8. NDV infectivity assays
2.8.1. FACS assay
Monolayers of control and cholesterol-depleted ELL-0 cells were
infected with 5 mois of NDV in OptiMEM for 1 h at room temperature.
Then, the viral inoculum was removed and complete medium was
added. At 24 h post-infection, cells were washed three times with
PBS and processed for FACS analysis as described above.
2.8.2. Fluorescent microscopy assays
Monolayers of control, cholesterol-depleted and cholesterol
replenished ELL-0 cells were infected with 1 moi of a recombinant
NDV (rNDV-F3aa-mRFP , that expresses a monomeric red fluores-
cent protein , kindly provided by Dr. Adolfo García-Sastre) for 1 h
at room temperature. After 24 h at 37 °C, the cells were observed
under an Olympus IX51 inverted fluorescence microscope with a
10× objective. Quantification of infectivity was accomplished by
measuring the area of red fluorescence (infected cells that express
the RFP) in pixels, referred to the area of nuclei (stained with Hoechst
33258) of the field in three to seven random fields. Areas were quan-
tified using the analysis tool in ImageJ software.
To analyze the effect of cholesterol removal on the course of viral
infection, ELL-0 cells were infected with the recombinant NDV at 1
moi. At different times post-infection, 10 mM of MβCD and
4 μg ml−1of lovastatin were added for 1 h at 37 °C, after which the
cells were washed and maintained in complete medium in the pres-
ence of lovastatin for 24 h at 37 °C. The percentage of infectivity
was calculated as the number of red fluorescent cells (infected cells)
out of the total number of cells in three random fields.
2.9. Lectin staining
Lectin staining of ELL-0 cells was performed by incubation with
FITC-labeled lectins. MβCD-treated or untreated ELL-0 cells were de-
tached from the plates by scraping and fixed with FACS lysing solu-
tion for 10 min at room temperature, washed with PBS and
incubated for 30 min at 4 °C either with 10 μg/ml FITC-labeled
Maackia amurensis lectin (FITC-MALI), which specifically recognizes
α2,3-bound sialic acids , or with FITC-labeled Sambucus nigra lec-
tin (FITC-SNA), which recognizes α2,6-bound sialic acids . Lectin
binding to cells was detected by FACS as above. For confocal visualiza-
tion of lectin staining, MβCD-treated or untreated ELL-0 cells were
washed once with OptiMEM and then fixed with 2.5% paraformalde-
hyde in PBS for 30 min at 4 °C and then incubated with 10 μg ml−1
FITC-MALI lectin for 30 min at 4 °C. After a short rinse with PBS,
cells were mounted in Prolong Gold antifade reagent with DAPI (Invi-
trogen) and viewed with a confocal microscope (Leica SP5 equipped
with a 488 nm argon laser for FITC and a 405 nm diode laser for
DAPI, 63× objective lens).
2.10. Fractionation of detergent-insoluble and -soluble membranes
Cells were fractionated into detergent-soluble and insoluble frac-
tions mainly as described previously [20,44,45]. Monolayers of ELL-
0 or HeLa cells grown on 60 mm plates were infected with 25 mois
of NDV for 1 h at 4 °C and, where indicated, shifted to 37 °C for an ad-
ditional hour. Cells were then washed in cold PBS and lysed in ice-
cold lysis buffer (10 mM Tris HCl, pH 7.6, 140 mM NaCl, 5 mM
EDTA, 1% sodium deoxycholate, 1% TX-100) containing a cocktail of
protease inhibitors for 30 min at 4 °C. The lysates were then passed
through a 20-gauge needle 20 times, and the nuclei and cellular
debris were pelleted by centrifugation at 12,000 rpm for 30 min.
