Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and Subsequent Trafficking through Early and Late Endosomes

Abstract

Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry mechanism of other filoviruses.

Full text

Cellular Entry of Ebola Virus Involves Uptake by a
Macropinocytosis-Like Mechanism and Subsequent
Trafficking through Early and Late Endosomes
Mohammad F. Saeed
1,2,3
, Andrey A. Kolokoltsov
1,2,3
, Thomas Albrecht
4
, Robert A. Davey
1,2,3
*
1 Department of Microbiology & Immunology, The University of Texas Medical Branch, Galveston, Texas, United States of America, 2 Galveston National Laboratory, The
University of Texas Medical Branch, Galveston, Texas, United States of America, 3 Institute of Human Infection and Immunity, The University of Texas Medical Branch,
Galveston, Texas, United States of America, 4 Department SK, Building 37, NASA, Houston, Texas, United States of America
Abstract
Zaire ebolavirus (ZEBOV), a highly pathogenic zoonotic virus, poses serious public health, ecological and potential
bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic
intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters
cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have
produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were
performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately
recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent
infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate
that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the
uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase
uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/
BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in
ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is
trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the
cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for
the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can
be applied to development of new therapeutics as well as provide potential insight into the trafficking and entry
mechanism of other filoviruses.
Citation: Saeed MF, Kolokoltsov AA, Albrecht T, Davey RA (2010) Cellular Entry of Ebola Virus Involves Uptake by a Macropinocytosis-Like Mechanism and
Subsequent Trafficking through Early and Late Endosomes. PLoS Pathog 6(9): e1001110. doi:10.1371/journal.ppat.1001110
Editor: Christopher F. Basler, Mount Sinai School of Medicine, United States of America
Received December 22, 2009; Accepted August 17, 2010; Published September 16, 2010
Copyright: ß 2010 Saeed et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the NIH, NIH/NIAID 5R01AI063513-02 and NIH NIAID 2 U54 AI057156-06, subproject RP21. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: radavey@utmb.edu
Introduction
Zaire ebolavirus (ZEBOV, Genbank:AF086833), a member of the
family Filoviridae, genus Filovirus, causes a highly fatal hemorrhagic
fever in humans and non-human primates. Over the past three
decades numerous human outbreaks have occurred in Central
Africa involving hundreds of cases with fatality rates ranging from
50–89% [1]. In addition, outbreaks of ZEBOV infection have been
implicated in deaths of tens of thousands of gorillas, chimpanzees
and duikers in Central and Western Africa posing a considerable
threat to the wildlife and ecology in those areas [2]. Due to a very
high case fatality rate in humans, significant transmissibility of the
virus, lack of effective preventive or therapeutic measures against
the disease, ZEBOV is considered a serious emerging viral
pathogen. Currently no specific therapy or vaccine is approved
for human or animal use against this pathogen.
As for other members of Filoviridae, ZEBOV is morphologically
distinct from other animal viruses. The virions are long and
filamentous with an average length of 800–1000 nm and a
diameter of about 80 nm but can form a variety of shapes ranging
from straight rods to closed circles [3]. Virions are surrounded by
a host cell-derived lipid envelope. The envelope contains virally-
encoded glycoprotein (GP) spikes composed of homotrimers of two
virally-encoded glycoproteins, GP1 and GP2. The approximately
19 kb single-stranded, negative-sense genomic RNA complexed
with nucleocapsid, VP35, VP30 and L proteins form the
nucleocapsid, while VP40 forms the matrix that underlies the
viral membrane [4].
Like all viruses, ZEBOV largely relies on host cell factors and
physiological processes for key steps of its replication cycle.
Identification of these processes and factors will not only allow a
better insight into pathogenic mechanism, but may identify novel
targets for future therapeutic development. As the first step of
replication, entry into the host cell is an attractive target for
therapeutic intervention as infection can be stopped before virus
replication disrupts cellular functions. However, the entry
mechanism of ZEBOV, and that of other large enveloped viruses
is very limited.
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Many enveloped viruses, including ZEBOV, require endocyto-
sis to infect cells. The internalized virus is transported through
successive endocytic vesicles to reach a vesicle/compartment
where conditions are conducive (low pH and/or presence of
proteolytic enzymes) for the GP to attain a suitable conformation
needed for membrane fusion [5,6,7]. Upon fusion of viral and
endocytic membranes, the capsid moves into the cell cytoplasm to
begin genome replication. Several distinct endocytic mechanisms
exist in mammalian cells. They are distinguished from each other
on a number of criteria including the size and morphology of
endocytic vesicles, the type of cargo they carry, the cellular factors
involved in their control and their origins and destinations [8].
Different viruses employ different routes of endocytosis, and the
route taken by a given virus largely depends on the receptor it
interacts with.
Clathrin-mediated endocytosis (CME) is the best understood
endocytic pathway. A number of viruses including Influenza A
(Genbank:M73524), Semliki forest (Genbank:X04129) and vesic-
ular stomatitis viruses (VSV; Genbank:J02428) employ this
pathway for entry [9,10]. The internalization of the virus-receptor
cargo occurs in specialized areas of cell membrane called clathrin-
coated pits (CCPs). CCPs are formed on the cytoplasmic face of
the plasma membrane through sequential assembly of proteins
including clathrin that form a cage-like structure lining the
cytoplasmic side of the pit. The pit then invaginates and buds from
the plasma membrane forming a clathrin-coated vesicle approx-
imately 120 nm in diameter containing the internalized cargo.
Subsequently, the vesicle sheds its clathrin coat, a prerequisite for
further trafficking and merging with other compartments. A
number of accessory, adaptor and signaling molecules participate
in this process, and provide a tight regulation of the pathway.
Some, such as accessory protein-2 (AP2) and Eps15, are
specifically associated with CCPs, while others such as dynamin,
which is responsible for vesicle budding from the plasma
membrane, are shared with other endocytic pathways [8].
Caveolin-mediated endocytosis (CavME), first observed for the
cellular uptake of simian virus 40 (SV40) [11], differs from CME in
terms of internalization mechanism and vesicular transport route.
Caveolae are flask-shaped invaginations in the plasma membrane
that are rich in caveolin protein, and are predominantly associated
with cholesterol-rich plasma membrane microdomains termed
lipid rafts. Therefore, extraction or perturbation of membrane
cholesterol severely impedes entry of viruses that use CavME.
Vesicles derived from CavME are indicated by the presence of
caveolin and are termed caveosomes. Other cellular factors such as
Eps15-related (Eps15R) protein are thought to be specific for
CavME, but as with CME, dynamin is still required for severing of
caveolae from the plasma membrane. Another distinguishing
feature is that caveloae are smaller than CCPs and have an
average diameter of approximately 60–80 nm [8].
Recently, the importance of macropinocytosis, as a distinct
endocytic uptake mechanism for virus infection, has started to be
realized for some viruses [9]. Macropinocytosis is associated with
membrane ruffles such as those formed by filopodia and
lamellipodia, which are outward extensions of the plasma
membrane driven by actin polymerization underneath the
membrane surface [12]. When a ruffle folds back upon itself a
cavity can be formed. Subsequent fusion of the distal end of the
loop with the plasma membrane results in formation of a large
vesicle called a macropinosome. These can range in size from 200
to 10,000 nm across and take up cargoes of similar dimensions [9].
Morphological and regulatory characteristics that distinguish
macropinocytosis from other endocytic processes have also begun
to emerge [8,9,13,14]. Macropinosomes are best characterized for
uptake of fluid phase markers such as high molecular weight
dextran and horse-radish peroxidase and is sensitive to inhibitors
of Na+/H+ exchangers, such as amilorides [14]. As for CavME,
they are dependent on cholesterol-rich lipid rafts, but dynamin is
not required. Instead, scission of macropinosomes appears to
require CtBP/BARS [9,15,16]. Other work indicates the involve-
ment of cell signaling factors PI3K, Akt, PKC, PLCc and PLC-A
2
that act to promote membrane ruffling by stimulating actin
remodeling through Rac and cdc42 [9].
All endocytic pathways used by viruses serve to deliver virus to
vesicles and compartments conducive to virus membrane fusion
and release of the core into the cell cytoplasm at a site where
replication proceeds optimally. Many endocytic pathways share
common features, such as acidification, yet each virus type appears
to prefer one trafficking pathway over others and misdirection into
alternative pathways can result in inhibition of infection. For most
enveloped viruses, the point at which membrane fusion occurs
appears to be at the early or late endosome stage. This evidence
has been gathered by comparing pH-sensitivity of the GP to
known pH of the endosome at different stages of maturation. More
recently, the use of dominant negative GTPases, that are involved
in endosomal maturation, have been used in determining virus exit
points from the endosome [17,18,19]. In general, the two methods
agree but provide little detail as to whether viruses have additional
requirements in terms of site of release other than the early or late
endosome.
