Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway.
ABSTRACT Ebola virus infection causes a highly lethal hemorrhagic fever syndrome associated with profound immunosuppression through its ability to induce widespread inflammation and cellular damage. Though GP, the viral envelope glycoprotein, mediates many of these effects, the molecular events that underlie Ebola virus cytopathicity are poorly understood. Here, we define a cellular mechanism responsible for Ebola virus GP cytotoxicity. GP selectively decreased the expression of cell surface molecules that are essential for cell adhesion and immune function. GP dramatically reduced levels of alphaVbeta3 without affecting the levels of alpha2beta1 or cadherin, leading to cell detachment and death. This effect was inhibited in vitro and in vivo by brefeldin A and was dependent on dynamin, the GTPase. GP also decreased cell surface expression of major histocompatibility complex class I molecules, which alters recognition by immune cells, and this effect was also dependent on the mucin domain previously implicated in GP cytotoxicity. By altering the trafficking of select cellular proteins, Ebola virus GP inflicts cell damage and may facilitate immune escape by the virus.
- SourceAvailable from: Pierre Becquart[Show abstract] [Hide abstract]
ABSTRACT: Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.PLoS ONE 06/2014; 9(6):e96360. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ebola virus (EBOV) causes a highly lethal hemorrhagic fever syndrome in humans and has been associated with mortality rates of up to 91% in Zaire, the most lethal strain. Though the viral envelope glycoprotein (GP) mediates widespread inflammation and cellular damage, these changes have mainly focused on alterations at the protein level, the role of microRNAs (miRNAs) in the molecular pathogenesis underlying this lethal disease is not fully understood. Here, we report that the mi-RNAs hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p were induced in human umbilical vein endothelial cells (HUVECs) following expression of EBOV GP. Among the proteins encoded by predicted targets of these miRNAs, the adhesion-related molecules tissue factor pathway inhibitor (TFPI), dystroglycan1 (DAG1) and the caspase 8 and FADD-like apoptosis regulator (CFLAR) were significantly downregulated in EBOV GP-expressing HUVECs. Moreover, inhibition of hsa-miR-1246, hsa-miR-320a and hsa-miR-196b-5p, or overexpression of TFPI, DAG1 and CFLAR rescued the cell viability that was induced by EBOV GP. Our results provide a novel molecular basis for EBOV pathogenesis and may contribute to the development of strategies to protect against future EBOV pandemics.Science China. Life sciences 09/2014; · 1.51 Impact Factor
- DARU Journal of Pharmaceutical Sciences 12/2014; 22(1):70. · 1.11 Impact Factor
JOURNAL OF VIROLOGY, Jan. 2005, p. 547–553
Vol. 79, No. 1
Ebola Virus Glycoprotein Toxicity Is Mediated by a
Dynamin-Dependent Protein-Trafficking Pathway
Nancy J. Sullivan,1Mary Peterson,1Zhi-yong Yang,1Wing-pui Kong,1
Heinricus Duckers,2† Elizabeth Nabel,2and Gary J. Nabel1*
Vaccine Research Center, National Institute for Allergy and Infectious Disease,1and
National Heart, Lung and Blood Institute,2National
Institutes of Health, Bethesda, Maryland
Received 26 April 2004/Accepted 23 August 2004
Ebola virus infection causes a highly lethal hemorrhagic fever syndrome associated with profound immu-
nosuppression through its ability to induce widespread inflammation and cellular damage. Though GP, the
viral envelope glycoprotein, mediates many of these effects, the molecular events that underlie Ebola virus
cytopathicity are poorly understood. Here, we define a cellular mechanism responsible for Ebola virus GP
cytotoxicity. GP selectively decreased the expression of cell surface molecules that are essential for cell
adhesion and immune function. GP dramatically reduced levels of ?V?3 without affecting the levels of ?2?1
or cadherin, leading to cell detachment and death. This effect was inhibited in vitro and in vivo by brefeldin
A and was dependent on dynamin, the GTPase. GP also decreased cell surface expression of major histocom-
patibility complex class I molecules, which alters recognition by immune cells, and this effect was also
dependent on the mucin domain previously implicated in GP cytotoxicity. By altering the trafficking of select
cellular proteins, Ebola virus GP inflicts cell damage and may facilitate immune escape by the virus.