The supernatants were layered at the bottom of a 40%–30%–5%
discontinuous OptiPrep gradient formed by overlaying 2 ml 40% (con-
taining the lysates), 6.5 ml 30% and 3.5 ml 5% of OptiPrep in buffer
lysis. Gradients were centrifuged at 34,000 rpm in a Beckman SW40
Ti rotor for 20 h at 4 °C. A total of 12 fractions of 1 ml were collected
from the top of the gradient by pipetting, after which they were ana-
lyzed for the presence of viral and cellular proteins by Western
2.11. Western blot analyses
Samples were separated by 10% sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) and transferred to PVDF mem-
branes (GE Healthcare). Membranes were blocked overnight at 4 °C
in TBS (50 mM Tris–HCl, pH 7.6, 150 mM NaCl) blocking buffer con-
taining 5% dry skimmed milk. Then, membranes were incubated
with individual primary rabbit polyclonal anti-NDV (1:5000 dilution),
rabbit polyclonal anti-caveolin N20 (1:300 dilution), mouse monoclo-
nal anti-β-tubulin (1:2000 dilution), and monoclonal anti-GAPDH
(1:2000 dilution) antibodies, for 1 h at room temperature. After ex-
tensive washes with blocking buffer, the membranes were incubated
for 1 h at room temperature with secondary anti-rabbit or anti-mouse
antibodies conjugated with horseradish peroxidase (1:5000 and
1:5000 dilution respectively, GE Healthcare), washed, and developed
with ECL Plus Western blotting reagent system (GE Healthcare).
2.12. MTT cell viability assay
Cell viability after lovastatin incubation for 24 h in the infectivity
experiments was assessed with a tetrazolium bromide colorimetric
assay. Briefly, Ell-0 were seeded in a 96-well plate and incubated for
24 h in the presence of 4 μg ml−1of lovastatin. Then, the culture me-
dium was removed and 100 μl of complete medium containing 10 μl
MTT (5 mg/ml in PBS) was added. The absorbance of each well was
measured at 620 nmon a Microplate Reader (Mutiskan Ex, Thermo Sci-
entific) with pure DMSO as a blank. Non-treated cells were used as a
control and relative cell viability (mean %±SD, n=3) was expressed
as ODlovastatin/ODcontrol×100%. The data indicate that lovastatin did
not elicit a negative effect on cell viability/proliferation, even higher
OD values (132.9%+/−17.6, mean±SD, n=3) being obtained.
To determine whether the removal of cholesterol from the target
membrane affected NDV entry into cells, ELL-0 cells were treated
with increasing concentrations of MβCD. MβCD treatment of cells
resulted in a dose-dependent reduction in the cellular cholesterol
content (Fig. 1a). Approximately, 85–90% of cellular cholesterol was
removed at a concentration of 10 mM MβCD. The fusion of NDV
with MβCD-treated ELL-0 cells was significantly reduced in a dose-
dependent manner, as determined by an R18 transfer assay (Fig. 1b
and Table 1). At a concentration of 5 mM MβCD (60% of cholesterol
reduction, Fig. 1a), viral fusion was reduced by about 25% (Fig. 1b
and Table 1), whereas the highest concentration of MβCD assayed,
(10 mM, which led to 80% of cholesterol reduction (Fig. 1a)) elicited
about 40% of NDV fusion inhibition (Fig. 1b and Table 1).
The effect of cholesterol depletion on NDV attachment was ana-
lyzed by flow cytometric assays. The binding of NDV to cholesterol-
depleted cells was reduced by about 50% as compared with the con-
trols in 10 mM MβCD-treated cells (Fig. 2a and Table 1). As a negative
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
control, the virus was inactivated by heating at 100 °C for 2 min. No
differences with samples in the absence of virus were detected, as
shown in Supplementary Fig. S2. To analyze whether cholesterol de-
pletion was affecting the expression of viral receptors at the cell sur-
face, after the MβCD treatment, cells were labeled with two
fluorescent-labeled lectins, FITC-MALI and FITC-SNA. The binding of
FITC-MALI to cells preincubated in the presence of sialidase from
Vibrio cholerae was reduced as assessed by fluorescence microscopy
(Supplementary Fig. S3) revealing the specificity of lectin binding to
sialoglycoconjugates. The degree of binding of both lectins to treated
cells was similar to that of control cells (Fig. 2b), indicating that after
cholesterol removal the receptor concentration was not modified at
the cell surface. Nevertheless, when we analyzed the lectin-labeled
cells by confocal microscopy, the FITC-MALI lectin-staining pattern
was altered (Fig. 2c and Supplementary Fig. S4): upon MβCD
treatment, the cell surface displayed patches enriched in sialic acid
compound (Fig. 2c), in agreement with previous reports [34,46].
