Download Newcastle disease virus may enter cells by caveolae

Journal of General Virology (2007), 88, 559–569
DOI 10.1099/vir.0.82150-0
Newcastle disease virus may enter cells by
caveolae-mediated endocytosis
Celia Cantı́n,34 Javier Holguera,3 Laura Ferreira, Enrique Villar
and Isabel Muñoz-Barroso
Correspondence
Isabel Muñoz-Barroso
Departamento de Bioquı́mica y Biologı́a Molecular, Universidad de Salamanca, Edificio
Departamental Lab. 108, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
[email protected]
Received 19 April 2006
Accepted 17 October 2006
The entry into cells of Newcastle disease virus (NDV), a prototype member of the paramyxoviruses,
is believed to occur by direct fusion at the plasma membrane through a pH-independent
mechanism. In addition, NDV may enter host cells by an endocytic pathway. Treatment of cells with
drugs that block caveolae-dependent endocytosis reduced NDV fusion and infectivity, the degree
of inhibition being dependent on virus concentration. The inhibitory effect was reduced greatly when
drugs were added after virus adsorption. Cells treated with methyl b-cyclodextrin, a drug that
sequesters cholesterol from membranes, reduced the extent of fusion, infectivity and virus–cell
binding; this indicates that cholesterol plays a role in NDV entry. Double-labelling
immunofluorescence assays performed with anti-NDV monoclonal antibodies and antibodies
against the early endosome marker EEA1 revealed the localization of the virus in these intracellular
structures. Using fluorescence microscopy, it was found that cell–cell fusion was enhanced at low
pH. It is concluded that NDV may infect cells through a caveolae-dependent endocytic pathway,
suggesting that this pathway could be an alternative route for virus entry into cells.
INTRODUCTION
Newcastle disease virus (NDV) is one of the causes of avian
respiratory diseases and economic losses in the poultry
industry. It is a member of the family Paramyxoviridae,
which includes enveloped, negative-stranded RNA viruses
that encode two transmembrane glycoproteins: an attachment protein, HN, and a fusion protein or F protein. Virus
adsorption occurs at the surface of the host cell membrane.
The viral attachment protein HN recognizes and binds to
sialic acid-containing molecules such as glycoproteins and
glycolipids (Ferreira et al., 2004) and has neuraminidase
and fusion-promoting activities. Following binding, the F
protein promotes fusion between the viral and cellular
membranes and the viral genome is delivered into the
cytoplasm.
Enveloped viruses enter the cell through two main pathways:
direct fusion between the viral envelope and the plasma
membrane; and receptor-mediated endocytosis. In the case
of paramyxoviruses, it has been established that the
membrane fusion process takes place at the host plasma
membrane in a pH-independent manner. Despite this, it has
been shown previously that fusion of NDV with cultured
cells is enhanced at acidic pH (San Roman et al., 1999),
3These authors contributed equally to this work.
4Present address: Estacion Experimental de Aula Dei, CSIC, Carretera
de Montaña 1005, 50059 Zaragoza, Spain.
0008-2150 G 2007 SGM
leading us to propose that NDV may also penetrate the cell
via an endocytic pathway in a pH-dependent process.
After binding of an enveloped virus to its cognate receptor at
the cell surface, membrane fusion is responsible for delivery
of the nucleocapsid into the cytoplasm. Receptor binding
and low pH have been considered to be the two main
triggering mechanisms responsible for conformational
changes in viral envelope glycoproteins leading to fusion.
Also, it has been shown that in the recently emerged
paramyxoviruses Nipah virus and Hendra virus, activation
of the F protein is accomplished by proteolysis after
endocytic entry (Diederich et al., 2005; Meulendyke et al.,
2005), similarly to the activation of the Ebola virus
glycoprotein (Chandran et al., 2005) and the spike
glycoprotein of severe acute respiratory syndrome coronavirus (Simmons et al., 2005). For pH-independent viruses
such as retroviruses or paramyxoviruses, activation of the
fusion protein occurs through interaction of the viral
glycoproteins with the receptor (Eckert & Kim, 2001). For
pH-dependent viruses such as togaviruses, rhabdoviruses,
orthomyxoviruses, alphaviruses and flaviviruses, low pH
triggers conformational changes after internalization into
endosomes (Lamb, 1993; Marsh & Helenius, 1989; White
et al., 1983). Recently, a new activation mechanism has
emerged, as described for the retrovirus avian sarcoma and
leukosis virus, in which the trigger needs a combination of
both mechanisms: an initial binding to receptors at neutral
pH, which then renders the viral glycoprotein sensitive to
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Printed in Great Britain
559
C. Cantı́n and others
low pH activation (Matsuyama et al., 2004; Melikyan et al.,
2004; Mothes et al., 2000).
The endocytic pathways used by animal viruses to enter host
cells include macropinocytosis, phagocytosis, the clathrinand caveolae-dependent pathways and some less wellcharacterized routes called the non-clathrin-, non-caveolaedependent pathways (Conner & Schmid, 2003; Pelkmans &
Helenius, 2003; Sieczkarski & Whittaker, 2002a; Marsh &
Helenius, 2006). Clathrin coats are known to be involved in
the entry of numerous enveloped and non-enveloped
viruses, e.g. the alphaviruses Semliki Forest virus and
Sindbis virus (DeTulleo & Kirchhausen, 1998; Marsh &
Helenius, 1980), rhabdoviruses such as vesicular stomatitis
virus (Matlin et al., 1982; Sun et al., 2005), influenza viruses
(Matlin et al., 1981; Roy et al., 2000), adenoviruses (Bartlett
et al., 2000), parvoviruses (Parker & Parrish, 2000), JC
polyomavirus (Pho et al., 2000), picornaviruses (Parker &
Parrish, 2000; Joki-Korpela et al., 2001) and bunyaviruses
(Jin et al., 2002). Caveolae are small, flask-shaped
invaginations in the plasma membrane with high levels of
cholesterol and glycosphingolipids and the integral membrane protein caveolin. These structures are used for the
internalization of viruses such as simian virus 40 (SV-40)
(Anderson et al., 1996; Pelkmans et al., 2001). The
picornavirus echovirus 1 (Marjomaki et al., 2002), Ebola
virus (Empig & Goldsmith, 2002) and BK polyomavirus
(Eash et al., 2004) have also been reported to enter via
caveolae. It has been proposed that murine polyomavirus
enters cells through non-clathrin-, non-caveolae-dependent
pathways (Gilbert & Benjamin, 2000). Additionally, the
human enterovirus EV-11 uses a novel entry pathway that is
dependent on lipid rafts (Stuart et al., 2002), and rotaviruses
may infect cells in a cholesterol- and dynamin-dependent,
but clathrin- and caveolae-independent manner (SanchezSan Martin et al., 2004). In addition to clathrin-dependent
entry, the internalization of influenza virus through a nonclathrin-dependent pathway has also been reported
(Sieczkarski & Whittaker, 2002b).