Ebola virus causes a highly pathogenic infection resulting in
rapid failure of multiple organ systems and in death in many
cases. The highest mortality rates are observed with the Zaire
subtype, one of four types identified to date (7, 14). Though the
pathogenesis of Ebola virus infection at the molecular level has
not been fully explained, the envelope glycoproteins likely con-
tribute to adverse events in the host (17, 18). The envelope
gene of Ebola virus gives rise to two products from a single
gene, a nonstructural secreted form of the glycoprotein, sGP,
encoded by the predominant transcript (?80%), and the virion
envelope glycoprotein, GP, the result of RNA editing during
formation of the message (?20%). The tight control of virion
GP synthesis may be necessary due to the ability of the full-
length glycoprotein to elicit cytopathic effects in cells targeted
by the virus (4, 17, 18). The mechanism by which GP causes
cytopathicity in target cells remains to be elucidated, but its
effects are evident from its ability to cause cell rounding and
detachment (18). Here we report that Ebola virus GP-induced
cytotoxicity involves a cellular trafficking pathway that is de-
pendent upon dynamin, a GTPase that mediates transport
MATERIALS AND METHODS
Expression vectors and cell lines. ADV-GP(Z), the recombinant adenovirus
expressing Zaire GP, was made according to the standard protocol for making
first-generation recombinant adenovirus (1) and has been described previously.
Expression vectors p1012, pGP(Z), and pGP?MUC contain a cytomegalovirus
enhancer promoter and have been described previously. The dynamin and K44E-
dynamin expression vectors are pcDNA3.1 based and were a generous gift from
P. Okamoto and R. Vallee and were received from D. Ganem (9). Human
umbilical vein endothelial cells (HUVEC) and EGM-2 culture medium were
obtained from BioWhittaker/Clonetics. 293 human embryonic kidney cells were
cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum (GIBCO). Transfections were performed using Fugene6 transfec-
tion reagent (Roche) according to the manufacturer’s instructions.
Flow cytometry and antibodies. HUVEC were infected with the indicated
vectors at a multiplicity of infection (MOI) of 500, or 293 cells were transfected
with plasmid vectors and analyzed by fluorescence-activated cell sorting for cell
surface expression of the indicated glycoproteins after 12 to 24 h. Cells were
collected after incubation with phosphate-buffered saline (PBS) (3 mM EDTA)
and incubated with control immunoglobulin (Ig), rabbit anti-sGP/GP serum
(generously provided by A. Sanchez), anti-integrin monoclonal antibodies
(Chemicon International, Inc., Temecula, Calif.), or an HLA class I antibody
(Biosource International) for 30 min on ice. The cells were washed twice with
ice-cold PBS containing 2.5% fetal bovine serum, incubated with fluorescein
isothiocyanate (FITC) (Jackson ImmunoResearch Laboratories, West Grove,
Pa.)- or phycoerythrin (Sigma)-conjugated secondary antibodies for 30 min on
ice, followed by washing. Analysis was conducted using a Becton Dickinson
4-color Calibur flow cytometer and FlowJo analysis software (Tree Star, Inc.).
Confocal microscopy. HUVEC were infected with adenovirus vectors in cham-
ber slides, fixed, and permeabilized 10 to 16 h postinfection. Cells were stained
using the primary antibodies described above for 1 h at room temperature,
washed, and incubated with Alexa 488-, Alexa 568-, or Alexa 594-conjugated
secondary antibodies (Jackson ImmunoResearch Laboratories). Images were
collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems,
Exton, Pa.) using a 63? oil immersion objective (NA 1.32, zoom X). Fluoro-
chromes were excited using an argon laser at 488 nm for FITC and a krypton
laser at 568 nm for Alexa 568. Detector slits were configured to minimize any
cross-talk between the channels. Differential interference contrast images were
collected simultaneously with the fluorescence images by use of a transmitted
light detector. Images were processed using Leica TCS-NT/SP (version 1.6.587),
Imaris 3.1.1 (Bitplane AG, Zurich, Switzerland), and Adobe Photoshop 5.5
(Adobe Systems) software.
Metabolic labeling and immunoprecipitation. Cells were metabolically labeled
with 75 ?Ci each of [35S]cysteine and [35S]methionine per 10-cm-diameter tissue
culture dish overnight. Radioactive medium was removed, and cells were
washed, lysed with NP-40 buffer (1% NP-40, 0.15 M NaCl, 10 mM Tris, pH 7.5),
and immunoprecipitated with antibodies against integrins and GP (described
above) or dynamin (Transduction Laboratories, San Diego, Calif.) by use of
immobilized protein G (Pierce, Rockford, Ill.). For some experiments metabolic
* Corresponding author: Mailing address: Vaccine Research Cen-
ter, National Institute for Allergy and Infectious Disease, National
Institutes of Health, 40 Convent Dr., MSC-3005, Bethesda, MD 20814.