The infectivity of NDV was probed by FACS-based assays after 24 h
of infection (Fig. 3a). Pre-treatment of ELL-0 cells with increasing
concentrations of MβCD prior to exposure to 5 mois of NDV resulted
in a reduction in viral infectivity, being about 50% less than that of
untreated control cells at the highest MβCD concentration assayed
(15 mM) (Fig. 3a and Table 1). In negative controls, the virus was
inactivated by heating at 100 °C for 2 min. No differences with sam-
ples in the absence of virus were detected, as shown in Supplementa-
ry Fig. S5. Additionally, cellular cholesterol was depleted at several
time points post-infectionby
(Fig. 3b). ELL-0 cells were infected with rNDV-F3aa-mRFP recombi-
nant NDV. Viral infectivity was monitored at 24 h post-infection, as
detailed in Materials and methods. The results showed that the addi-
tion of MβCD at 1 h post-infection did not exert any inhibitory effect
on NDV. Moreover, treatment with MβCD at 20 min post-infection
only had a moderate effect (Fig. 3b). These results suggest that cho-
lesterol is required during the entry of NDV into the host cell during
the first stages of viral infectivity.
To further check that the inhibitory effects of MβCD treatment on
NDV activities described above were due to the removal of cellular
cholesterol, the effect of cholesterol replenishment was analyzed
(Fig. 4 and Table 2). After the depletion of cellular cholesterol by
pre-treatment with 10 mM MβCD, cholesterol-depleted cells were in-
cubated in the presence of MβCD previously complexed to cholester-
ol, acting as a cholesterol donor to cells . The addition of 200 μM
of exogenous cholesterolalmost completely
treatment with 10 mM MβCD
Fig. 1. Effect of cellular cholesterol removal on NDV fusion. (a) Uninfected avian ELL-0 cell monolayers were incubated in the presence of increasing concentrations of MβCD for 1 h
at 37 °C. Cholesterol was extracted and quantified as described in Materials and methods. Data are means±standard deviations of three independent experiments. (b) R18-labeled
NDV was bound to MβCD-treated cells and untreated cells (control) for 1 h at 4 °C, after which they were allowed to fuse for 1 h at 37 °C. Fusion was assessed by transfer of the R18
red dye to the cell membrane. As a control of non-specific dye transfer, NDV was inactivated by incubation at 100 °C for 2 min (NDV-R18 inact). In these samples, non-significant
dye transfer was observed in both untreated and MβCD-treated cells.
Quantification of the effect of cholesterol removal on NDV activities.a
Concentration of MβCD
5 mM10 mM 15 mM
ND: not determined.
aPercentage of activity from control untreated cells.
bData are means±SD of three or four independent experiments.
cData from those as shown in Fig. 3a are means of two independent experiments.
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
cholesterol levels to those of the controls (Fig. 4a). Our data revealed
a correlation between the degree of cholesterol replacement (Fig. 4a)
and the degree of recovery of NDV activities (Fig. 4 and Table 2). Viral
fusion and infectivity were recovered by almost 100% in comparison
with the control (Fig. 4b, d and Table 2); viral binding was recovered
up to 70% (Fig. 4c). In sum, the recovery of viral activities following
cholesterol replenishment strongly supports the notion that the in-
hibitory effects of MβCD treatment on NDV activities would be due
specifically to cholesterol depletion per se, and that this effect
would be reversible.