The acidic pH activation of the fusion activity of NDV and
the partial blocking effect of lysosomotropic agents on NDV
fusion have been reported previously (San Roman et al.,
1999). In the present study, the suggestion that endocytosis
might play a role in NDV entry is addressed directly.
Different approaches were used to dissect the entry
mechanism of NDV, i.e. utilization of drugs that specifically
disrupt the clathrin- or caveolae-dependent pathways and
studies of the co-localization of NDV with markers of the
endosomal network. In addition to pH-independent fusion
with the plasma membrane, our results seem to point to the
use of the caveolae-endocytic route by NDV, which delivers
virus particles to early endosomes.
METHODS
Reagents and antibodies. Chlorpromazine, phorbol 12-myristate
13-acetate (PMA), methyl b-cyclodextrin (MbCD) and nystatin were
from Sigma. CMFDA, CMPTX, octadecylrhodamine B chloride
560
(R18), Hoechst 33258, Alexa fluor 488 donkey anti-mouse IgG and
Alexa fluor 568 rabbit anti-goat IgG were from Molecular Probes.
Monoclonal antibodies against NDV HN protein (anti-HN mAb)
and against respiratory syncytial virus (RSV) F protein (22A4) were
a generous gift from A. Garcı́a-Sastre (Mount Sinai School of
Medicine, New York). Goat polyclonal anti-EEA1 antibody was
from Santa Cruz Biotechnology.
Cells and viruses. The lentogenic ‘Clone 30’ strain of NDV was
obtained from Intervet Laboratories. Virus was grown and purified
mainly as described previously (Garcia-Sastre et al., 1989). COS-7,
HeLa and Vero cells were obtained from the ATCC and maintained
in Dulbecco’s modified Eagle medium (DMEM) supplemented with
21
21
L-glutamine (580 mg l ), penicillin-streptomycin (100 U ml –
100 mg ml21) and heat-inactivated fetal bovine serum (FBS; 10 %
for COS-7 and HeLa cells and 5 % for Vero cells).
NDV infection in cell culture and syncytia assays. At 12–24 h
after plating, cells were infected with NDV at an m.o.i. of 1 in
DMEM containing 2 % FBS. The virus was left to be adsorbed for
1 h at 37 uC. At 12 h post-infection (p.i.), trypsin digestion of the
precursor F0 was accomplished as described previously (Young et al.,
1997) and cells were incubated for an additional 4–12 h. Cells were
then fixed with methanol and stained with Giemsa. The number of
syncytia (cells containing more than three nuclei) was determined in
20 random areas of the well.
Virus titration. Virus titres were calculated in plaque formation
assays in Vero cells as described previously (San Roman et al.,
2002). Plaque numbers were counted under a light microscope;
NDV infectivity was expressed as p.f.u. ml21.
Effect of drugs on NDV fusion and infection. At 12–24 h after
plating, cells were pretreated with various concentrations of drugs
(MbCD for 30 min and PMA, nystatin or chlorpromazine for
45 min). Cells were then infected as described above with NDV at
different m.o.i. Drugs were maintained in the cultures throughout
all incubation periods or removed before virus infection. Drugs were
added after virus adsorption where stated. Cell viability was determined by Trypan blue exclusion. Viabilities of cells treated with
PMA (10 mM) and nystatin (10 mg ml21) were greater than 90 and
82 % for 45 min and 24 h drug treatment, respectively; those for
MbCD (5 mM) were greater than 90 and 64 % for 30 min and 12 h
drug treatment, respectively. For chlorpromazine, about 90 % cell
viability was observed after 45 min with 5 mg chlorpromazine ml21.
Owing to the notorious toxicity of this drug, longer periods of
chlorpromazine treatment were not considered.
Virus binding assays. Purified NDV was labelled with the fluores-
cent probe octadecylrhodamine (R18) essentially as described previously (Hoekstra et al., 1984; San Roman et al., 1999). R18-labelled
NDV (R18-NDV; 20 mg) was allowed to bind to 3.56106 cells at
4 uC in 400 ml PBS, pH 7.4, over 20 min, stirring periodically. After
incubation, unbound virus was removed by centrifugation at 1000 g
for 5 min at 4 uC. The distribution of R18 between the cell pellet
and supernatant, corresponding to bound and free virus, respectively, was determined after the addition of Triton X-100 at a final
concentration of 1 % (v/v).
Immunofluorescence labelling and confocal microscopy.
HeLa cells grown on glass coverslips were untreated or pretreated
with drugs at the stated concentrations as described above. Cells
were then infected with NDV or RSV at an m.o.i. of 10 for 1 h. At
30 min p.i. (confocal assays) or 15 h p.i. (infectivity assays), cells
were washed three times with PBS++ (PBS containing 1 mM
MgCl2 and 0.1 mM CaCl2) and fixed in 2.5 % paraformaldehyde for
30 min. Following this, cells were washed three more times with
PBS++ and permeabilized with 0.1 % Triton X-100 for 10 min.
Cells were then incubated in blocking solution (PBS++ containing
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Journal of General Virology 88
Endocytic entry of NDV into host cells
1 % BSA) for 30 min, after which they were incubated with primary
antibody for 1 h (anti-HN mAb, dilution 1 : 200; goat anti-EEA1,
dilution 1 : 100). After washing three times with PBS++, cells were
incubated with the appropriate Alexa fluor-labelled secondary antibody for 45 min [Alexa fluor 488 donkey anti-mouse (1 mg ml21) or
Alexa fluor 568 rabbit anti-goat (10 mg ml21)]. Nuclei were stained
with Hoechst 33258 (10 mg ml21). After washing with blocking solution and a short rinse in ultrapure water, cells were mounted in
Mowiol and viewed with a confocal microscope (Zeiss LSM 510)
equipped with a 488 nm argon laser and a 543 nm helium-neon
laser (636 objective lens for co-localization assays and 406 objective lens for infectivity assays).