Currently, a detailed understanding of ZEBOV endocytosis and
trafficking is lacking. Each of the previous studies on understand-
ing ZEBOV entry pathway have indicated involvement of
different pathways, including CME [20,21], CavME [20,22] and
a Rho GTPase-dependent pathway that may suggest involvement
of macropinocytosis [23]. These conflicting findings may be due to
the use of surrogate models of ZEBOV such as pseudotyped
retroviruses, which are morphologically and biochemically distinct
from wild type filamentous ZEBOV and/or reliance on one
analytical approach, such as use of pharmacological agents, which
are likely to act on more than one cellular target [24].
Here we have used multiple independent approaches employing
replication-competent, infectious ZEBOV and/or morphological-
ly comparable virus-like particles (VLPs). We have examined the
contribution of each endocytic pathway to ZEBOV entry and
infection by quantitative analysis. The work involves measuring
the impact of drugs, siRNA and/or expression of well character-
ized dominant negative (DN) mutants of cell trafficking proteins on
virus entry and infection. We also use fluorescently-labeled virus-
like particles (VLPs) to follow virus internalization and trafficking
Author Summary
Filoviruses, including Zaire ebolavirus (ZEBOV), are among
the most pathogenic viruses known. Our understanding of
how these viruses enter into host cells is very limited. A
deeper understanding of this process would enable the
design of better targeted antiviral therapies. This study
defines in detail, key steps of ZEBOV cellular uptake and
trafficking into cells using wild type virus as well as the
host factors that are responsible for permitting virus entry
into cells. Our data indicated that the primary mechanism
of ZEBOV uptake is a macropinocytosis-like process that
delivers the virus to early endosomes and subsequently to
late endosomes. These findings aid in our understanding
of how filoviruses infect cells and suggest that disruption
of macropinocytosis may be useful in treatment of
infection.
Entry Pathway of Ebola Virus
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through different endocytic compartments. The product of
combining all these approaches provides, for the first time, an
accurate and detailed description of ZEBOV uptake mechanism.
Our data clearly indicate that wild type ZEBOV enters and infects
Vero and HEK293T cells independently of clathrin, caveolae and
dynamin. Instead, virus entry required the presence of cholesterol
in the cell membrane and was inhibited by the amiloride
derivative, EIPA. A marked induction in fluid-phase uptake was
also observed shortly after virus binding to cells and internalized
virus particles showed significant colocalization with high
molecular weight dextran. In addition, inhibition of p53-activated
kinase (Pak1) or CtBP/BARS resulted in significant reduction in
virus entry and infection. Importantly, the virus particles appear to
stimulate uptake through this pathway directly by promoting
localized actin polymerization and this is consistent with our
previous work where the GP triggered the PI3 kinase signaling
cascade and Rac1 activity [25]. No evidence for involvement of
clathrin- or caveolae-dependent endocytosis was seen. Instead, the
primary mechanism of virus uptake appears closely related to
macropinocytosis. Subsequent to internalization, the virus utilizes
the conventional endolysosomal pathway and is trafficked through
early and late endosomes before membrane fusion takes place.
This study provides novel information regarding ZEBOV entry,
and is likely to be useful in understanding the entry mechanism of
other filoviruses.
Results
Clathrin and caveolar endocytosis are not involved in
ZEBOV virus entry
A recent study suggested that Ebola virus uses CME for cellular
entry [21], while an earlier study had implicated both CME and
CavME [20,21]. However, these studies utilized either pseudo-
typed virus, which due to morphological and/or biochemical
differences may not accurately depict the ZEBOV entry pathway,
and/or relied solely on the use of pharmacological agents, which
may alter multiple processes important for membrane trafficking.
To more closely examine the role of the each endocytic pathway
we used replication-competent infectious virus and morphologi-
cally comparable ZEBOV virus-like particles (VLPs) to determine
the impact of specific dominant-negative (DN) forms of Eps15
(OMIM:600051) and caveolin-1 (cav-1, OMIM:601047) on
infection and VLP uptake into cells. Eps15 and caveolin-1 (cav-
1) are required for the formation and trafficking of CME and
CavME vesicles respectively, and their DN forms inhibit the
respective endocytosis with high specificity [26]. HEK293T cells
were transfected with plasmid encoding GFP alone or GFP-tagged
forms of DN-Eps15 or DN-Cav1, and subsequently infected with
ZEBOV. Infected cells were detected by immunofluorescence
staining for ZEBOV matrix protein (VP40) protein and the
proportion of cells that co-expressed the transfected protein and
ZEBOV VP40 (infection marker) was calculated. It was found that
the proportion of ZEBOV-infected cells in cultures expressing
DN-Eps15-GFP or DN-Cav1-GFP was not significantly different
(P.0.05) to that in cultures expressing GFP alone, indicating that
neither DN protein had a significant impact on ZEBOV infection
(Fig. 1A). This lack of effect of either DN protein on ZEBOV entry
was confirmed using a sensitive contents-mixing entry assay that
measures virus-endosomal membrane fusion by monitoring
luciferase release from VLPs into the cell cytoplasm [25].
Expression of either DN protein failed to have any significant
effect on entry of ZEBO-VLP (P.0.05; Fig. 1B). As control, VLPs
bearing VSV envelope glycoprotein (VSV-VLP) were used. VSV
is known to use CME for cellular entry [27]. Entry of VSV-VLP
was significantly inhibited only in cells expressing DN-Eps15
(Fig.1B, P,0.001). To confirm that DN-Cav1 expression
impacted caveolar endocytosis, murine leukemia virus 10A1
(MLV-10A1) infection and cholera toxin B subunit (CTxB,
Pubchem:53787834) uptake were measured as both processes
are known to require CavME [28,29]. 10A1 infection of cells
expressing DN-Cav-1 was reduced by half (Fig. 1C) and is
consistent with previously reported observations [28]. Uptake of
CTxB (Fig. 1D) was more strongly inhibited, as cells expressing
DN-Cav1 had little CTxB inside the cytoplasm, indicating that
DN-Cav1 was functional, blocking caveolar endocytosis.
As a further test, colocalization of internalized GFP-labeled
ZEBO-VLPs (gfpZEBO-VLP) with established markers of CME
(clathrin light chain A; OMIM:118960 and transferrin;
OMIM:190000) or CavME (caveolin-1) pathways was examined.
Confocal microscopy revealed no significant colocalization of
gfpZEBO-VLP with any of the markers used (Fig. 1E). Similar
results were obtained when Vero cells were used (not shown).
Taken together, the above findings indicated that neither CME
nor CavME plays a major role in entry and infection of ZEBOV
into HEK293T or Vero cells.
Dynamin is not required for ZEBOV entry
Dynam in (OMIM:602377) is a large GTP ase and plays a
critical role in numerous endocytic pathways including CME and
CavME as well as some of the non-clathrin/non-caveolin-
dependent (NC) pathways [8]. Dynamin acts by mediating the
release of newly-formed endocytic vesicles from the plasma
membrane. To determine ZEBOV dependence on dyna min, the
effect of dynasore (Pubchem:56437635), a potent and specific
dynam in inhibitor [30] was tested. A recombinant infectious
ZEBOV that enco des GFP (gfpZEBO V) was used. This virus is
comparable to wild-type ZEBOV in terms of replication and
cytopathic effects (CPE) in cultured cells but has been engineered
to express GFP as an infection marker [31]. As control, a
recombinant infectious VSV that encoded red fluorescent protein
(rfpVSV) was used . Dynasore treatment of Vero cells greatly
reduced rfpVSV infection but failed to have any significant effect
on infection by gfpZEBOV even at the highest concentration
tested (Fig. 2A, B). Similar results were obtained in HEK293T
cells (not shown). This result was confirmed using the VLP -based
entry assay. Just like the gfpZEBOV, entry of ZEBO-VLP was
unaffected at any of the doses used while VSV-VLPs were
strongly inhibited by dynasore in a dose-dependent manner
(Fig. 2C). To ensure that dynasore inhibited dynamin-mediated
endocytosis, its effect on internalization of transferrin (CME
marker) or CTxB (CavME marker) was determined. Confocal
microscopy revealed that trea tment reduced internalization of
both markers by .80% and 96% res pectively in Vero cells
(Fig. 2D).