The effect of MβCD treatment on the synthesis of viral proteins in
NDV-infected cells was also analyzed (Fig. 5). In these experiments,
Fig. 2. Effect of cellular cholesterol depletion on NDV binding and receptor expression.
(a) NDV at a moi of 5 was allowed to bind to MβCD-treated and -untreated cells for 1 h
at 4 °C. Then, the binding of NDV to target cells was analyzed by a FACS-based immu-
noassay (see Materials and methods). The negative control corresponds to cells in the
absence of virus without MβCD (red) or with MβCD (black). (b) 10 mM MβCD-treated
or -untreated ELL-0 cells were incubated either with 10 μg ml−1of FITC-MALI lectin or
with FITC-SNA lectin, and analyzed by FACSs. Negative control: cells without lectin nor
MβCD. (c) Untreated control cells, lovastatin-treated and MβCD-incubated cells were
stained with FITC-MALI lectin and analyzed under confocal microscopy as detailed in
Materials and methods.
Fig. 3. Effect of cholesterol removal from cells on NDV infectivity. (a) NDV at 5 mois
was allowed to bind to MβCD-treated and -untreated cells for 1 h at 4 °C, and was
then transferred to 37 °C. At 24 h post-infection, viral infectivity was analyzed by a
FACS-based immunoassay, probing with anti-F and anti-HN primary antibodies (see
Materials and methods). The negative control corresponds to cells in the absence of
virus with or without MβCD. (b) Effect of cholesterol depletion on the course of NDV
entry. Monolayers of ELL-0 cells were infected with a 10−4dilution of the recombinant
rNDV-F3aa-mRFP NDV, meaning a moi of 1. At different times post-infection, 10 mM of
MβCD and lovastatin were added for 1 h at 37 °C. After 24 h at 37 °C, infectivity was
quantified as the number of red fluorescent cells out of the total number of cells in a
fluorescent microscopy assay, and is referred to control untreated cells. Preincubation
data refer to cells incubated with 10 mM of MβCD for 1 h at 37 °C before virus infec-
tion. Data are means±SD of three independent experiments.
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
viral protein synthesis was assayed by immunoblot analysis using a
rabbit polyclonal anti-NDV antibody. ELL-0 cells were infected with
NDV at 5 mois. At 24 h post-infection, cell extracts were analyzed
by Western blot as detailed in Materials and methods. As can be
seen, in 10 mM MβCD-treated cells the synthesis of viral F and NP
proteins was reduced. The addition of cholesterol to MβCD-treated
cells prior to virus infection led to an increase in the synthesis of
viral proteins as compared to MβCD-treated ELL-0 cells. The levels
of cellular GAPDH were unaffected by MβCD treatment or virus
Fig. 4. Effect of cellular cholesterol replenishment on NDV activities. (a) Cholesterol-depleted cells were incubated with increasing concentrations of cholesterol for 1 h at 37 °C.
Cholesterol was extracted and quantified as described in Materials and methods. Data are expressed as percentages of total cholesterol with respect to untreated control cells.
Data are means±standard deviations of three independent experiments. (b) The fusion activity of R18-labeled NDV with replenished-cholesterol cells was analyzed by fluorescence mi-
croscopy,asdetailedinthe legendtoFig.1b.(c)Aftercellularcholesterolreplenishment,NDVatamoiof5was allowedtobindcellsfor1 hat4 °C.Then,thebindingofNDVtotargetcells
was analyzed by a FACS-based immunoassay as detailed in Materials and methods. The negative control corresponds to cells in the absence of virus. (d) Viral infectivity was analyzed by
infecting control, cholesterol-depleted and cholesterol replenished ELL-0 cells with 1 moi of the recombinant rNDV-F3aa-mRFP for 1 h at room temperature. After 24 h at 37 °C, the cells
were observed under an Olympus IX51 inverted fluorescence microscope with a 10× objective. Quantification of infectivity was accomplished by measuring the area of red fluorescence
(infected cells that express the RFP) in pixelsreferred to the area of nuclei (blue fluorescence)of the field inthree to seven random fields. Areas were quantified using the analysis tool in
Quantification of the effect of cholesterol replenishment on NDV activities.a
MβCD concentration+cholesterol concentration
10 mM 10 mM+100 μM 10 mM+200 μM
aData are percentages of activity from control untreated cells.