Cell–cell fusion assays. Target cells (non-infected COS-7 cells)
were labelled with the cytoplasmic dye CMTPX (red emission; excitation/emission 577/602 nm) at a concentration of 10 mM for
45 min at 37 uC. At 12 h p.i., effector cells (NDV-infected COS-7
cells) were labelled with the cytosolic probe CMFDA (green emission; excitation/emission 492/517 nm) at a concentration of 10 mM
for 45 min at 37 uC. After labelling, the two cell populations were
mixed in the ratio 3 : 1 (effector : target) in complete medium and
co-cultured for 3 h at 37 uC. The F0 precursor was then proteolytically activated as described above and, immediately after, cells were
incubated in PBS at different pH values for 1, 2, 5 or 10 min. The
pH was returned to around neutral with complete medium and cells
were incubated for an additional 2 h at 37 uC. Dye distribution was
then monitored microscopically using an inverted Olympus IX51
microscope with the U-MNG2 filter cube for rhodamine probes and
the U-MNB2 filter cube for fluorescein probes. The extent of fusion
was calculated as the mean of a and b, where a is 1006(number of
positive cells for both dyes/total number of cells positive for
CMTPX) and b is 1006(number of positive cells for both dyes/total
number of positive cells for CMFDA).
RESULTS
Effect of inhibitors of endocytosis on NDV
infectivity, fusion and binding
To characterize further the entry mechanism of NDV into
host cells, the effect of several inhibitors of clathrin- or
caveolae-dependent endocytosis on NDV infectivity and
fusion was analysed. Cells were pretreated in the presence of
several concentrations of different drugs: PMA, an inhibitor
of caveolae-mediated endocytosis that acts by constitutively
phosphorylating caveolin (Anderson, 1993); nystatin, a
steroid-binding antibiotic that is also used to inhibit
caveolae uptake since cholesterol is important for
caveola maintenance and dynamics (Anderson, 1993;
Lisanti et al., 1993; Rothberg et al., 1990); and MbCD, a
cholesterol-sequestering agent that depletes cholesterol in
cell membranes, thus preventing the formation of both
clathrin-coated vesicles and caveolae (Rodal et al., 1999;
Subtil et al., 1999). The cationic amphiphilic compound
chlorpromazine, which prevents the recycling of clathrin
(Hunt & Marshall-Carlson, 1986; Wang et al., 1993), was
used as a specific inhibitor of clathrin-mediated endocytosis.
As can be seen from the data in Fig. 1, pre-incubation of cells
with PMA, nystatin or MbCD elicited a reduction in virus
infectivity. This decrease was detected when the drugs were
present throughout the incubation periods (Fig. 1a) and
when they were removed before virus infection (Fig. 1b),
http://vir.sgmjournals.org
pointing to a specific effect of the drugs. Moreover, in some
drug-treated cells, virus bound to the cell surface without
entering the cell. However, when cells were treated with
chlorpromazine, no decrease in virus infectivity was detected
(Fig. 1b). Additionally, the reduction in virus infectivity was
quantified with a plaque reduction assay in Vero cells, as
detailed in Methods. Cells treated with PMA (10 mM) and
nystatin (10 mg ml21) showed a reduction of about 70 % in
virus infectivity compared with the controls, whereas NDV
infectivity in cells treated with MbCD (10 mM) was reduced
by about 54 % (data not shown). These data were confirmed
by those relating to the effect of nystatin, PMA and MbCD on
virus fusion. Syncytium formation between NDV-infected
cells was inhibited in COS-7 cells (Fig. 2a) and HeLa cells
(Fig. 2b). Additionally, the drug effect was dose-dependent.
Nystatin and PMA showed the highest degree of syncytium
reduction in COS-7-infected cells (Fig. 2a), which was
similar to the effect observed with nystatin in HeLa-infected
cells (Fig. 2b). Nevertheless, fusion was reduced to a greater
extent by PMA in COS-7-infected cells than in HeLa cells.
The inhibitory effect was greatly reduced when drugs were
added after virus adsorption (Fig. 2a, indicated by asterisks),
thus confirming their effect on virus entry. Unlike data
reported for other viruses that enter cells mainly by
endocytosis, none of the drugs studied here completely
abolished NDV fusion or infectivity at the concentrations
assayed. To discard some other drug effects in the cells due to
long incubation periods, syncytium formation in NDVinfected HeLa cells was analysed when drugs were removed
before virus infection. For PMA and MbCD, similar levels of
syncytium inhibition were detected, whereas the degree of
inhibition by nystatin was slightly reduced (data not shown).
Moreover, cell–cell fusion induced by the paramyxovirus
RSV was not abolished by PMA or nystatin treatment of
HeLa cells: pretreatment of cells with 10 mM PMA or 10 mg
nystatin ml21 elicited 0 and 8 % fusion inhibition,
respectively, as assessed by a syncytium assay (Fig. 3).
These results confirm the specific effect of the drugs.
To test whether the inhibitory effect of the drugs was affected
by virus concentration, COS-7 cells were infected with NDV
at different concentrations (m.o.i. of 0.1, 1 and 10). Results
(Fig. 2c) show that NDV fusion was inhibited by the drugs at
the three virus concentrations assayed. Nevertheless, the drug
effect was less pronounced at the lowest virus concentration,
eliciting approximately 20–30 % fusion inhibition compared
with more than 50 % fusion inhibition at m.o.i. of 1 and 10,
with almost no difference between the two highest virus
concentrations studied. These results suggest that NDV might
use endocytic routes of entry more extensively when larger
amounts of virus are present, suggesting that endocytosis is a
less specific route of entry (DeTulleo & Kirchhausen, 1998;
Stuart et al., 2002) and, hence, that other routes are available
for the virus. Nevertheless, endocytosis might be used at low
virus concentrations, i.e. m.o.i. of 0.1.
The extent of NDV binding to COS-7 cells previously
treated with PMA, nystatin, MbCD and chlorpromazine was
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
561
C. Cantı́n and others
(a)
Hoechst
NDV-HN
Merge
(b)
No drug
No drug
PMA
(10 mM)
PMA
(10 mM)
Nystatin
_
(10 mg ml 1)
Nystatin
_
(10 mg ml 1)
MbCD
(5 mM)
MbCD
(10 mM)
Hoechst
NDV-HN
Merge
Chlorpromazine
_
(10 mg ml 1)
Fig. 1. Effect of several inhibitors of endocytosis on NDV infectivity. (a) At 15 h p.i., cells were treated for
immunofluorescence as detailed in Methods. Indirect immunofluorescence infection (NDV-HN) along with Hoechst 33258
counter-stain for the cell nuclei is shown. Drugs were maintained in the cultures throughout all incubation periods. (b) Cells
were pretreated with 10 mM PMA, 10 mg nystatin ml”1, 10 mM MbCD or 10 mg chlorpromazine ml”1 for 30 min (MbCD) or
45 min (PMA, nystatin and chlorpromazine) and then cells were treated as in (a), except that the drug was removed before
virus infection.
determined using the spectrofluorimetric assay described in
Methods. As can be deduced from the data summarized in
Table 1, pretreatment of cells with PMA and nystatin did
not significantly modify the degree of NDV binding to cells,
meaning that the inhibition shown in Figs 1 and 2 was not
exerted on the virus–cell binding step. Nevertheless, preincubation of cells in the presence of 10 mM MbCD led to a
significant reduction in the extent of virus–cell binding,
suggesting an important role for cholesterol in virus–cell
binding.