As a further test of the dynamin independence of ZEBOV
infection cells were made to express a DN form of dynamin-2
(Dyn2-K44A) and VLP entry assays were performed. As with
dynasore, there was a significant drop in the entry of VSV-VLP in
cells transfected with Dyn2-K44A (P,0.05). In contrast, the entry
of ZEBO-VLPs actually increased significantly (P,0.05), suggest-
ing that the suppression of dynamin function may enhance entry
of ZEBOV (Fig. 2E). Furthermore, the majority of GFP labeled
ZEBO-VLPs (gfpZEBO-VLPs) did not colocalize with endoge-
nous dynamin at any point up to 60 min after cell contact (Fig. 2F).
These findings indicated that cell entry of ZEBOV is independent
of dynamin and that dynamin activity may actually redirect virus
to a non-productive pathway.
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Figure 1. Clathrin and caveolar endocytosis are not required for ZEBOV entry. (A) Regulators of clathrin and caveolae-mediated
endocytosis are not important for ZEBOV infection. The role of proteins important for endocytosis in ZEBOV infection was assessed using
dominant negative (DN) effector proteins. HEK293T cells were transfected with plasmids encoding GFP, DN-Eps15-GFP or DN-Cav1-GFP. Twenty-four
h post-transfection cells were inoculated with wild-type ZEBOV (MOI = 0.2). Cells were fixed 48 h later, stained for nuclei using DAPI and for ZEBOV
VP40 matrix protein using a specific rabbit antiserum followed by Alexa
633
secondary antibody. Images were taken by fluorescence microscopy and
analyzed as described in the methods. To quantitate the infection dependency of ZEBOV on expression of each construct, the proportion of cells that
were expressing each GFP-tagged fusion protein and infected by ZEBOV was calculated as a fraction of the total cell population. The data were
averaged for all replicates (.5) and normalized to that seen in cells transfected with GFP alone. (B) To measure the impact of expression of each GFP-
tagged protein on the virus entry step into cells, a contents mixing assay was performed. Cells were transfected as above and then used in the assay
36 h after transfection. Both ZEBO-VLPs and VSV-G pseudotyped particles were used as indicated. Measurements were made at 3 h, at which time the
contents mixing signal peaked in untreated cells (peak is at 2–3 h post cell binding). Measurements were normalized to untransfected cells. The
results are mean 6 st.dev. of 3 independent experiments. (C) To test DN-Cav1 efficacy, HEK293 cells were transfected with plasmids encoding GFP or
DN-Cav1 tagged with GFP. Thirty six hours after transfection cells were infected with a recombinant 10A1 MLV virus encoding a truncated CD4
receptor as a marker for infection. 36 h after the infection cells were stained for CD4 expression with anti-CD4 antibody conjugated to PE (red) and
cells expressing CD4 and the GFP-tagged protein by microscopy. Data were analyzed as described in the methods and in (A). (D) Cholera toxin B
subunit uptake is blocked in cells expressing DN-Cav-1. As an additional test of DN-Cav-1 efficacy, the impact of expression on cholera toxin
subunit B (CTxB) uptake was measured. HEK293T cells were transfected with plasmid encoding GFP (left panel) or GFP-tagged DN-Cav1 protein (right
panel). Thirty-six h after transfection cells were incubated with fluorescently-labeled CTxB for 30 or 60 min, fixed and imaged. Images were taken by
confocal microscopy with a mid z-section shown. Green = GFP or DN-Cav1; Red = CTxB. (E) ZEBO-VLPs do not associate with markers of
caveolae or clathrin-coated endosomes. Vero cells were preincubated with gfpZEBO-VLPs at 16uC (to prevent endocytosis) for 15 min to allow
virus attachment. Excess virus was then removed and the temperature raised to 37uC (to initiate endocytosis) prior to fixation at indicated times. For
caveolin-1 and clathrin light chain A, permeabilized cells were stained with anti-Cav1 antibody or anti-CLCA antibody followed by Alexafluor
594
-
conjugated secondary antibody. For transferrin, Alexafluor
594
-labeled transferrin was added to cells during incubation with the VLPs. DAPI was used
to stain nuclei (blue). Images were taken by confocal microscopy with a mid z-section shown. Green = gfpZEBO-VLPs; Red = indicated endocytic
marker.
doi:10.1371/journal.ppat.1001110.g001
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Figure 2. ZEBOV entry does not require dynamin activity. (A) Dynasore does not affect ZEBOV infection. Vero cells pretreated with the
indicated doses of dynasore were incubated with either GFP-expressing infectious ZEBOV (gfpZEBOV, top panel) or RFP-expressing infectious VSV
(rfpVSV, middle panel) in the continued presence of the drug (MOI = 0.1). After 24 h, cells were washed and fixed with 10% formalin and images
taken using an epifluorescence microscope. Phase-contrast microscopy was also performed to ensure cell monolayers were intact (bottom panel). (B)
The bar graph shows quantitation of data shown in (A). For this, green fluorescent cells were counted using Cell Profiler software (Broad Inst., MA)
and normalized to the average of the untreated control. At least 4 sets of images were analyzed. Solid bars represent infection by gfpZEBOV and
open bars represent rfpVSV. Similar results were obtained with HEK293T cells (not shown). (C) Dynasore does not affect cell entry of ZEBO-
VLPs. Entry assays were performed with HEK293T cells after pre-incubation with dynasore for 1 h. Cells were then challenged with luciferase
containing ZEBO-VLP (solid bars) or VSV-VLP (open bars) for 3 h in the continued presence of the drug. Subsequently, cells were washed and
incubated with luciferase assay buffer and luciferase activity was measured. The results are expressed as luciferase activity relative to that in untreated
cells. The data represents average 6 st.dev. of 3 independent experiments each performed in duplicate. ( D) Dynasore blocks CTxB and
transferrin uptake. To confirm the activity of dynasore, Vero cells (untreated or pre-treated with 50
mM dynasore) were incubated with
Alexafluor
594
-labeled cholera toxin B subunit (CTxB) or transferrin (both red). After 1 h, cells were fixed and analyzed by fluorescence microscopy for
uptake of each marker as indicated. Nuclei (blue) were stained with DAPI. The bar graph (right panel) shows the relative amount of each probe taken
up by cells, which was determined by calculating the mean pixel intensity of the probe signal per unit area and expressed as the average of 10 cells.
(E) Dominant negative dynamin does not affect ZEBO-VLP cell entry. Effect of DN-dynamin on VLP entry was determined by using HEK293T
cells transfected with plasmid encoding GFP alone or DN dynamin2 (K44A)-GFP fusion protein (DN-Dyn2-GFP). Twenty-four h post-transfection, cells
were incubated with ZEBO-VLP or VSV-VLP for 3 h. Luciferase activity was then measured in each sample and expressed relative to that in control
Entry Pathway of Ebola Virus
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ZEBOV entry associates with lipid rafts and requires free
membrane cholesterol
Many of the cellular endocytic pathways including CavME,
macropinocytosis and certain NC pathways occur in cholesterol-
rich membrane microdomains such as lipid rafts. An earlier study
suggested that lipid rafts may play a role in ZEBOV infection [32].
Consistent with this, we found significant co-localization of
gfpZEBO-VLPs with lipid rafts during entry (Fig. 3A). Further-
more, methyl-b cyclodextrin (Pubchem:3889506) or nystatin
(Pubchem:6433272), which disrupt lipid rafts by extracting or
sequestering cholesterol out of the plasma membrane, respectively
were able to block gfpZEBOV infection in a dose-dependent
manner (Fig. 3B). Similar effects of both drugs were observed
when tested using the ZEBO-VLP-based entry assay (not shown).
These data indicated that cholesterol-rich lipid raft domains are
the likely site of ZEBOV entry.
ZEBOV entry and infection are sensitive to amiloride
treatment
The above findings clearly indicated that ZEBOV uptake
occurs through a dynamin-independent, lipid raft-dependent, non-
clathrin/non-caveolar endocytic mechanism but remains choles-
terol dependent. Macropinocytosis is one pathway that is known to
be cholesterol-dependent, but independent of clathrin, caveolin
and dynamin and has been shown important for uptake of
vaccinia virus into cells as well as bacteria [9]. Also, our previous
work indicated involvement of PI3K and Rac1 (a rho family
GTPase) in ZEBOV entry and infection [25]. Work by others had
also indicated involvement of Rho GTPase in Ebola virus entry
[23]. Each of these signaling proteins is also thought to be
important for macropinocytosis [13,33].