Phone: (301) 496-1852. Fax: (301) 480-0274. E-mail: firstname.lastname@example.org.
† Present address: Dept. of Cell Biology & Genetics, Erasmus Uni-
versity, 3015 GE Rotterdam, The Netherlands.
labeling was omitted, and immunoprecipitated material was analyzed after so-
dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by West-
Expression of GP in vasculature and peroxidase perfusion experiments. Por-
cine carotid arteries were infected with 2.5 ? 108PFU of ADV-?E1 or ADV-GP
(Zaire)/ml in PBS for 1 h at room temperature. Vessels were then incubated with
M199 medium containing 20% fetal calf serum for 48 h in a tissue culture
incubator. Subsequently, the vessels were divided and processed for peroxidase
perfusion experiments to determine permeability changes of the vessels or for
scanning electronic microscopy as described previously (18). Briefly, for perfu-
sion the vessels were perfused with 200 U of peroxidase (P8250; Sigma)/ml in
PBS for 3 min at a constant Hg pressure (90 ? 2 [standard error of the mean]
mm), fixed in 4% paraformaldehyde–10% glutaraldehyde for 2 h at 4°C, and
incubated in diaminobenzidine solution (2 ng/ml) for 1 h.
Down-regulation of cell surface proteins by GP is selective.
Expression of Ebola virus GP disrupts cell adhesion in 293
embryonic kidney cells or HUVEC and is followed by cell
death in 24 to 48 h (18). We therefore examined whether GP
could alter cell surface expression of adhesion molecules.
HUVEC were infected with replication-defective adenovirus
vectors with no insert (ADV-?E1), the negative control, or
with an insert encoding Ebola virus GP (ADV-GP). After 24 h,
cells infected with ADV-GP showed rounding and detachment
from the substratum whereas ADV-?E1 cells were unchanged,
as reported previously (18). Because cadherin, ?2?1, and
?V?3 are representative cell surface molecules known to me-
diate cell adhesion via their interaction with receptors on
neighboring cells or the extracellular matrix, their expression
levels were measured by flow cytometry (Fig. 1A). Cell surface
?V?3 levels were dramatically reduced in GP-expressing cells,
in contrast to the results seen with cadherin, which was simi-
larly expressed in endothelial cells infected with ADV-?E1 or
ADV-GP. The ?2?1 heterodimer expression was also unaf-
fected by GP, whereas the ?1 subunit was shown to be down-
modulated in 293 cells (16), suggesting specificity of GP effects.
Ebola virus GP contains a region approximately 200 amino
acids in length that is rich in serines and threonines, in simi-
larity to mucin proteins, and this mucin-like region is respon-
sible for GP-mediated cell rounding and cytotoxicity (18). Ex-
pression of the GP lacking mucin (ADV-GP?MUC), which is
functionally active (18), abolished GP-mediated cell detach-
ment and also completely reversed the decrease in ?V?3 levels
(Fig. 1B). These data suggested that one effect of Ebola virus
GP expression in endothelial target cells was disruption of cell
adhesion through an effect on the ?V?3 integrin.
GP alters recycling of ?V?3. To define the mechanism by
which GP reduced ?V?3 expression, we examined the effect of
Ebola virus GP on ?V?3 turnover. Pulse-chase experiments
([35S]cysteine-methionine) were conducted using HUVEC in-
fected with the ADV-?E1 or ADV-GP adenovirus vector to
label the intracellular pool of ?V?3, and its degradation was
monitored. Over a 19-h period, total cell-associated ?V?3 lev-
els remained constant in cells infected with ADV-GP and were
comparable to the levels in cells infected with empty vector
(ADV-?E1) (Fig. 1C), despite the marked decrease in cell
surface expression. The ?V?3 bands at 19 h in the control cells
are somewhat lighter than those seen at the same time point in
the GP-expressing cells. However, the difference in levels of
band intensity (by scanning densitometry) is less than twofold.
These data suggested that the reduction of cell surface ?V?3
levels was independent of its total turnover within the cell and
that GP affected its transport or recycling.