Co-localization of NDV and the endocytic
pathways
To provide further confirmation that NDV may enter the
host cell via endocytic pathways, immunofluorescence
labelling with antibodies against the HN protein of NDV
and the early endosomal antigen 1 marker protein EEA1 was
used together with confocal microscopy. After 30 min
of infection, a punctate staining pattern with a strong
562
co-localization of HN protein and EEA1 was found, thus
confirming that HN is targeted to early endosomes. (Fig. 4a,
no drug). No co-localization was observed when cells were
pretreated with PMA, nystatin or MbCD, although NDV
HN co-localized with EEA1 after chlorpromazine treatment
(Fig. 4a). Co-localization of RSV and EEA1 was not detected
at 30 min p.i. (Fig. 4b).
Effect of pH on NDV-induced cell–cell fusion
Using the spectrofluorimetric technique of R18 dequenching, it has been shown previously (San Roman et al., 1999)
that the fusion of NDV with COS-7 cells is enhanced at
acidic pH. This effect was confirmed using fluorescence
microscopy techniques. The pH dependence of cell–cell
fusion induced by NDV was studied. For this, NDV-infected
COS-7 cells were used as effector cells and non-infected
COS-7 cells were used as target cells. Effector cells were
labelled with the green cytoplasmic fluorescent probe
CMFDA, whereas target cells were labelled with the red
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Journal of General Virology 88
Endocytic entry of NDV into host cells
cytosolic fluorescent probe CMTPX, as described in
Methods. Following this, the two populations of cells were
mixed and pre-incubated at different pH values for the
indicated times, with an additional 2 h incubation in
complete medium (pH 7.4) at 37 uC. Fusion was monitored
by aqueous dye transfer redistribution analysis with
fluorescence microscopy, scoring those cells showing both
red and green fluorescence as fusion-positive events. As seen
in Fig. 5, pre-incubation for 5 min at low pH increased the
number of fusion-positive events, pointing to activation at
acidic pH. The time dependence of activation at acidic pH is
shown in Fig. 6. Data are from images similar to those shown
in Fig. 5. Fusion is expressed as the number of fusionpositive events over the total number of cells averaged in ten
microscopic fields, as detailed in Methods. At the three times
assayed, pre-incubation of cells at acidic pH elicited an
increase in fusion of more than 20 % compared with preincubation at neutral pH. Additionally, a slight increase in
fusion was detected between 1 and 5 min incubation at both
acidic and neutral pH values. These data confirm the
activation of NDV-triggered membrane fusion at acidic pH.
DISCUSSION
Fig. 2. Effect of several inhibitors of endocytosis on NDV
fusion. COS-7 cells (a) or HeLa cells (b) were pretreated with
PMA, nystatin (Nys.) or MbCD as in the legend to Fig. 1 and
then infected with NDV at an m.o.i. of 1 under the continuous
presence of the drug. At 18–24 h p.i., cells were fixed and
Giemsa-stained; the extent of fusion inhibition was expressed
as the percentage reduction in the number of syncytia from the
control (untreated cells). Data are means±SD of four independent experiments. For data labelled with asterisks (*) in (a),
drug was added after virus adsorption; these data are means±range of values from two independent experiments. (c)
COS-7 cells were pretreated with PMA (5 mM), nystatin
(5 mg ml”1) or MbCD (10 mM) as outlined in the legend to
Fig. 1 and then infected with NDV at m.o.i. of 0.1, 1 or 10
under the continuous presence of the drug. The extent of
fusion inhibition was calculated as described for (a) and (b).
Data are means±SD of three independent experiments.
http://vir.sgmjournals.org
Viruses need to exploit the host cell machinery for their
replication and propagation. Although it has been established that the paramyxovirus envelope, including that of
NDV, fuses directly with the plasma membrane, it has been
suggested previously that, in addition to this route, NDV
might penetrate the cell by endocytosis (San Roman et al.,
1999). The aim of the present work was to investigate this
issue further and to address the endocytic mechanism(s) of
NDV entry using pharmacological agents and co-localization assays. NDV binding, fusion and infection were studied
in the presence of drugs known to inhibit particular
endocytic routes. The caveolae-pathway inhibitors tested,
PMA, nystatin and MbCD, exerted a partial blockade of
NDV infectivity and fusion (Figs 1 and 2), suggesting
involvement of the caveolae pathway in NDV entry and the
existence of different simultaneous entry pathways.
Moreover, when drugs were added after virus adsorption,
the inhibitory effect was greatly reduced (Fig. 2a, indicated
by asterisks), thus confirming that the drug effect was
mainly exerted in the early steps of virus–cell interactions.
The lack of an inhibitory effect on NDV infectivity exerted
by the clathrin pathway-disturbing drug chlorpromazine
(Fig. 1), together with the absence of the blockage of NDV
transfer to endosomes after chlorpromazine treatment
observed in our co-localization assays (Fig. 4a), suggest
that NDV does not enter cells via clathrin-mediated
endocytosis. Since chlorpromazine is highly toxic, it was
not possible to have long periods of incubation with this
drug. Accordingly, further experiments should be performed to assess the absence of the role of clathrin in the
entry of NDV into cells.
Our binding experiments revealed that the inhibitory
effect on NDV fusion and infectivity of the caveolae
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
563
C. Cantı́n and others
NDV
RSV
No virus
No drug
Nystatin
Fig. 3. Effect of nystatin and PMA on NDVand RSV-induced cell–cell fusion. COS-7
cells (NDV) or HeLa cells (RSV) were pretreated with 10 mM PMA or 10 mg nystatin
ml”1 as described in the legends to Figs 1
and 2 and then infected with NDV or RSV
at an m.o.i. of 1 under the continuous presence of the drug. At 24 h p.i., cells were
fixed and Giemsa-stained for syncytia analysis. Arrows indicate syncytia.
PMA
pathway-disturbing drugs nystatin and PMA took place at a
post-binding stage (Table 1). Nevertheless, when COS-7
cells were treated with the cholesterol-depleting agent
MbCD, the binding of NDV to cells was considerably
reduced (Table 1), in contrast to the data reported for other
viruses such as poliovirus (Danthi & Chow, 2004) and the
effect of the sterol-binding antibiotic nystatin reported here
(Table 1). This suggests that cell-membrane cholesterol is
involved in virus–cell interactions at the cell surface, in
addition to the interference in the endocytic pathway
discussed above. As a possible explanation, NDV receptors
might be associated with cholesterol-rich plasma membrane
microdomains, such as lipid rafts. Alterations to such
structures after drug treatment might modify the accessibility of receptor molecules to the virus or may affect their
native conformation. Moreover, modifications of cholesterol levels could alter bilayer fluidity and/or the integrity of
564
membrane lipid microdomains, hindering lateral diffusion
within the membrane (Danthi & Chow, 2004) and,
consequently, recruitment of the viral receptors and coreceptors needed for optimum virus–cell binding.