To assess the involvement of macropinocytosis, the effect of
EIPA (5-(N-ethyl-N-isopropyl amiloride; Pubchem:1795) on ZE-
BOV infection was determined. EIPA, an amiloride, is a potent
and specific inhibitor of Na
+
/H
+
exchanger activity important for
macropinosome formation [34,35,36,37]. Consistent with this
activity, EIPA caused a significant reduction (.80%) in the uptake
of high molecular weight dextran, a marker of macropinosomes
(Fig. 4A and B). When tested in Vero cells, a dose-dependent
inhibition of gfpZEBOV infection was observed in the presence of
EIPA (Fig. 4C, top panels; Fig. 4D), while infection by VSV was
not significantly affected (Fig. 4C, middle panels; Fig. 4D). As
used, EIPA had no significant cytotoxic effect as assessed by cell
monolayer integrity (Fig. 4C, bottom panels). Counting of infected
cells revealed that VSV infection was inhibited by 30% but
increasing the dosage of the drug did not further reduce infection,
indicating a small portion of VSV infection may occur through an
EIPA-sensitive pathway. In contrast, the majority of ZEBOV
infection inhibition was dosage dependent, potent and indicative
of inhibition of a single uptake pathway (Fig. 4D). Similar results
were observed in HEK293T cells (data not shown).
To rule out the possibility that EIPA blocked ZEBOV infection
at a post-entry step, the VLP entry assay was used. Here, EIPA
treatment had no significant effect on entry of VSV-VLP (P,0.05)
while the level of ZEBO-VLP entry was inhibited similarly to that
seen for infectious virus (Fig. 4E). The impact of EIPA on virus
binding to cells was also tested. Cells were pretreated with EIPA
and then incubated with luciferase-containing ZEBO-VLPs for
10 min on ice. Unbound particles were washed away and then the
amount of VLP associated with cells was measured by lysis in non-
ionic detergent to release virus-encapsulated luciferase. Compared
to DMSO-treated (control) cells, no significant difference was
observed in luciferase activity in samples that were treated with
EIPA, indicating that ZEBO-VLP binding to cells was unaffected
(Fig. 4F). Finally, to directly visualize the effect of EIPA on virus
uptake, Vero cells treated with DMSO or EIPA were incubated
with gfpZEBOV-VLPs. Confocal microscopy revealed that there
was a marked drop (3.5-fold) in gfpZEBO-VLP uptake in cells
treated with EIPA as compared to that in DMSO-treated cells
(Figs. 4G and H).
(untransfected) cells. The data represents average 6 st.dev. of 3 independent experiments, each performed in duplicate. (F) ZEBO-VLPs do not
colocalize with endogenous dynamin. VLP colocalization with dynamin was tested by binding gfpZEBO-VLPs to HEK293T cells at 16uC for
15 min. Cells were then shifted to 37uC to allow VLP uptake and fixed at 15 or 60 min. They were then permeabilized and stained with anti-dynamin-
2 antibody and Alexafluor
594
-conjugated secondary antibody. Images were taken with a confocal microscope with mid z-sections shown. Nuclei
(blue) were stained with DAPI. Green = gfpZEBO-VLPs; Red = endogenous dynamin-2.
doi:10.1371/journal.ppat.1001110.g002
Figure 3. Cholesterol-enriched lipid raft microdomains are
important for ZEBOV entry. (A) ZEBO-VLPs associate with lipid
rafts. Vero cells were incubated with gfpZEBO-VLP (green) at 37uC for
15 min and unbound virus was removed by washing. Lipid rafts were
visualized by first incubating the cells with Alexafluor
594
-labeled CTxB
(red) followed by coalescing the small raft domains with anti-CTxB
antibody. The samples were then fixed and images taken by confocal
microscopy. A mid z-section of the cells is shown. Insets i and ii are
enlarged images of the indicated areas. (B) Cholesterol sequestering
drugs inhibit ZEBOV infection. Vero cells were pretreated with the
indicated concentrations of methyl-b cyclodextrin or nystatin for 1 h.
Cells were then washed extensively to remove the drugs, and gfpZEBOV
was added at an MOI of 0.1. After 24 h, cells were washed and fixed.
Images were then taken with a 106 objective lens. The number of foci
of infected (gfp-expressing) cells were counted for 4 images per sample
in duplicate. The average number of foci is indicated 6 st.dev. Similar
results were obtained with HEK293T cells (not shown).
doi:10.1371/journal.ppat.1001110.g003
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Figure 4. ZEBOV uptake and infection is inhibited by EIPA and VLP uptake is associated with dextran containing vesicles. (A) EIPA
inhibits dextran accumulation into vesicles. Vero cells were treated with DMSO or EIPA (50 uM) for 30 min. Subsequently, cells were incubated
with Alexafluor
594
-labeled dextran (1 mg/ml) in the presence of the inhibitor. After 30 min, cells were washed, fixed and observed by confocal
microscopy. Nuclei (blue) were stained with DAPI. Images were taken using a 1006 oil immersion objective lens. (B) Accumulation of dextran in cells
was analyzed by counting the total number of macropinocytic vesicles (occupying .0.25
mm
2
in images) relative to the area occupied by the cell. (C)
EIPA blocks ZEBOV infection. Vero cells were pre-treated with the indicated concentrations of EIPA, followed by incubation with gfpZEBOV (top
panel) or rfpVSV (middle panel) each at MOI of 0.1 in the continued presence of the drug. Control cells received DMSO instead of the drug. After 24 h,
cells were washed and fixed. Virus infection was determined by counting fluorescent foci. Cell monolayer integrity was confirmed by phase-contrast
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ZEBOV induces fluid phase uptake and colocalizes with
internalized dextran
Vaccinia virus was shown to induce fluid phase uptake and
exhibit colocalization with fluid phase markers such as dextran
[36]. To see if a similar induction of fluid phase uptake was seen
with ZEBOV, dextran and virus were incubated together on cells
and examined by confocal microscopy. Starting within 10 min and
continuing until at least 60 min post-binding, at any one time,
approximately 20% of internalized VLPs overlapped with dextran
(Fig. 4I). An additional 20–30% of the remaining VLPs were also
found juxtaposed to vesicles containing dextran, indicating a close
association with this compartment. Furthermore, while performing
these experiments we observed that cells incubated with ZEBO-
VLPs appeared to have more dextran containing vesicles than
intact cells. Indeed, when studied in detail, a (2–3 fold) increase in
the number of dextran-containing vesicles per cell was seen after
incubation with ZEBOV as compared to cells incubated with VSV
or medium alone (Fig. 4J). A similar outcome was seen for cells
incubated with VLPs (not shown).
Activity of p53-activated kinase 1 (Pak1) is required for
ZEBOV entry
The above data indicated that cellular uptake of ZEBOV
primarily occurs through a pathway that has characteristics of
macropinocytosis. Another hallmark of macropinocytosis is its
dependence on the activity of Pak1 [9]. Therefore, the role of Pak1
in entry of ZEBOV was investigated. First, we measured the effect
of siRNA-mediated suppression of endogenous Pak1 and found
that the infection of gfpZEBOV was significantly reduced in cells
transfected with Pak1 siRNA (Fig. 5A). To confirm, cells
transfected with plasmid encoding wild-type Pak1 or DN Pak1
were challenged with gfpZEBOV. The infection of gfpZEBOV
was reduced .95% in cells expressing the DN Pak1 protein than
in cells expressing the wild-type Pak1 protein (Fig. 5B). A similar
effect of DN Pak1 was observed when ZEBO-VLPs were tested in
the entry assay (not shown). The protein CtBP/BARS, is also
known as important for macropinocytosis [15,16] and substitutes
for dynamin in promoting vesicle scission from the plasma
membrane. Again, siRNA were used to suppress expression and
the impact on infection measured. Two independent siRNA were
able to suppress expression of CtBP/BARS by .70% and 80%
respectively (Fig. 5C, left panels). Infection was also reduced by
50% and 90% respectively (Fig 5C, right). This observation
indicated that the suppression of CtBP/BARS expression must
cross a threshold before becoming limiting to ZEBOV infection
but plays an important role. Both sets of data support roles for
Pak1 and CtBP/BARS in ZEBOV infection.