bMeans±SD of three or four independent experiments.
cData from experiments as those shown in Fig. 4d, means of two independent
Fig. 5. Effect of depletion and replenishment of cellular cholesterol on viral protein ex-
pression in NDV-infected ELL-0 cells. Cells were either untreated or treated with differ-
ent concentrations of MβCD for 1 h at 37 °C, after which they were incubated or not in
the presence of exogenous cholesterol for an additional hour at 37 °C and infected with
NDV at 5 mois. Total cell protein extracts were separated by SDS-PAGE and analyzed by
Western blot, using polyclonal anti-NDV antibodies. The viral protein band belonging
to F and NP is shown. As a control, the expression of cellular GAPDH was analyzed.
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
infection. These results indicated that the synthesis of viral proteins
was reduced in cholesterol-depleted cells, in support of the infectivity
inhibition data shown in Fig. 3. We observed a certain stimulatory
effect of protein expression in the presence of 200 μM exogenous cho-
lesterol, which correlates with some enhancement of viral infectivity
(Table 2). This enhancement might be related to the increase of
cholesterol in the cell membrane above normal values (Fig. 4a),
although further research is needed to clarify the significance of this
Since it is reported that MβCD-driven removal of cholesterol led to
a disruption of the cholesterol-rich DRMs of lipid rafts, we next
wished to determine whether NDV might localize to these membrane
microdomains during entry. We analyzed the association of viral pro-
teins with isolated DRMs from ELL-0 cells by treatment with TX100 at
low temperatures, followed by discontinuous OptiPrep flotation gra-
dients, as detailed in Materials and methods. As a control, caveolin,
a protein raft marker, was mainly present in the detergent-resistant
fractions 5 and 6, whereas β-tubulin was mainly found in the
detergent-soluble membrane fractions 11 and 12 (Fig. 6a). For some
samples, NDV was bound to cells for 1 h at 4 °C (Fig. 6a, HN 4 °C);
for other samples, after virus binding for 1 h at 4 °C, cells were shifted
to 37 °C for an additional hour (Fig. 6a, HN 4 °C/37 °C). Then, the cells
were lysed and layered at the bottom of a 40%–30%–5% OptiPrep den-
sity gradient. Fractions were collected from the top of the gradient
and analyzed by Western blotting, probing with a polyclonal anti-
NDV antibody. We found that after virus binding at 4 °C, HN was dis-
tributed in both the low density Triton-insoluble fractions 5 and 6 as
well as in the detergent-soluble fractions. Nevertheless, after virus–
cell samples shifting to 37 °C, the association of HN to raft fractions
was limited to fraction 6. These results were also corroborated in HeLa
cells, where HN protein was distributed in both the raft and non-raft
fractions (Fig. 6b, HN 4 °C/37 °C). After MβCD incubation of cells
and wasmainly associated withthe non-raft fractions. After thereplen-
ishment of cholesterol (Fig. 6b, HN 10 mM MβCD+200 μM Cho), the
distribution of HN in the flotation gradient fractions was as observed
in the case of the control cells (Fig. 6b HN 4 °C/37 °C).
Cholesterol and lipid raft domains have been implicated in several
stages of the life cycle of different animal virus families (reviewed in
[9,11]). The involvement of lipid rafts in viral assembly and budding
has been demonstrated mainly for enveloped viruses, including HIV
and influenza [9,47]. Additionally, cholesterol and lipid rafts have
been implicated in viral entry [9,10], a physical association between
viral glycoproteins and cellular membrane lipid rafts having been
reported [21,48]. For viruses that use caveolar/lipid raft endocytic
pathways (for a review of viral entry by endocytosis, see ), the de-
pendence of the viruses on cholesterol has been related to the integ-
rity of their portal of entry, i.e., lipid rafts.