The use of endocytic pathways in NDV entry was further
assessed by direct examination by confocal microscopy
(Fig. 4a), which confirmed our data concerning drug action
on NDV fusion and infectivity discussed above. NDV colocalized with the early endosome marker EEA1 at
30 min p.i. (Fig. 4a, no drug). The absence of co-localization of HN protein with EEA1 after treatment with caveolae
pathway-disturbing drugs further supports the idea that the
drug inhibitory effect shown here was related to endocytic
entry of the virus. These data suggest that, after internalization, virus particles would be trafficked through early
endosomes.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Journal of General Virology 88
Endocytic entry of NDV into host cells
Table 1. Effect of inhibitors of endocytosis on NDV binding
to COS-7 cells
Control, untreated COS-7 cells. Data are means±SD of three independent experiments.
Inhibitor
Control
PMA (10 mM)
Nystatin (10 mg ml21)
MbCD (10 mM)
Chlorpromazine (10 mg ml21)
NDV binding (%)
43.3±4.1
40.3±2.4
37.7±2.1
6.3±1.5*
35.3±3.2
*P<0.001 (paired t-test).
In summary, our results suggest that NDV may use the
caveolae-mediated endocytic pathway. Additionally, it may
be possible that NDV uses other non-clathrin-, noncaveolae-mediated endocytosis routes. As summarized in
the Introduction, for most viruses that penetrate the cell
through endocytosis, the use of only one specific endocytic
pathway has been described, clathrin- or caveolae-dependent. Data are now emerging that support the notion of
viruses using more than one pathway for entry. The
enterovirus EV-11 uses both the clathrin and caveolae
pathways at high m.o.i. (Stuart et al., 2002); in addition to
clathrin-coated pits, influenza virus may penetrate the cell
through undetermined non-clathrin-, non-caveolae-dependent pathways (Sieczkarski & Whittaker, 2002b) or caveolar
endocytosis (Nunes-Correia et al., 2004). SV-40, which
represents the prototype of caveolar endocytosis entry, may
use an alternative route of entry in the absence of caveolin, in
a pathway independent of clathrin and dynamin (Damm
et al., 2005). Epstein–Barr virus (Miller & Hutt-Fletcher,
1992), herpes simplex virus (Nicola et al., 2003; Milne et al.,
2005), human immunodeficiency virus (Daecke et al., 2005)
and Vaccinia virus (Townsley et al., 2006) enter the cell
through direct fusion at the plasma membrane or through
endocytosis, in most cases in a cell-specific manner. For
NDV, our data seem to point to the existence of more than
one active entry pathway into a single cell type, i.e. direct
fusion with the plasma membrane and caveolae-mediated
endocytosis. The type of pathways exploited may be
explained in terms of the use of different virus receptors
(Sieczkarski & Whittaker, 2002b). It has been shown
previously that NDV binds to different sialic acid
compounds such as gangliosides and N-glycoproteins
(Ferreira et al., 2004). The presence of two different sialic
acid-binding sites in the NDV receptor-binding protein HN
has been reported (Zaitsev et al., 2004). The pliable catalytic
site, site I, exhibits both neuraminidase and receptorbinding activities, whereas the second binding site, site II,
shows only receptor-binding activity. Mutations in site II
have revealed that it is not essential for virus infection, but
enhances it (Bousse et al., 2004). Certain molecules that
interact with HN site I would activate site II and promote
continued receptor binding, although binding to site I does
http://vir.sgmjournals.org
not imply binding to site II (Porotto et al., 2006). The case
may be that, once activated, site II would recognize specific
receptors for endocytosis. It could also be speculated that a
population of site II would bind ligands in the absence of
binding to site I, leading to endocytic entry. Future work will
address this issue. Another interesting possibility is that
endocytic entry might be governed through interaction of
the F protein with the cell surface independently of the HN
protein (Sergel et al., 2000; Russell et al., 2001; Cobaleda
et al., 2002).
Structurally, the fusion proteins of enveloped viruses have
been classified into at least two classes: class I proteins,
whose prototype is influenza virus HA protein; and class II
proteins, whose prototype is the tick-borne encephalitis
virus glycoprotein E (Jardetzky & Lamb, 2004; Schibli &
Weissenhorn, 2004; Kielian & Rey, 2006). The fusion
glycoproteins of paramyxoviruses belong to class I. The
triggering of class I fusion proteins can be accomplished by
lowering the pH (for pH-dependent viruses) or by binding
to receptor(s) (for pH-independent viruses). Recently, a
third type of activation mechanism has been proposed that
combines both of the above mechanisms: an initial
triggering of the protein by receptor binding at neutral
pH, followed by acidic pH activation (Earp et al., 2003;
Mothes et al., 2000; Barnard et al., 2004; Matsuyama et al.,
2004). Additionally, pH-independent viruses that have been
thought to fuse directly at the plasma membrane may be
internalized through a pH-independent endocytic pathway
(Daecke et al., 2005). Consequently, the pH-independent
classification of some viruses might well deserve reappraisal.
Here, it has been shown that fusion between NDV-infected
and non-infected cells occurs both at acidic and neutral pH
(Figs 5 and 6), although a greater degree of fusion is
observed at acidic pH. It has been reported previously (San
Roman et al., 1999) that incubation of cells with the
lysosomotropic weak base ammonium chloride partially
prevents NDV from fusing with COS-7 cells. This observation is consistent with pH-dependent virus fusion along the
endocytic route. In the present work, the pre-incubation of
cells at acidic pH (Fig. 6) elicited an increase in fusion of
about 20 % compared with controls. This activation
occurred at least 1 min after exposure to acidic pH. In
addition to NDV, the SER paramyxovirus has a pHdependent entry mechanism (Seth et al., 2003), exhibiting
fusion at acidic pH but, unlike NDV, not at neutral pH.
Additionally, cleavage activation of the F protein of the
recently emerged Nipah virus and Hendra virus occurs after
internalization of the protein by clathrin-mediated endocytosis (Diederich et al., 2005; Meulendyke et al., 2005).