GP effects on cell surface proteins are sensitive to brefeldin
A. Brefeldin A, a fungal metabolite, is known to block protein
trafficking between the endoplasmic reticulum and Golgi com-
plex, as well as recycling. To determine whether the effects of
GP were mediated by a brefeldin-sensitive pathway, HUVEC
were infected with ADV-?E1 or ADV-GP and treated with
this drug for 10 h after infection. Treatment with brefeldin A
completely blocked the ability of GP to induce cell rounding
and detachment, and these cultures appeared to be similar to
those of control cells infected with the empty vector (ADV-
?E1). In untreated cells that express GP, rounding was ob-
served in approximately 50% of the cells (Fig. 2A). Moreover,
brefeldin A was able to block GP cytopathicity in intact blood
vessels. Porcine carotid arteries were surgically removed and
infused with an adenovirus vector expressing Ebola virus GP
(Fig. 2B). In the absence of brefeldin A, GP destroyed the
integrity of the endothelial cell layer of the vessels, exposing
the basement membrane, as previously shown (18). Brefeldin
A reversed the GP cytopathic effect completely. The results
with brefeldin A suggested that the ability of GP to cause
down-regulation of ?V?3 and disruption of blood vessel integ-
rity was dependent upon the presence of a brefeldin-sensitive
?V?3 and GP colocalize and interact biochemically. We
next examined whether there was a relationship between the
subcellular localizations of ?V?3 and GP by use of confocal
microscopy. HUVEC were infected with ADV-GP and
costained with a monoclonal antibody against ?V and a poly-
clonal antiserum raised against GP. This analysis revealed that
GP and ?V colocalized in the perinuclear region and within a
small area at the plasma membrane (Fig. 3A). The colocaliza-
tion of GP and ?V suggested that GP might directly cause the
redistribution of ?V within cells. Coimmunoprecipitation stud-
ies were therefore performed to determine whether the two
molecules interact biochemically. 293 cells were transfected
with an empty, control vector or with a GP expression vector
plasmid, and cell lysates were immunoprecipitated with a
monoclonal antibody against ?V integrin or an isotype control.
These samples were run on SDS-PAGE and immunoblotted
with a polyclonal antiserum to Ebola virus GP. The monoclo-
nal antibody specific for ?V, in contrast to a control antibody,
was able to coimmunprecipitate GP (Fig. 3B, left panel). We
then performed the converse experiment in which an antibody
to ?V integrin was used to coprecipitate GP (Fig. 3B, middle
panel). It is noteworthy that GP?MUC, when expressed at
levels similar to those seen with wild-type GP (Fig. 3B, right
panel), which does not down-regulate ?V, did not coprecipi-
tate ?V. These results demonstrated a protein association be-
tween Ebola virus GP and ?V integrin that likely contributes to
the intracellular redistribution of ?V.
GP-mediated down-regulation of ?V?3 requires functional
dynamin. The biosynthesis of mammalian ?V integrins is slow;
full maturation to the biologically active form requires 20 h.
Once on the cell surface, integrins accumulate within microdo-
mains called caveolae or lipid rafts (8). Recycling of molecules
that reside in lipid rafts is influenced by the GTPase dynamin,
which mediates pinching off of transport vesicles from the
cytoplasmic face of rafts (12). K44E, a mutant form of dy-
548SULLIVAN ET AL.J. VIROL.
FIG. 1. Effect of Ebola virus GP on cell surface adhesion molecules and down-regulation of ?V integrins. (A) Selective down-regulation of cell
surface adhesion molecules by Ebola virus GP. HUVEC were infected with the indicated vectors at an MOI of 500 and analyzed by fluorescence-
activated cell sorting for cell surface expression of the indicated glycoproteins after 15 h. Cells were collected after incubation with PBS (3 mM
EDTA) and incubated with anti-integrin anti-bodies (solid lines) as indicated for 30 min on ice. The cells were washed once with ice-cold PBS and
incubated with FITC-conjugated sheep anti-mouse IgG for 30 min on ice, followed by washing. A matched isotype antibody was used as a negative
control in each case (dashed line). Results are representative of three independent experiments. (B) Down-regulation of ?V integrin in 293 cells.