In the present study, we have demonstrated that NDV requires cell
membrane cholesterol for optimal infection of ELL-0 cells. This is sup-
ported by several lines of evidence: i) MβCD treatment of cells par-
tially inhibited NDV binding, fusion and infectivity; ii) the effect of
cholesterol removal was reversible; iii) the effect of MβCD treatment
following NDV absorption was not significant, suggesting that choles-
terol must be essential during the initial virus entry stages; iv) choles-
terol depletion led to a reduction in viral protein synthesis in infected
cells; and v) the partial association of the HN NDV attachment protein
to lipid DRMs.
Among their different functions, membrane rafts exert a concen-
trating effect on proteins. Receptors and coreceptors of several virus-
es have been reported to be associated with lipid rafts on cell
membranes such, as CD4 for HIV-1 . During viral attachment, pro-
tein recruitment would facilitate protein interactions, leading to viral
entry either through a concentration of viral receptors in lipid rafts
and/or through a concentration of viral envelope proteins facilitating
the formation of the viral protein fusion scaffold . The depen-
dence of the binding of HIV-1 envelope gp120 glycoprotein to lipid
rafts on cholesterol strongly supports the idea that raft-colocalized
receptors would be directly involved in virus entry [50–52]. Similarly,
for NDV it could be speculated that rafts might act as a platform for
local concentrations of receptors and coreceptors, such as glycosphin-
golipids and/or glycoproteins. During the very early binding steps, a
percentage of HN would colocalize to lipid rafts (Fig. 6a, HN 4 °C)
shifting to non-raft fractions as the viral–cell interaction progresses
(Fig. 6a, HN 4 °C/37 °C). This could explain the negative effect of cho-
lesterol removal on NDV binding. The partial colocalization of viral
proteins to DRMs during binding suggests a weak or partial interac-
tion with rafts, as proposed for other viruses [21,22]. The different
patterns of lectin staining in control and cholesterol-depleted cells
suggest that a pool of sialic acid glycoconjugates recognized by NDV
might be associated with lipid rafts and that could be modified after
cholesterol removal, whereas sialic acid concentrations at the cell sur-
face would not be modified (Fig. 2). Surprisingly, the data presented
here differ from our previous data, which showed that most HN was
located in the DRM fractions after viral binding to COS-7 cells .
These differences might be cell-dependent, ELL-0 avian fibroblasts
being the natural viral host. Additionally, the formation and
Fig. 6. Distribution of NDV HN protein in DRMs. Membrane microdomains were isolat-
ed from ELL-0 (a) or HeLa (b) cells by OptiPrep density gradient centrifugation as de-
tailed in Materials and methods. Caveolin and β-tubulin were used as positive and
negative controls respectively for lipid raft association. (a) NDV alone, distribution of
HN protein of 5 mois of purified NDV; HN (4 °C) is from cells incubated with 25 mois
of NDV for 1 h at 4 °C; in HN (4 °C/37 °C), cells were incubated NDV for 1 h at 4 °C
and then shifted to 37 °C for 1 h. (b) In HN (4 °C/37 °C), cells were incubated with
NDV for 1 h at 4 °C and then shifted to 37 °C for 1 h; in HN (10 mM MβCD), prior to
viral infection the cells were treated with 10 mM MβCD; in HN (10 mM MβCD
+200 μM cholesterol), cells were incubated with 10 mM MβCD and then with
200 μM cholesterol prior to viral infection. HN was detected by Western blot probing
with an anti-NDV polyclonal antibody.
J.J. Martín et al. / Biochimica et Biophysica Acta 1818 (2012) 753–761
enlargement of a fusion pore requires the concerted action of several
viral proteins . Therefore, the disruption of lipid rafts might hin-
der the assembly of the viral envelope glycoproteins needed for the
formation of the fusion scaffold . In this sense, it has been pro-
posed that the increase in the antiviral potency of cholesterol-
tagged-peptides derived from the C-terminal heptad repeat regions
of viral fusion proteins, including those of paramyxoviruses, would
be related to the preconcentration or targeting of these peptides to
lipid rafts [55,56].