Since low pH does not seem to be a strict requirement for
NDV fusion, acidic pH activation would mean the existence
of an alternative mechanism to neutral pH activation, as
proposed previously (San Roman et al., 1999). According to
our hypothesis, NDV fuses at both neutral and acidic pH,
depending on the route of entry. At neutral pH, some viruses
bound to the cell surface could become endocytosed and
then fusion-activated in the endosome compartments.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
565
C. Cantı́n and others
Fig. 4. Co-localization of NDV and RSV
with the early endosome marker EEA1. (a)
Untreated cells (no drug) or cells treated
with different drugs were simultaneously
probed with anti-HN mAb and anti-EEA1
goat antibody at 30 min p.i. with NDV (m.o.i.
of 10). Bound antibody was traced with
Alexa fluor 488 donkey anti-mouse (left) or
Alexa fluor 568 rabbit anti-goat (middle).
Cells were examined using a confocal microscope as described in the text. (b) Cells
infected with RSV at an m.o.i. of 10 were
simultaneously
probed
with
anti-F
22A4 mAb and anti-EEA1 goat antibody at
30 min p.i. and examined by confocal microscopy as described above.
Several possibilities may be invoked to explain the detected
enhancement of NDV fusion at acidic pH (Figs 5 and 6).
Thus, it could be argued that triggering of the conformational changes required for fusion might be accelerated at
low pH. This would imply that HN fusion promotion
activity (Porotto et al., 2003) and/or the population of F that
is activated independently of HN (Sergel et al., 2000; Russell
et al., 2001; Cobaleda et al., 2002) would be enhanced once
the endocytic compartments have been reached. Since
566
paramyxovirus neuraminidases have an acidic optimum pH
(4.8–5.5) (Ryan et al., 2006), an acidic pH in the endosome
would enhance the neuraminidase activity of HN at site I and
also, hypothetically, the conformational changes of HN
required for site II to be activated (Porotto et al., 2006). Since
site II exhibits greater receptor avidity than site I (Porotto
et al., 2006), the increase in virus–receptor contact would be
responsible for the higher extent of fusion observed when the
pH is lowered experimentally (Figs 5 and 6).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Journal of General Virology 88
Endocytic entry of NDV into host cells
CMTPX
CMFDA
Merge
pH 7.4
pH 5
Fig. 5. Effect of pH on NDV-mediated cell–cell fusion. Effector
cells (NDV-infected COS-7 cells) and target cells were labelled
with the green fluorescent probe CMFDA and the red fluorescent probe CMPTX, respectively. The two cell populations were
mixed in a proportion of 3 : 1 (effector : target) and co-cultured
for 3 h at 37 6C. F protein was then activated proteolytically as
described in the text. Immediately after activation, cells were
incubated in PBS at pH 5.0 or 7.4 for 5 min. The pH was then
returned to around neutral with complete medium and cells
were incubated for an additional 2 h at 37 6C. Images were
collected using a 406 objective and dye distribution was monitored under an inverted microscope. Rhodamine (left), FITC
(middle) and merge (right) images are shown. Fusion-positive
events (cells labelled with both dyes) are marked (*) in the
merge field.
Fig. 6. Time dependence of the effect of pH on NDV-mediated
cell–cell fusion. Effector and target cells were labelled and cocultured as described in the legend to Fig. 5. After protein F
activation, cells were incubated in PBS at pH 5.0 (filled bars)
or pH 7.4 (open bars) for 1, 2 or 5 min. pH was returned to
around neutral with complete medium and cells were incubated
for an additional 2 h at 37 6C. Dye distribution was monitored
microscopically and the extent of fusion was scored as
described in the text. Data are means±range of values from
two independent experiments.
REFERENCES
Anderson, R. G. (1993). Caveolae: where incoming and outgoing
messengers meet. Proc Natl Acad Sci U S A 90, 10909–10913.
The general conclusion of our work is that NDV would be
able to enter and infect cells through different pathways; in
addition to direct fusion with the plasma membrane, a
certain percentage of virions would be endocytosed to
endosomes, where fusion would occur. Our concept of virus
entry is becoming increasingly more complex and why and
how viruses are able to use different pathways remain open
questions. Viruses could have adapted to the multiple
mechanisms and pathways that cells have for internalizing
different molecules, exploiting different entry routes.
Moreover, the same virus may have different preferences,
depending on the cell line. Knowledge of virus entry is
crucial for the development of antiviral agents, as well as for
the use of viruses as tools in gene therapy to deliver genes
to cells.
Anderson, H. A., Chen, Y. & Norkin, L. C. (1996). Bound simian virus
40 translocates to caveolin-enriched membrane domains, and its
entry is inhibited by drugs that selectively disrupt caveolae. Mol Biol
Cell 7, 1825–1834.
Barnard, R. J., Narayan, S., Dornadula, G., Miller, M. D. & Young, J. A.
(2004). Low pH is required for avian sarcoma and leukosis virus
Env-dependent viral penetration into the cytosol and not for viral
uncoating. J Virol 78, 10433–10441.
Bartlett, J. S., Wilcher, R. & Samulski, R. J. (2000). Infectious entry
pathway of adeno-associated virus and adeno-associated virus
vectors. J Virol 74, 2777–2785.
Bousse, T. L., Taylor, G., Krishnamurthy, S., Portner, A., Samal, S. K.
& Takimoto, T. (2004). Biological significance of the second receptor
binding site of Newcastle disease virus hemagglutinin-neuraminidase
protein. J Virol 78, 13351–13355.
Chandran, K., Sullivan, N. J., Felbor, U., Whelan, S. P. &
Cunningham, J. M. (2005). Endosomal proteolysis of the Ebola
virus glycoprotein is necessary for infection. Science 308, 1643–1645.
Cobaleda, C., Muñoz-Barroso, I., Sagrera, A. & Villar, E. (2002).
ACKNOWLEDGEMENTS
This work was partially supported by grants from the Spanish Fondo
de Investigaciones Sanitarias (FIS; PI021848 and PI051796) (cofinanced by the European Union FEDER Funds within the FEDERFSE 2000-2006 framework) to E. V. and Junta de Castilla y Leon
(SA037A05) to I. M. B. We thank Dr E. Dı́ez Espada and Dr J. A.
Rodrı́guez from Intervet Laboratories (Salamanca, Spain) for providing the lentogenic ‘Clone 30’ strain of NDV and Dr Adolfo Garcı́aSastre for providing anti-HN and anti-RSV F mAbs. Thanks are also
due to N. Skinner for language corrections and proofreading of the
manuscript.
http://vir.sgmjournals.org
Fusogenic activity of reconstituted Newcastle disease virus envelopes:
a role for the hemagglutinin-neuraminidase protein in the fusion
process. Int J Biochem Cell Biol 34, 403–413.
Conner, S. D. & Schmid, S. L. (2003). Regulated portals of entry into
the cell. Nature 422, 37–44.
Daecke, J., Fackler, O. T., Dittmar, M. T. & Krausslich, H. G. (2005).
Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry. J Virol 79, 1581–1594.
Damm, E. M., Pelkmans, L., Kartenbeck, J., Mezzacasa, A.,
Kurzchalia, T. & Helenius, A. (2005). Clathrin- and caveolin-1-
independent endocytosis: entry of simian virus 40 into cells devoid
of caveolae. J Cell Biol 168, 477–488.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
567
C. Cantı́n and others
Danthi, P. & Chow, M. (2004). Cholesterol removal by methyl-beta-
Matlin, K. S., Reggio, H., Helenius, A. & Simons, K. (1982). Pathway
cyclodextrin inhibits poliovirus entry. J Virol 78, 33–41.
DeTulleo, L. & Kirchhausen, T. (1998). The clathrin endocytic
of vesicular stomatitis virus entry leading to infection. J Mol Biol 156,
609–631.
pathway in viral infection. EMBO J 17, 4585–4593.
Matsuyama, S., Delos, S. E. & White, J. M. (2004). Sequential roles of
Diederich, S., Moll, M., Klenk, H.-D. & Maisner, A. (2005). The Nipah
receptor binding and low pH in forming prehairpin and hairpin
conformations of a retroviral envelope glycoprotein. J Virol 78,
8201–8209.
virus fusion protein is cleaved within the endosomal compartment.
J Biol Chem 280, 29899–29903.
Earp, L. J., Delos, S. E., Netter, R. C., Bates, P. & White, J. M. (2003).
The avian retrovirus avian sarcoma/leukosis virus subtype A reaches
the lipid mixing stage of fusion at neutral pH. J Virol 77, 3058–3066.
Eash, S., Querbes, W. & Atwood, W. J. (2004). Infection of vero cells
by BK virus is dependent on caveolae. J Virol 78, 11583–11590.
Eckert, D. M. & Kim, P. S. (2001). Mechanisms of viral membrane
fusion and its inhibition. Annu Rev Biochem 70, 777–810.
Empig, C. J. & Goldsmith, M. A. (2002). Association of the caveola
vesicular system with cellular entry by filoviruses. J Virol 76,
5266–5270.
Ferreira, L., Villar, E. & Muñoz-Barroso, I. (2004). Gangliosides and
N-glycoproteins function as Newcastle disease virus receptors. Int
J Biochem Cell Biol 36, 2344–2356.
Garcia-Sastre, A., Cabezas, J. A. & Villar, E. (1989). Proteins of
Newcastle disease virus envelope: interaction between the outer
hemagglutinin-neuraminidase glycoprotein and the inner nonglycosylated matrix protein. Biochim Biophys Acta 999, 171–175.
Gilbert, J. M. & Benjamin, T. L. (2000). Early steps of polyomavirus
entry into cells. J Virol 74, 8582–8588.
Hoekstra, D., de Boer, T., Klappe, K. & Wilschut, J. (1984).
Fluorescence method for measuring the kinetics of fusion between
biological membranes. Biochemistry 23, 5675–5681.
Hunt, R. C. & Marshall-Carlson, L. (1986). Internalization and
recycling of transferrin and its receptor. Effect of trifluoperazine on
recycling in human erythroleukemic cells. J Biol Chem 261,
3681–3686.
Jardetzky, T. S. & Lamb, R. A. (2004). Virology: a class act. Nature
427, 307–308.
Jin, M., Park, J., Lee, S., Park, B., Shin, J., Song, K. J., Ahn, T. I., Hwang,
S. Y., Ahn, B. Y. & Ahn, K. (2002). Hantaan virus enters cells by clathrin-
dependent receptor-mediated endocytosis. Virology 294, 60–69.
Joki-Korpela, P., Marjomaki, V., Krogerus, C., Heino, J. & Hyypia, T.
(2001). Entry of human parechovirus 1. J Virol 75, 1958–1967.
Kielian, M. & Rey, F. A. (2006). Virus membrane-fusion proteins:
more than one way to make a hairpin. Nat Rev Microbiol 4, 67–76.
Lamb, R. A. (1993). Paramyxovirus fusion: a hypothesis for changes.
Virology 197, 1–11.
Lisanti, M. P., Tang, Z. L. & Sargiacomo, M. (1993). Caveolin forms a
hetero-oligomeric protein complex that interacts with an apical GPIlinked protein: implications for the biogenesis of caveolae. J Cell Biol
123, 595–604.
Marjomaki, V., Pietiainen, V., Matilainen, H., Upla, P., Ivaska, J.,
Nissinen, L., Reunanen, H., Huttunen, P., Hyypia, T. & Heino, J. (2002).
Internalization of echovirus 1 in caveolae. J Virol 76, 1856–1865.
Marsh, M. & Helenius, A. (1980). Adsorptive endocytosis of Semliki
Melikyan, G. B., Barnard, R. J., Markosyan, R. M., Young, J. A. &
Cohen, F. S. (2004). Low pH is required for avian sarcoma and
leukosis virus Env-induced hemifusion and fusion pore formation
but not for pore growth. J Virol 78, 3753–3762.
Meulendyke, K. A., Wurth, M. A., McCann, R. O. & Dutch, R. E.
(2005). Endocytosis plays a critical role in proteolytic processing of
the Hendra virus fusion protein. J Virol 79, 12643–12649.
Miller, N. & Hutt-Fletcher, L. M. (1992). Epstein-Barr virus enters B
cells and epithelial cells by different routes. J Virol 66, 3409–3414.
Milne, R. S., Nicola, A. V., Whitbeck, J. C., Eisenberg, R. J. & Cohen,
G. H. (2005). Glycoprotein D receptor-dependent, low-pH-indepen-
dent endocytic entry of herpes simplex virus type 1. J Virol 79,
6655–6663.
Mothes, W., Boerger, A. L., Narayan, S., Cunningham, J. M. & Young,
J. A. (2000). Retroviral entry mediated by receptor priming and low pH
triggering of an envelope glycoprotein. Cell 103, 679–689.
Nicola, A. V., McEvoy, A. M. & Straus, S. E. (2003). Roles for
endocytosis and low pH in herpes simplex virus entry into HeLa and
Chinese hamster ovary cells. J Virol 77, 5324–5332.
Nunes-Correia, I., Eulalio, A., Nir, S. & Pedroso de Lima, M. C.
(2004). Caveolae as an additional route for influenza virus
endocytosis in MDCK cells. Cell Mol Biol Lett 9, 47–60.
Parker, J. S. & Parrish, C. R. (2000). Cellular uptake and infection by
canine parvovirus involves rapid dynamin-regulated clathrinmediated endocytosis, followed by slower intracellular trafficking.
J Virol 74, 1919–1930.