293 cells were transfected with a plasmid encoding vector control, Ebola virus GP, or Ebola virus GP(?MUC). Both floating and adherent cells
were collected 18 h after transfection, using 3 mM EDTA to remove adherent cells. Flow cytometry was performed on cells double stained for
Ebola virus GP and ?V integrin as described in Materials and Methods. Results are shown for events in the live cell gate. (C) Analysis of ?V
integrin degradation by metabolic labeling. HUVEC cells were infected with adenovirus vectors expressing the indicated inserts and metabolically
labeled with [35S]cysteine and [35S]methionine. At 8 h postinfection, cells were washed and harvested either immediately (8 h) or after further
incubation in unlabeled medium (11 and 19 h). After harvesting, cells were lysed in NP-40 buffer, immunoprecipitated with an ?V?3 antibody,
clone LM609 (Chemicon International), and analyzed by SDS-PAGE on 4 to 15% gels.
VOL. 79, 2005EBOLA VIRUS TOXICITY INVOLVES CELLULAR TRAFFICKING 549
namin, contains a mutation in the GTP binding domain that
renders the molecule nonfunctional and causes it to interfere
with the normal function of wild-type dynamin in a dominant-
negative manner (9). We employed dynamin K44E to investi-
gate the possibility that GP reduces cell surface ?V integrin
through a dynamin-dependent transport pathway. 293 cells
were cotransfected with expression vectors encoding GP and
dynamin K44E, and the amount of ?V integrin on the cell
surface was measured by flow cytometry. Dynamin K44E alone
had no effect on basal ?V cell surface expression in vector
control-transfected cells (data not shown); however, dynamin
K44E reversed approximately 40% of the GP-mediated de-
crease in surface ?V integrin levels (Fig. 4A).
whether Ebola virus GP could also interact with dynamin. 293
cells were transfected with GP, and cell lysates were immuno-
FIG. 2. Brefeldin A inhibits the cytopathic effects of GP in cell culture and in porcine arteries. (A) Effect of brefeldin A on Ebola virus GP
cytopathicity. HUVEC cells were infected by ADV-?E1 or ADV-GP at an MOI of 500. At 6 h after infection, brefeldin A was added (?) or the
cells were left untreated (?). At 16 h after infection, pictures were taken. The percentage of rounding was calculated by counting the cells of three
slides. (B) Cultured porcine arteries infected with ADV-GP. Peroxidase perfusion analysis to determine permeability changes was performed in
the absence (None) (0 ng/ml) or presence (Brefeldin A) of brefeldin A treatment (100 ng/ml) as described in Materials and Methods. (C) Scanning
electron micrograph of porcine arteries infected with ADV-GP and treated with brefeldin A (Brefeldin A) or left untreated (None). Vessels were
infected with ADV-GP as described in Materials and Methods and processed for scanning electron microscopy (11).
550SULLIVAN ET AL.J. VIROL.
precipitated with a monoclonal antibody against dynamin, fol-
lowed by Western blotting with a polyclonal antiserum to
Ebola virus GP. GP coprecipitated with dynamin, indicating
that there was also a colocalization of the two molecules (Fig.
4B). In contrast to the interaction of ?V integrin with both
mature GP and an intermediate form, dynamin coprecipitates
with only the mature GP. These results suggested that Ebola
virus GP down-regulated ?V integrin through its ability to
influence the dynamin protein-trafficking pathway. Through its
interaction with dynamin and ?V integrin, GP may redirect the
normal cycling of one or both of these molecules in a way that
alters the steady-state levels of ?V integrin on the cell surface.
Down-regulation of MHC-I by Ebola virus GP. Many viruses
have evolved to circumvent immune responses by modulating
proteins essential for immune signaling and function. Major
histocompatibility complex class I (MHC-I) molecules are tar-
geted by several pathogens, including cytomegalovirus, human
immunodeficiency viruses, and herpesviruses (13). One of
these viruses, Kaposi’s sarcoma-associated herpesvirus (5), re-
duces cell surface expression of MHC-I molecules through a
dynamin-dependent pathway (6). Since the down-regulation of
?V integrin is dynamin dependent, we investigated whether
Ebola virus GP also reduced MHC-I expression and thus al-
tered protective immunity to Ebola virus. To test this possibil-
ity, cell surface expression of MHC-I was determined in 293
cells transfected with a control or GP expression vector by flow
cytometry. At 24 h after transfection, cells expressing Ebola
virus GP exhibited a dramatic reduction in cell surface MHC-I
levels (Fig. 4C). In similarity to ?V integrin, down-modulation
was dependent on the mucin domain of GP and was partially
reversed by the dominant-negative dynamin, K44E (Fig. 4C).