Actin and actin regulatory factors play a role in ZEBOV
entry
Macropinocytosis is heavily actin-dependent. Actin is required
for the formation of plasma membrane ruffles in macropinosome
formation, as well as trafficking of macropinosomes into the cell
[38]. Ligands that utilize macropinocytosis often promote changes
in the cell actin dynamics by regulating various cellular proteins
involved in controlling F-actin assembly and disassembly. Arp2
protein is an integral component of a multi-protein complex that
serves as a nucleation site for de novo actin assembly. We observed a
significant increase in the size of Arp2-containing complexes
shortly after ZEBOV binding to cells (Fig. 5D, E). Analysis of the
data indicated a 2-fold increase in the number of large
(.0.25
mm
2
) Arp2-containing complexes (Fig. 5D). A similar
outcome was seen with cells incubated with VLPs (not shown) and
a significant proportion of VLPs were associated with Arp2
complexes (Fig. 5F). Further support for a role of actin in ZEBOV
entry came from the observation that gfpZEBO-VLPs were
associated with F-actin foci within the interior of the cell but this
was not seen for VSV-VLPs (Fig. 5G). Similarly, the gfpZEBO-
VLPs were also seen associated with vasodilator-stimulated
phosphoprotein (VASP), an actin-associated protein that promotes
actin nucleation (Fig. 5H). In each of these cases, VLPs and
staining for each marker often did not completely overlap. Instead
VLP and actin or VASP often were closely juxtaposed and is
consistent with nucleation occurring around vesicles containing
microscopy (bottom panel). (D) Quantitation of data shown in (C). Solid bars represent gfpZEBOV and open bars represent rfpVSV. Data were
normalized to the average number of foci seen for untreated cells. Similar results were obtained when HEK293T cells were used (not shown). (E)
EIPA-mediated block is at the entry step of infection. The mechanism of the EIPA-mediated inhibition of infection was examined by
performing entry assays. HEK293T cells were pre-treated with the indicated concentrations of EIPA for 1 h followed by incubation with ZEBO-VLP
(solid bars) or VSV-VLP (open bars) for an additional 3 h in the continued presence of the drug. Subsequently, cells were washed, and luciferase
activity was measured for each sample. The results are expressed as luciferase activity relative to that in control (DMSO-treated) cells. The data
represents average 6 st.dev. of 3 independent experiments each performed in duplicate. (F) EIPA does not affect ZEBO-VLP binding to cells.
HEK293 cells were pre-treated with EIPA (50
mM) for 1 h at 37uC, followed by incubation with ZEBO-VLPs for 10 min at room temperature. Cells were
then washed to remove unbound virus, resuspended in luciferase assay buffer containing triton X-100 detergent, and luciferase activity was
measured. Data were normalized to luciferase activity in vehicle-treated samples. Each data point represents mean 6 st.dev. of 3 experiments. (G)
EIPA treatment inhibits cellular uptake of ZEBO-VLPs. Vero cells were treated with DMSO or EIPA (50
mM) for 30 min. Subsequently, cells were
incubated with gfpZEBO-VLP (green) in the presence of the inhibitor. After 30 min, cells were washed, fixed and the cell periphery was visualized by
staining with phalloidin (red), staining cortical actin. Images were taken by confocal microscopy using 1006 oil immersion objective lens. Only the
mid-optical section representing the cell interior is shown. (H) VLP uptake was quantified by counting the total number of internalized VLPs in cells in
each image (4–6 cells/image). A total of 10 images were analyzed for each sample. The data are presented as average number of VLPs per cell 6
st.dev. (I) Internalized ZEBO-VLPs colocalize with dextran. HEK293T cells were incubated with gfpZEBO-VLP at 16uC. After 15 min, samples
were washed and incubated with Alexafluor
594
-labeled dextran 10,000 W (1 mg/ml) at 37uC. At indicated time intervals, cells were fixed and analyzed
by confocal microscopy. Each image represents a mid optical section. Arrowheads indicate examples of association between gfpZEBO-VLPs (green)
with dextran-containing vesicles (red). Nuclei (blue) were stained with DAPI. Quantitation of VLP colocalization (right panel). Multiple sections of
each image were analyzed for VLPs that exhibited colocalization with dextran and their number expressed as percent of total VLPs in those sections.
At least 10 images (5–6 cells/image) were analyzed for each sample. Mean 6 st.dev. are shown. Note: this is likely an underestimate of the association
as VLPs out of the plane or close to but not completely overlapping dextran positive vesicles were not counted. (J) ZEBOV induces dextran
uptake by cells. Vero cells were incubated with replication-competent VSV or ZEBOV (MOI = 5) for 15 min. Control cells were incubated with
growth medium alone (none). Cells were then washed and incubated with medium containing fluorescently-labeled dextran 10,000 MW (1 mg/ml).
After 30 min, cells were washed and fixed. Images were then taken by confocal microscopy using a 1006 oil immersion objective lens, and the
number of dextran-containing vesicles in individual cells were counted. For each sample, at least 10 images ($25 cells) representing randomly
selected fields were analyzed. The data represent mean 6 st.dev. of dextran-containing vesicles/cell. A similar outcome was observed when HEK293T
cells were incubated with ZEBO-VLP (not shown).
doi:10.1371/journal.ppat.1001110.g004
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Figure 5. Actin and actin regulatory proteins are important for ZEBOV infection. (A) Suppression of Pak1 by siRNA blocks ZEBOV
infection. HEK293 cells were transfected with siRNA targeting Pak1 (two distinct siRNA, i and ii, used) or non-targeting siRNA. Expression of Pak1 was
evaluated by Western blot using an appropriate antibody (Cell Signaling Technology, MA) and relative peak intensity determined by densitometry
using a Typhoon scanner and associated software (GE Biosciences, NJ). The impact of Pak1 suppression on gfpZEBOV infection was then determined
and expressed relative to untransfected controls. (B) DN Pak1 reduces ZEBOV infection. HEK293T cells were transfected with plasmids encoding
b-galactosidase (b-gal) or myc/GST-tagged forms of wt Pak1 or DN-Pak1. 36 h later, cells were infected with gfpZEBOV and after 24 h were fixed and
stained for myc or GST tags using appropriate primary and secondary antibodies. Cells were then imaged and analyzed as in the methods. The
proportion of cells that were expressing each tagged protein and infected by ZEBOV was calculated as a fraction of the total cell population and
expressed relative to the infection seen for cells transfected with plasmid encoding b-galactosidase. (C) Suppression of CtBP/BARS by siRNA
blocks ZEBOV infection. HEK293 cells were transfected with siRNA targeting CtBP/BARS (two used, i and ii) or non-targeting or firefly luciferase
(luc) targeting siRNA. Expression levels were determined by evaluating immunofluorescent staining intensity of CtBP/BARS in nuclei of each cell
(CtBP/BARS is predominantly localized to cell nucleus) and normalizing to the nuclear stain, DAPI and untransfected controls. The left panel shows
portion of microscope image with cell nuclei stained with DAPI or CtBP/BARS antibody and center panel shows quantitation of staining from 20,000
cells. Right panel shows impact on infection by ZEBOV-GFP. (D) ZEBOV induces Arp2-nucleation. Vero cells were incubated in medium without
virus or replication-competent infectious ZEBOV (MOI = 5) for the indicated time. Subsequently cells were washed, fixed, permeabilized and stained
for Arp2 protein using a specific antibody. The number and apparent size of Arp2 complexes was analyzed using the Analyze particles function of
ImageJ software (http://rsbweb.nih.gov/ij/). While total number of Arp2 clusters did not change, the size distribution was altered by ZEBOV
incubation with cells. This was expressed as the number of Arp2 complexes of the size ranges indicated (area occupied in image) relative to the total
number of complexes (*-P,0.05, **-P,0.01). (E) Images showing Arp2 nucleation. Arp2 (red), DAPI stained nuclei (blue). Images were taken by
confocal microscopy using a 1006 oil immersion objective lens. (F) ZEBO-VLPs associate with Arp2 complexes. Vero cells were incubated with
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the VLP. These observations suggested that the virus actively
promotes actin assembly and associates with actin-based structures
to facilitate its uptake and/or trafficking.