Pelkmans, L. & Helenius, A. (2003). Insider information: what
viruses tell us about endocytosis. Curr Opin Cell Biol 15, 414–422.
Pelkmans, L., Kartenbeck, J. & Helenius, A. (2001). Caveolar
endocytosis of simian virus 40 reveals a new two-step vesiculartransport pathway to the ER. Nat Cell Biol 3, 473–483.
Pho, M. T., Ashok, A. & Atwood, W. J. (2000). JC virus enters human
glial cells by clathrin-dependent receptor-mediated endocytosis.
J Virol 74, 2288–2292.
Porotto, M., Murrell, M., Greengard, O. & Moscona, A. (2003).
Triggering of human parainfluenza virus 3 fusion protein (F) by the
hemagglutinin-neuraminidase (HN) protein: an HN mutation
diminishes the rate of F activation and fusion. J Virol 77,
3647–3654.
Porotto, M., Fornabaio, M., Greengard, O., Murrell, M. T., Kellogg, G. E.
& Moscona, A. (2006). Paramyxovirus receptor-binding molecules:
engagement of one site on the hemagglutinin-neuraminidase protein
modulates activity at the second site. J Virol 80, 1204–1213.
Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., van Deurs, B. &
Sandvig, K. (1999). Extraction of cholesterol with methyl-beta-
Forest virus. J Mol Biol 142, 439–454.
cyclodextrin perturbs formation of clathrin-coated endocytic vesicles.
Mol Biol Cell 10, 961–974.
Marsh, M. & Helenius, A. (1989). Virus entry into animal cells. Adv
Rothberg, K. G., Ying, Y. S., Kamen, B. A. & Anderson, R. G. (1990).
Virus Res 36, 107–151.
729–740.
Cholesterol controls the clustering of the glycophospholipidanchored membrane receptor for 5-methyltetrahydrofolate. J Cell
Biol 111, 2931–2938.
Matlin, K. S., Reggio, H., Helenius, A. & Simons, K. (1981).
Roy, A. M., Parker, J. S., Parrish, C. R. & Whittaker, G. R. (2000).
Infectious entry pathway of influenza virus in a canine kidney cell
line. J Cell Biol 91, 601–613.
Early stages of influenza virus entry into Mv-1 lung cells:
involvement of dynamin. Virology 267, 17–28.
Marsh, M. & Helenius, A. (2006). Virus entry: open sesame. Cell 124,
568
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
Journal of General Virology 88
Endocytic entry of NDV into host cells
Russell, C. J., Jardetzky, T. S. & Lamb, R. A. (2001). Membrane
fusion machines of paramyxoviruses: capture of intermediates of
fusion. EMBO J 20, 4024–4034.
Simmons, G., Gosalia, D. N., Rennekamp, A. J., Reeves, J. D.,
Diamond, S. L. & Bates, P. (2005). Inhibitors of cathepsin L prevent
severe acute respiratory syndrome coronavirus entry. Proc Natl Acad
Sci U S A 102, 11876–11881.
Ryan, C., Zaitsev, V., Tindal, D. J., Dyason, J. C., Thomson, R. J.,
Alymova, I., Portner, A., Itzstein, M. & Taylor, G. (2006). Structural
Stuart, A. D., Eustace, H. E., McKee, T. A. & Brown, T. D. (2002). A
analysis of a designed inhibitor complexed with the hemagglutininneuraminidase of Newcastle disease virus. Glycoconj J 23, 135–141.
novel cell entry pathway for a DAF-using human enterovirus is
dependent on lipid rafts. J Virol 76, 9307–9322.
Sanchez-San Martin, C., Lopez, T., Arias, C. F. & Lopez, S. (2004).
Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M. A., Keen, J. H. &
McGraw, T. E. (1999). Acute cholesterol depletion inhibits clathrin-
Characterization of rotavirus cell entry. J Virol 78, 2310–2318.
San Roman, K., Villar, E. & Muñoz-Barroso, I. (1999). Acidic pH
coated pit budding. Proc Natl Acad Sci U S A 96, 6775–6780.
enhancement of the fusion of Newcastle disease virus with cultured
cells. Virology 260, 329–341.
Sun, X., Yau, V. K., Briggs, B. J. & Whittaker, G. R. (2005). Role of
San Roman, K., Villar, E. & Muñoz-Barroso, I. (2002). Mode of
the clathrin-mediated endocytosis during vesicular stomatitis virus
entry into host cells. Virology 338, 53–60.
action of two inhibitory peptides from heptad repeat domains of the
fusion protein of Newcastle disease virus. Int J Biochem Cell Biol 34,
1207–1220.
Townsley, A. C., Weisberg, A. S., Wagenaar, T. R. & Moss, B. (2006).
Schibli, D. J. & Weissenhorn, W. (2004). Class I and class II viral
Wang, L. H., Rothberg, K. G. & Anderson, R. G. (1993). Mis-assembly
fusion protein structures reveal similar principles in membrane
fusion. Mol Membr Biol 21, 361–371.
of clathrin lattices on endosomes reveals a regulatory switch for
coated pit formation. J Cell Biol 123, 1107–1117.
Sergel, T. A., McGinnes, L. W. & Morrison, T. G. (2000). A single amino
acid change in the Newcastle disease virus fusion protein alters the
requirement for HN protein in fusion. J Virol 74, 5101–5107.
J., Kielian, M. & Helenius, A. (1983). Membrane
fusion proteins of enveloped animal viruses. Q Rev Biophys 16,
151–195.
Seth, S., Vincent, A. & Compans, R. W. (2003). Activation of fusion
Young, J. K., Hicks, R. P., Wright, G. E. & Morrison, T. G. (1997).
by the SER virus F protein: a low-pH-dependent paramyxovirus
entry process. J Virol 77, 6520–6527.
Analysis of a peptide inhibitor of paramyxovirus (NDV) fusion using
biological assays, NMR, and molecular modeling. Virology 238,
291–304.
Sieczkarski, S. B. & Whittaker, G. R. (2002a). Dissecting virus entry
via endocytosis. J Gen Virol 83, 1535–1545.
Vaccinia virus entry into cells via a low-pH-dependent endosomal
pathway. J Virol 80, 8899–8908.
White,
Sieczkarski, S. B. & Whittaker, G. R. (2002b). Influenza virus can
Zaitsev, V., von Itzstein, M., Groves, D., Kiefel, M., Takimoto, T.,
Portner, A. & Taylor, G. (2004). Second sialic acid binding site in
enter and infect cells in the absence of clathrin-mediated endocytosis.
J Virol 76, 10455–10464.
Newcastle disease virus hemagglutinin-neuraminidase: implications
for fusion. J Virol 78, 3733–3741.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 14 May 2017 00:45:44
569