As expected, the decrease in surface MHC-I levels caused by
GP expression also reduced recognition of GP-expressing tar-
get cells by allogeneic effector T cells (data not shown).
Ebola virus typically avoids clearance by the host immune
response in humans, resulting in uncontrolled replication,
FIG. 3. Subcellular distribution of Ebola virus GP and biochemical interaction with ?V integrin. (A) Cellular distribution of Ebola virus GP
and ?V integrin by confocal microscopy. HUVEC were infected with ADV-?E1 or ADV-GP at an MOI of 300 for 1 h. At 10 h later, the cells
were fixed, permeabilized, and stained for the indicated protein by use of specific monoclonal antibodies followed by secondary staining with Alexa
488 (green [GP])- or Alexa 568 (red [?V])- conjugated antibodies. The “GP??V” panel shows an overlay of the red and green panels. GP (green)
and ?V (red) colocalized in the perinuclear region (solid arrow) and within a small area at the plasma membrane (dashed arrow). The far right
panel shows the distribution of ?V integrin in control cells not expressing GP. Slides were analyzed by confocal microscopy as described in
Materials and Methods. (B) Coprecipitation of Ebola virus GP and ?V integrin. 293 cells were transfected with Ebola virus GP (pGP), Ebola virus
GP lacking the mucin domain (p?MUC), or empty vector (Control) and incubated for 20 h. Supernatant cells were collected and pooled with
adherent cells harvested by incubation with 3 mM EDTA. Lysis was performed with NP-40 buffer, and immunoprecipitation was performed using
an isotype control mouse IgG (mIgG) or a monoclonal antibody against ?V integrin. Immunoprecipitated proteins were separated by SDS–4 to
15% PAGE, and gels were immunoblotted using an antibody specific for Ebola virus GP. The left panel shows coprecipitation of GP by ?V integrin
antibody. The middle panel shows the inverse coprecipitation of ?V integrin with GP antibody. The right panel shows the total amount of GP in
the cell lysates.
VOL. 79, 2005EBOLA VIRUS TOXICITY INVOLVES CELLULAR TRAFFICKING551
damage to host cells, and, ultimately, fatal organ destruction.
Though some patients who mount a strong, early immune
response can recover from infection (3), the fatality rates re-
main high. An understanding of Ebola virus pathogenic mech-
anisms facilitates the development of antiviral and immune
therapies, which are presently unavailable. Though previous
reports have suggested that Ebola virus GP alters expression of
cell surface proteins (15, 16), the specificity and mechanisms of
these effects have not been defined. The findings presented
here demonstrate that Ebola virus GP exerts this effect
through a specific mechanism involving a pathway that is de-
pendent upon the GTPase dynamin. Through its interaction
with dynamin, GP disrupts the normal intracellular trafficking
of cell surface proteins that are essential for cell attachment,
viability, and immune signaling. The functional interactions
shown herein appear to indicate a direct binding of GP to
dynamin and a specific subset of cell surface molecules. How-
ever, Ebola virus GP might also influence trafficking in partic-
FIG. 4. Interaction of Ebola virus GP with dynamin and inhibition of cell surface class I MHC expression. (A) Dominant-negative dynamin
inhibition of ?V integrin down-regulation. 293 cells were transfected with a vector encoding Ebola virus GP without (GP) or with (GPK?44E)
dynamin K44E as indicated. Empty vector was transfected in the absence of dynamin K44E. Floating and adherent cells were harvested 18 h after
transfection, pooled, and double stained with ?V integrin and GP antibodies, followed by secondary staining as described in Materials and
Methods. Results are shown for events in the live-cell gate. (B) Coprecipitation of GP with dynamin. 293 cells were transfected with Ebola virus
GP (pGP) or empty vector (Control) and incubated for 20 h. Supernatant cells were collected and pooled with adherent cells harvested by
incubation with 3 mM EDTA. Lysis was performed with NP-40 buffer, and immunoprecipitation was performed using a monoclonal antibody
against dynamin or an unrelated control antibody (?1 integrin). Immunoprecipitated proteins were separated by SDS–4 to 15% PAGE, and gels
were immunoblotted using an antibody specific for Ebola virus GP. (C) Down-regulation of MHC-I molecules and dynamin dependence. 293 cells
were transfected with a vector control (Control), Ebola virus GP (GP), Ebola virus GP lacking the mucin domain (GP?MUC), or Ebola virus GP
plus dynamin K44E. Floating and adherent cells were harvested and double stained with antibodies against MHC-I and GP (left three panels) and
against MHC-I alone, in GP-positive cells, in the absence (light line) or presence (bold line) of dynamin K44E (right panel). Analysis is shown for
events in the live-cell gate.