Intracellular trafficking of ZEBOV proceeds through early
and late endosomes
The above findings indicated that ZEBOV is primarily
internalized by a macropinocytosis-like pathway in Vero and
HEK293 cells. However, the subsequent trafficking route to the
site of penetration into the cytoplasm remained unknown. We
found that fluorescently-labeled ZEBOV particles significantly co-
localize with early endosomal antigen-1 (EEA1; OMIM:605070)
shortly after incubation with cells (Fig. 6A). At any time up to
60 min after the start of incubation, more than 30% of VLPs were
associated with this marker (Fig. 6B). This confirmed a role for
endocytic uptake into cells and suggested that following internal-
ization, ZEBOV is delivered to an EEA1-positive compartment,
likely sorting endosomes. Typically, the cargo from EEA1-positive
compartments is delivered to early endosomes followed by
trafficking to late endosomes. These vesicles are characterized by
the presence of Rab5 (OMIM:179512) and Rab7 (OMIM:602298)
GTPases on the cytoplasmic face of the vesicle, respectively, which
play a key role in regulating their trafficking. Consistent with a role
for early and late endosomes and in contrast to the lack of effect of
DN Eps15 and Cav-1 expression, GFP-tagged DN Rab5 or DN-
Rab7 resulted in significant reduction (P,0.001 for each) in
infection by gfpZEBOV (Fig. 6C). To determine if the effect was
due inhibition of virus entry, VLP entry assays were performed. As
compared to the negative control (GFP alone), wild-type Rab5 had
no significant effect on entry of either ZEBO-VLP or VSV-VLPs,
while there was .50% reduction in entry of both ZEBO-VLPs in
cells transfected with either DN-Rab5 or DN-Rab7. The level of
entry inhibition seen for ZEBO-VLP was similar to that of VSV-
VLPs (Fig. 6C) and indicated that like VSV, ZEBOV is taken up
gfpZEBO-VLPs (green) for 30 min and then fixed, permeabilized and stained for Arp2 (red) using appropriate antibodies. (G) ZEBO-VLPs associate
with actin foci and (H) VASP protein during cell entry. Vero cells were incubated with fluorescently-labeled ZEBO-VLPs or VSV-VLPs (green).
After 30 min, cells were washed, fixed and permeabilized. For actin staining, cells were incubated with medium containing fluorescently-labeled
phalloidin (red). For VASP staining, cells were incubated with anti-phospho-VASP antibody, followed by fluorescently-labeled secondary antibody
(red). Arrowheads indicate representative examples of VLP colocalization with actin or VASP. All Images were taken by confocal microscopy using a
1006 objective lens.
doi:10.1371/journal.ppat.1001110.g005
Figure 6. ZEBOV trafficking involves early and late endosomes. (A) ZEBO-VLPs colocalize with vesicles bearing early endosomal
antigen-1 (EEA1) shortly after internalization. HEK293T cells were incubated with fluorescently-labeled ZEBO-VLPs (green) for 10 min at 16uC.
After washing to remove unbound VLPs, fresh growth medium was added to cells, which were then incubated at 37uC. At the indicated time cells
were fixed, permeabilized and stained for EEA1 (red). Nuclei (blue) were stained with DAPI. Images were taken by confocal microscopy using a 1006
oil immersion objective lens. A representative image of mid-optical z-section is shown for each time point. (B) Quantitation of VLP
colocalization. VLPs colocalized with EEA1 were counted and expressed as percent of total VLPs in image sections. At least 10 images (5–6 cells/
image) were analyzed for each sample. Mean 6 st.dev. are presented in the data. (C) ZEBOV requires Rab5 and Rab7 function. HEK293T cells
were made to express GFP or GFP-tagged forms of DN Rab5, DN Rab7 by plasmid transfection. Twenty-four h post-transfection cells were incubated
with wild-type ZEBOV for 48 h. Cells were fixed after 36 h and immunostained for ZEBOV VP40 matrix protein as a marker of infection. Nuclei were
stained with DAPI and images were taken by fluorescence microscopy. Image analysis was performed using Cell Profiler software (Broad Inst. MA) as
described in methods. The proportion of cells that were expressing each GFP-tagged fusion protein and infected by ZEBOV was calculated as a
fraction of the total cell population and averaged for all replicates (.5). Data were normalized to that seen in cells transfected with GFP alone. (D)
Rab5 and Rab7 function is necessary for the cell entry step of infection. To determine the step of infection that was affected by each DN
protein, entry assays were performed using HEK293Tcells expressing GFP, or GFP-tagged forms of wild-type Rab5, DN Rab5 or DN Rab7. Cells were
incubated with VSV-VLP (open bars) or ZEBO-VLP (solid bars) for 3 h. Subsequently, luciferase activity was measured in each sample and expressed
relative to that in control (untransfected) cells. The data represents average 6 st.dev. of 3 independent experiments, each performed in duplicate.
doi:10.1371/journal.ppat.1001110.g006
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by Rab5-dependent early and Rab7-dependent late endosomes.
However, this does not mean that both virus types are present in
the same vesicle population but that similar trafficking proteins are
required at this stage of endocytosis. Currently, it is unknown
whether ZEBOV envelope fusion occurs in late endosomes or
further trafficking to a different compartment is needed.
Discussion
Endocytosis offers an efficient way for viruses to cross the
significant physical barrier imposed by the plasma membrane and
to traverse the underlying cortical matrix. Viruses have also
evolved to target distinct endocytic pathways that are capable of
delivering the capsid into the cell cytoplasm at sites suitable to
initiate replication and to avoid destructive compartments like the
lysosome. Understanding the pathway of virus entry and
deciphering the mechanism regulating it is important for
understanding viral pathogenesis as virus entry into host cell is
the first critical step in pathogenesis of infection.
While there is ample evidence that ZEBOV enters cells through
endocytosis in a pH-dependent manner [6,7,20], the specific
endocytic and trafficking pathways have not been clearly defined.
Previous studies to elucidate the ZEBOV entry pathway have
produced conflicting findings. Most of these studies relied on the
use of retrovirus-based pseudotypes in which the Ebola virus GP is
coated onto the surface of a retrovirus capsid containing a
recombinant genome. The use of this system overcomes the need
for high bio-containment but suffers from not having native virus
morphology, GP density, and other biochemical characteristics.
One early study on ZEBOV uptake using pseudotyped virus
indicated caveolae as important [22] but later work indicated that
caveolin activity was not required [39]. In contrast, a recent study
concluded that clathrin-mediated endocytosis was the major entry
pathway for ZEBOV [21]. While multiple approaches were used,
including dominant-negative mutant expression and siRNA to
specifically disrupt clathrin-mediated endocytosis, the key data was
obtained using lentivirus-based retroviral pseudotypes. In com-
parison, previous work using wild type virus [20] implicated both
clathrin and caveolar endocytosis in entry of ZEBOV. However,
only pharmacological inhibitors were used in this study and drug
specificity was not examined, making interpretation difficult.
Indeed, the only evidence of clathrin involvement in infection was
provided using chlorpromazine. Chlorpromazine is a useful drug
and there is ample evidence indicating that it disrupts clathrin-
coated pits, but it has recently been demonstrated to also interfere
with biogenesis of large intracellular vesicles such as phagosomes
and macropinosomes [24].
Here, by combining distinct and independent approaches we
have performed a detailed analysis of each major endocytic
pathway and have obtained, a clear and accurate picture of how
ZEBOV enters the cell and identified important cellular proteins
that are required. Careful assessment of specificity and function-
ality of each pathway was performed and correlated to infection
and virus uptake. Replication-competent infectious ZEBOV, as
well as ZEBO-VLPs (which are morphologically similar to
infectious ZEBOV and contain the native matrix protein in
addition to GP) were used to study the virus entry mechanism.
Drugs were used to inhibit pathways but issues of specificity and
pleiotropy were assessed by testing the function of each pathway
after treatment. This was done by using independent markers such
as transferrin, CTxB and high molecular weight dextran for CME,
CavME and macropinocytic uptake respectively. We also assessed
the association of fluorescent VLPs with each marker as well as
markers of each endocytic compartment being examined.
Furthermore, highly specific dominant-negative mutants and/or
siRNAs were also used to corroborate the data obtained by
pharmacological inhibitors. Importantly, throughout this work a
sensitive contents-mixing virus entry assay was used in discrimi-
nating against blocks in virus entry versus blocks in downstream
steps in the infection cycle. This is particularly important to do
when using drugs that often affect multiple cellular functions. It is
noteworthy that in each case, virus infection with wild type or the
GFP-expressing ZEBOV correlated exactly with the outcomes of
the VLP-based assays. This approach gives a highly detailed view
of the mechanism of ZEBOV uptake into cells.
Unlike previous studies [20,21,22], we found no evidence for
the involvement of either CME or CavME in ZEBOV entry and
infection. However, there was strong association of fluorescently-
labeled ZEBO-VLPs with lipid rafts, and a marked reduction of
ZEBOV infection by MBCD or nystatin, as reported previously
[32]. This signified that cholesterol-rich lipid raft domains are
required for productive entry of the virus. However, cholesterol-
rich membrane microdomains play important roles in many forms
of endocytosis including caveolae-dependent, non-clathrin/non-
caveolar pathways, and macropinocytosis [38,40]. Our previous
work indicated that entry of ZEBOV was dependent on signaling
through PI3K and Rac1 [25], which are important regulators of
macropinocytosis [38]. Work by others also showed that Rho
GTPases play a role in ZEBOV uptake [23]. Each of these cellular
signaling proteins are known to be important in macropinocytosis.