Cholesterol removal and/or lipid raft disruption negatively affect
the caveolae- and lipid raft-mediated endocytosis that is used as
pathway of entry by several viruses [57–59]. Cholesterol has also
been implicated as a regulatory factor in the entry of viruses that
use clathrin and non-clathrin non caveolar/lipid raft endocytic path-
ways . Recently, Melikyan's team  have demonstrated that
HIV productively infects cells via clathrin-mediated endocytosis,
which helps to explain the dependence of HIV on target membrane
cholesterol . Previously, we have reported the colocalization of
NDV with caveolin and with the early endosome marker EEA1, lead-
ing us to propose that a certain percentage of the virus manages to
penetrate the cell through caveolin-dependent endocytic pathways
. In this context, it is possible that removal of the cholesterol
could negatively affect NDV entry by altering endocytic pathways.
The absence of a total blockade of virus–cell fusion and infectivity
after MβCD treatment (Table 1), as well as the partial association of
HN with DRM fractions, may suggest that NDV entry would take
place in both raft and non-raft environments. Nevertheless, further
research into how the association of NDV with lipid rafts facilitates
viral entry is required. Our data indicate a stronger effect of cholester-
ol removal on NDV binding than on fusion (Table 1). Moreover, after
cholesterol replenishment, fusion and infectivity were almost
completely recovered, whereas binding was not (Table 2). Therefore,
it could be argued that some non-productive binding sites were per-
manently removed after MβCD treatment, whereas productive bind-
ing sites were recovered after cholesterol replenishment, allowing
viral fusion and infectivity to occur efficiently.
NDV entry might also depend on the presence of cholesterol not re-
lated to lipid rafts, but also on membrane fluidity; the stabilization the
local bilayer bending that takes place during membrane fusion ;
or on some specific requirementfor viral protein activities . In addi-
tion, raft association might be required for virus-induced signal path-
ways leading to virus entry and replication [63,64]. In Sendai virus,
another paramyxovirus, the role of AKT1 and Raf/MEK/ERK cascades
during viral fusion has been recently reported . Moreover, it has
been reported that cholesterol removal alters the actin cytoskeleton
, which might negatively affect viral interaction with cells since
cortical actin has been implicated in virus entry .
For some viruses, including paramyxoviruses , the depen-
dence on cholesterol did not appear to correlate with the interaction
with lipid rafts in the target membranes but with the cholesterol con-
tent of the viral envelope . Additionally, some viruses require cho-
lesterol in both viral and cell membranes . Cholesterol is the
major neutral lipid component of the NDV envelope present at a
high cholesterol/phospholipid ratio . Nevertheless, Morrison's
team  have reported that viral envelope cholesterol is not essen-
tial for NDV infectivity, whereas cellular lipid rafts have been impli-
cated in viral assembly and release.
In sum, our data show that cholesterol in the target membrane is
required for optimal NDV fusion and infectivity, suggesting that the
association of NDV proteins with lipid rafts might enhance viral
entry, although further studies are needed to understand the mecha-
nism(s) involved in the cholesterol dependence of NDV entry. A clear
understanding of the role of lipid membrane components in viral
entry will be useful for designing antiviral agents and vaccines.
Supplementary data to this article can be found online at doi:10.
This work was partially supported by grants from Junta de Castilla
y León (SA009A08) to I.M.B. and from the Fondo de Investigaciones
Sanitarias (FIS) (PI08/1813) cofinanced by FEDER funds from the EU
to E.V. We thank Dr. Adolfo García-Sastre for providing recombinant
rNDV-F3aa-mRFP NDV, and anti-HN and polyclonal anti-NDV anti-
bodies. Thanks are also due to N. Skinner for language corrections.
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