552SULLIVAN ET AL.J. VIROL.
ular regions of the cell surface membrane in which these spe-
cific sets of molecules reside.
We now have several lines of evidence to suggest that ?V
down-regulation is highly dependent on GP-trafficking within
the cell. For example, both transmembrane deletion and alter-
nate soluble forms of GP have no effect on ?V levels, implying
that residence of GP at the plasma membrane (or at least
within membrane compartments that are in equilibrium with
the plasma membrane) is necessary for the effect. The brefel-
din A results reinforce this interpretation, because brefeldin A
acts between the endoplasmic reticulum and Golgi complex to
inhibit membrane traffic and therefore likely prevents GP from
accessing specific membrane compartments or, perhaps, im-
pedes posttranslational modifications of GP that occur late in
trafficking and that are required for the effect. It is noteworthy
that the GP-induced cytopathic effects require cell surface ex-
pression of Ebola virus GP (16). A previous report suggested
that global down-regulation of cell surface molecules is exerted
by GP (15), but our present experiments and those of others
(15, 16) demonstrate that the expression of at least some cell
surface molecules is unaffected. With human kidney 293T cells,
minimal to nonexistent down-regulation was observed for sev-
eral ?-integrins (16), whereas ?1 integrin is unaffected in
HUVEC (15) but is down-regulated in 293T cells (15).
These disparate results suggest that Ebola virus GP does not
cause global down-regulation but, rather, is linked to particular
groups of molecules depending on cell type. Moreover, specific
intracellular trafficking pathways that affect cell surface expres-
sion seem to be particularly relevant to down-regulation.
Brefeldin A prevents Ebola virus GP from accessing cell sur-
face microcompartments, and dynamin influences cycling to
and from these same areas of the cell. We have observed that
GP expression appears to be lost from the cell surface at time
points when ?V down-regulation is maximal. A likely explana-
tion for this observation is that at late stages, down-regulation
of GP is linked to removal of ?V from the cell surface. Our
biochemical and confocal results indicate that these two mol-
ecules reside in close proximity to one another. Therefore, a
mechanism (e.g., increased endocytosis) that down-regulates
?V may be expected to also influence cell surface levels of GP.
The results presented herein provide a mechanism by which
Ebola virus GP might influence groups of molecules, specified
by particular cell trafficking pathways or by microlocalization at
the cell membrane.
Unlike many enveloped viruses, the envelope glycoprotein
gene of Ebola virus does not exhibit the high sequence vari-
ability that readily allows immune escape. These results sug-
gest that Ebola virus has evolved in its natural host a mecha-
nism that allows immune evasion in humans. Through its
effects on specific cell surface molecules, Ebola virus disrupts
several processes essential for immune activation and recogni-
tion, such as cell trafficking and antigen presentation. Adhe-
sion and accessory proteins such as ?V are involved in immune
cell homing and signaling (2), and down-regulation by GP may
further affect homing, acute inflammation, and costimulation
that would reduce both innate and acquired immune re-
sponses. At the same time, the cytotoxic effects of GP on
macrophage and endothelial cell function affect the function of
inflammatory cells (10, 19) and disrupt the integrity of the
vasculature (18). Together, these cytotoxic effects are likely
responsible for the inflammatory dysregulation, immune sup-
pression, and vascular dysfunction that are hallmarks of lethal
Ebola virus infection.
We thank Ati Tislerics, Karen Stroud, Toni Garrison, Brenda Hart-
man, and Tina Suhana for manuscript preparation, Judy Stein for
discussions and comments, and Owen Schwartz (National Institutes of
Health-National Institute of Allergy and Infectious Diseases) for as-
sistance with confocal microscopy. We are grateful to Patricia Oka-
moto, Richard Vallee, and Donald Ganem for kindly supplying the
dominant-negative dynamin expression vector. We also thank mem-
bers of the Roederer and Seder labs (National Institutes of Health,
Vaccine Research Center, NIAID) for helpful discussions.
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