Macropinocytosis is also distinguished from the other pathways
principally by criteria that include actin-dependent structural
changes in the plasma membrane, regulation by PI3K, PKC, Rho
family GTPases [9,13,33], Na
+
/H
+
exchangers, Pak1, actin, actin
regulatory factors, involvement of CtBP/BARS [14,38] as well as
ligand-induced upregulation of fluid phase uptake and colocaliza-
tion of the internalized ligand with fluid phase markers [14,36,37].
In our examination of ZEBOV entry mechanism, we found that
EIPA, a potent and specific inhibitor of the Na
+
/H
+
exchanger
[34,35,36,37] blocked ZEBOV infection and entry. Furthermore,
ZEBOV caused significant induction of dextran uptake (a fluid
phase marker) and the internalized virus particles colocalized with
dextran. Pak1 regulates macropinocytosis by promoting actin
remodeling and macropinosome closure through phosphorylation
of proteins LIMK and CtBP/BARS, respectively [9,16]. We found
that suppression of both Pak1 and CtBP/BARS activity by siRNA
or expression of a DN form of Pak1 reduced virus entry and
infection.
Actin plays a central role in formation and trafficking of
macropinosomes. Actin remodeling is a key event during
macropinocytosis and is often triggered by stimuli that promote
macropinocytosis. Arp2, among other actin regulatory proteins,
has been implicated in macropinocytosis [9]. Arp2 also plays an
important role in actin remodeling. It is an integral component of
a large multi-protein complex that forms in response to stimuli that
trigger actin assembly, and serves as a nucleation site for assembly
of actin monomers to form F-actin [41]. We observed a significant
increase in the size of the Arp2-containing complexes shortly after
ZEBOV binding to cells, indicating stimulation of actin nucleation
by the virus. The increase in Arp2 nucleation paralleled an
increase in large dextran containing vesicles inside cells corre-
sponding to macropinosomes. This activity appears to be
associated with the ZEBOV glycoprotein as VLPs were also
capable of inducing a similar increase in Arp2 nucleation and
dextran uptake (data not shown). Additionally, we found marked
association of fluorescently-labeled ZEBO-VLPs with F-actin foci,
as well as with the Arp2-containing complexes and actin-
regulatory protein, VASP, that resides in membrane ruffles and
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promotes actin foci formation. Together, these data provide
evidence for a role of actin in ZEBOV entry and suggest that the
virus can actively promote localized actin remodeling to facilitate
its uptake through macropinocytosis or a similar mechanism.
Despite using multiple approaches, we found no evidence for a
role of dynamin in ZEBOV entry. Dynamin is a large GTPase that
is involved in scission of newly-formed endocytic vesicles at the
plasma membrane [42,43]. Dynamin-independent entry of
ZEBOV further ruled out roles for clathrin or caveolae-mediated
pathways as both require dynamin activity [38,44,45]. In contrast,
the majority of studies suggest that macropinocytosis is indepen-
dent of dynamin [9]. Recently a novel mechanism has been
described for scission of shigatoxin-containing vesicles in which
Arp2-dependent actin-triggered membrane reorganization directly
leads to vesicle severance [46]. As indicated above, we observed a
marked increase in the size of Arp2 complexes shortly after
incubation with ZEBOV and a significant association of ZEBO-
VLPs with these and F-actin foci but it is unclear if this resulted in
membrane scission. In addition, several reports have indicated that
C-terminal binding protein (CtBP/BARS), originally identified as
a nuclear transcription factor, likely replaces dynamin in scission of
nascent macropinosome from the plasma membrane [15,16]. As
discussed above, suppression of CtBP/BARS by siRNA reduced
infection and is consistent with the requirement for macropino-
cytosis in ZEBOV infection.
Interestingly, ZEBOV VP40-based VLPs bearing VSV enve-
lope glycoprotein were found to enter cells through clathrin-
mediated endocytosis, as has been reported for the wild-type virus
[27]. This suggested that the choice of the internalization pathway
is primarily determined by envelope glycoprotein specificity. This
is in contrast to a study in influenza virus, where profound
differences were seen in the entry characteristics of early passage
filamentous virus compared to the laboratory grown spherical
isolates that tend to use clathrin-mediated endocytosis [47]. These
data indicated a more pronounced role of virion morphology on
the choice of endocytic pathway. The apparent reason for this
discrepancy is not clear but may relate to the differences in
biological characteristics of the viruses and/or cell types used in
the two studies.
Overall, our data provide strong evidence that in HEK293T
and Vero cells infection by ZEBOV occurs by a process that is
closely related to macropinocytosis. We cannot say that entry
occurs exclusively by this pathway, but that its disruption blocks
the majority of infection and particle uptake. Our work also
indicates that clathrin and/or caveolar endocytosis play at most,
only a minor role in infection by wild type virus.
A few other viruses as well as bacteria, require macropinocytosis
to establish infection [14]. Each uses different mechanisms to
induce macropinocytosis. Vaccinia virus has been shown to trigger
macropinocytosis by mimicking apoptotic bodies [36]. In contrast,
Coxsackie virus and adenovirus activate macropinocytosis by
binding to the cell surface proteins occludin and integrin aV,
respectively [48,49]. The mechanism by which ZEBOV triggers
macropinocytosis is currently unknown but likely involves GP
interaction with cell receptors. Axl (a receptor tyrosine kinase) and
integrin bI have been suggested to act as virus receptors [50,51].
Although, the role of Axl or integrin bI has not been studied in the
context of macropinocytosis, there is evidence that several other
receptor tyrosine kinases and integrins can trigger macropinocy-
tosis [52,53,54]. Therefore, it will be important to analyze the role
of Axl and/or integrin bI in this context.
After formation, macropinosomes traffic further into the
cytoplasm and may acquire new markers and/or undergo
heterotypic fusion with other vesicles of the classical endolysosomal
pathway thereby successively transferring the cargo to more acidic
compartments such as early and late endosomes [38,55].
Consistent with this, we found that ZEBO-VLPs co-localized
with EEA1-positive vesicles soon after binding [25]. Interestingly,
the timing of colocalization of VLPs with EEA1 positive vesicles
coincided with their appearance in dextran-containing macro-
pinosomes (within 10 min after binding). Possible explanations
may be that the macropinosomes acquire EEA1 shortly after
formation or that they undergo prompt fusion with EEA1 positive
vesicles. Our data also provided evidence that ZEBOV infection
and entry was dependent on Rab5 and Rab7 function, indicating
the involvement of early as well as late endosomes in ZEBOV
uptake and infection. While a role of early endosomes in Ebola
virus entry has not been previously reported, our finding that
ZEBOV is trafficked to late endosomes is consistent with prior
studies that showed inhibition of Ebola pseudovirion infection by
dominant-negative Rab7 [56] and proteolytic processing of Ebola
GP1 by late endosome-resident cathepsins [6,7]. However, it is
important to note that many distinct endocytic vesicles associate
with Rab5 and Rab7 during maturation but differ by the ligands
they carry [57]. This explains why transferrin, a marker of CME,
was never seen associated with ZEBOV containing vesicles, even
though both require Rab5 and Rab7 for endocytosis.
The intracellular trafficking of the macropinosome is not well
understood and existing data provide evidence both for and
against the involvement of classical endolysosomal pathway [58].
However, little mechanistic information is available with respect to
virus entry by macropinocytosis. Prior to our work only one study
analyzed trafficking in any detail, using vaccinia virus and found
that virus particles did not colocalize with markers of classical
endolysosomal pathway [59]. This difference is likely due to the
fact that ZEBOV requires transport to an acidic compartment for
membrane fusion while vaccinia virus, which is a pH-independent
virus, may undergo nucleocapsid release prior to fusion of
macropinosomes with more acidic compartments of the endoly-
sosomal pathway. Our findings now add novel and valuable
information regarding macropinosome trafficking mechanism in
general and in the context of virus entry.
In conclusion, the evidence presented here demonstrates that
ZEBOV utilizes a macropinocytosis-like pathway as the primary
means of entry into HEK293T and Vero cells. Once taken up by
endocytosis, virus trafficking occurs through early and then late
endosomes; however, the exact site where envelope fusion and
nucleocapsid release occur is unknown. We do not know if
ZEBOV and other filoviruses follow the same pathway into other
cell types, like macrophages, that are thought to be a primary
target for infection. However, most cell types are capable of
macropinocytosis and it is likely that the same or a similar pathway
will be used. These findings are important as they not only
provided a detailed understanding of ZEBOV entry mechanism,
but also identified novel cellular factors that may provide new
potential targets for therapies against this virus. It will be
important to determine if other filoviruses share the same
pathway. If so, it may be possible to develop broad-spectrum
therapies that temporarily block this pathway in cells.
Materials and Methods
Cells and culture
Human Embryonic Kidney HEK293T and Vero cells were
maintained in Dulbecco’s modified Eagle’s (DMEM) medium
supplemented with 10% fetal bovine serum (Gemini Bioproducts,
GA), 1% non-essential amino acids (Sigma, MO) and 1%
penicillin-streptomycin solution (Sigma, MO).
Entry Pathway of Ebola Virus
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Reagents and antibodies
All pharmacological inhibitors were purchased from Calbio-
chem (San Diego, CA) or Sigma (St. Louis, MO). Stock solutions
were prepared either in water, DMSO, or methanol, as per
manufacturer’s recommendation, and stored at 280uC in small
aliquots. Alexafluor-labeled reagents including cholera toxin B
subunit, transferrin, dextran (10,000 MW) and secondary anti-
bodies were from Invitrogen (Eugene, OR). Specific antibodies
against clathrin light chain, caveolin, dynamin, cholera toxin B,
Arp2, CtBP/BARS, phospho-VASP and Pak1 were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) or Cell
Signaling Technology (Beverly, MA). siRNA were from Qiagen
(Valencia, CA) and transfections were performed using Dharma-
fect transfection reagent 1 according to the manufacturer’s
(Dharmacon, Lafayette, CO) instructions. 4–6 pmol of siRNA
were used per transfection of cells in 0.1 ml of medium per well of
a 96-well plate. Assays were performed 48 h after transfection.
Plasmid constructs
All plasmids were prepared using Qiagen kits or by CsCl
gradient centrifugation following standard procedures. The
plasmid encoding VSV-G envelope glycoprotein (pLP-VSVG)
was purchased from Invitrogen. Construction of the plasmid
encoding Nef-luciferase fusion protein (pCDNA3-nef-luc) has been
described previously [60]. Plasmids encoding ZEBOV matrix
protein (VP40), ZEBOV envelope glycoproteins were kindly
provided by Christopher Basler (Mount Sinai School of Medicine),
Paul Bates (University of Pennsylvania), and Luis Mayorga
(Universidad Nacional de Cuyo, Argentina) respectively. Plasmids
expressing dominant-negative Eps15, caveolin-1, and dynamin-2
K44A have been described previously [19]. The Pak1 expression
plasmids were obtained from Addgene (Cambridge, MA).
Production of virus-like particles (VLPs) containing
Nef-luciferase fusion protein
ZEBOV-VLPs were produced by co-transfecting HEK293T
cells with plasmids encoding ZEBOV matrix (VP40) protein,
ZEBOV envelope glycoproteins, and Nef-luciferase fusion protein
using the calcium phosphate method. For VSV-VLP, plasmid
encoding ZEBOV glycoproteins was replaced with one encoding
VSV-G. Cell culture supernatant was collected 48 h after
transfection and cell debris was cleared by centrifugation
(1,200 rpm for 10 min at 4uC). Subsequently, VLPs were purified
by centrifugation (25,000 rpm in SW28 rotor for 3.5 h at 4uC)
through a 20% (w/v) sucrose cushion in PBS. The VLP pellet was
resuspended in 0.01 volume of DMEM, aliquoted and stored at
4uC. Assays were performed within 2–3 days after purification of
VLPs.
VLP entry assay
HEK293T cells were used for contents mixing assays to measure
nucleocapsid release into the cell cytoplasm. The cells were removed
from plates by trypsin treatment, pelleted by centrifugation and then
resuspended in fresh medium. Cells (10
6
per assay point) were
mixed with nef-luciferase containing VLPs in a volume of 0.2 ml
and incubated at 37uC on a rotating platform for 3 h. Subsequently,
the cells were washed 2–3 times with DMEM to remove the
unbound VLPs and the final cell pellet was resuspended in 0.1 ml of
luciferase assay buffer lacking detergent (Promega, WI). Luciferase
activity was then measured using a Turner Design TD 20/20
luminometer and expressed as counts/sec.
To study drug activity on virus entry, cells were pre-treated with
drug for 1 h, followed by incubation with VLPs in the continued
presence of the drug. Virus entry was then measured as described
above. For measuring the effect of ectopic gene expression, cells
were transfected with the control plasmid or one encoding the
protein of interest. Cells were then used for entry assays 36 h after
transfection. Typical transfection efficiency was 50–70%.
Cultivation of ZEBOV and determination of virus titer
Wild type ZEBOV (Mayinga strain) was provided by Michael
Holbrook (UTMB, TX) and the recombinant virus encoding GFP
(gfpZEBOV) was from Heinz Feldman (NIH, Rocky Mountain
Laboratory, MT). The virus was cultivated on Vero-E6 cells by
infection at an MOI of approximately 0.1. All infected cells
expressed GFP approximately 24 h post-infection. Culture
supernatants were collected after 7 d and clarified by centrifuga-
tion at 2000 x g for 15 min. Virus titer was determined by serial
dilution on Vero-E6 cells. Cells were incubated with virus for 1 h
and then overlaid with 0.8% tragacanth gum in culture medium.
10 d post-infection cells were fixed with formalin, and stained with
crystal violet 10 d post-infection for plaque counting. All
experiments with ZEBOV were performed under biosafety level
4 conditions in the Robert E. Shope BSL-4 Laboratory at UTMB.
Virus infection assays
Cells were pre-treated with inhibitors for 1 h and then
incubated with gfpZEBOV at 37uC for 2 h (except in the case
of MBCD and nystatin, where cells were washed to remove the
inhibitors prior to incubation with the virus). Subsequently, the
unbound virus particles were removed by washing with PBS, and
cells incubated in fresh growth medium. Twenty-four h later, cells
were washed and fixed with 10% formalin for 48 h. Images were
taken by epifluorescence microscopy and infected foci counted.
Counting was performed using the Cell Profiler software package
[61]. The processing pipeline used by the software is available
upon request.
Immunofluorescence staining and microscopy of VLP
uptake into cells
HEK293T or Vero-E6 cells were cultivated overnight on
chambered coverglass slides (Nunc, Rochester, NY) at a density of
50%. The following day, cells were incubated with GFP-tagged
ZEBO-VLPs. Cells were then washed three times in DMEM and
fixed in 4% fresh paraformaldehyde in PBS. After one wash in
PBS residual paraformaldehyde was neutralized by addition of
0.1 M glycine buffer, pH 7.4 and cells were permeabilized using
0.1% Triton X-100 for 1 min at room temperature. For
immunofluorescence, cells were incubated with the appropriate
primary antibody, typically diluted 1:200 in PBS. After washing in
PBS, the cells were then incubated with the indicated secondary
Alexafluor conjugated secondary antibody. Cells were imaged
using a Nikon TE Eclipse inverted microscope with a 1006 oil
immersion lens or a Zeiss LSM 510 confocal microscope in the
UTMB optical imaging core.
Analysis of the impact of ectopic gene expression on
infection by replication competent viruses
Cells were transfected with plasmids encoding GFP-tagged
forms of the protein of interest. For work with Pak1, myc-tagged or
GST-tagged expression constructs were also used. After 24 h, the
cells were challenged with wild type ZEBOV. After an additional
48 h, the cells were fixed in formalin. Cells were then stained for
ZEBOV VP40 using a rabbit polyclonal antiserum (Ricardo
Carrion, Southwest Foundation for Biomedical Research, San
Antonio, TX) followed by an Alexa
633
secondary antibody. For
Entry Pathway of Ebola Virus
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Pak1 work, cells were also stained for myc-tag or GST-tag using
the corresponding primary and a fluorescently labeled secondary
antibody. Cell nuclei were also stained using DAPI (Invitrogen).
Images were taken using an epifluorescence microscope and the
intensity of GFP fluorescence and VP40 staining was evaluated on
a cell per cell basis using the Cell profiler software package [61].
For this, cells were first identified by DAPI staining of the cell
nuclei. Then cytoplasmic fluorescence intensity for GFP and VP40
staining was determined. The algorithm pipeline used for this part
of the analysis is available from R. Davey upon request. The
output data, which gives intensities on a scale of 0 to 1, was
converted to a scale of 0–1024 using Excel. This data file was then
converted to a text file and processed using A2FCS software,
which is part of the MFI/FCS Verification Suite (Purdue
University) and is available at http://www.cyto.purdue.edu/
flowcyt/software/Catalog.htm. This conversion makes the data
accessible to conventional FACS analysis software. The data were
then analyzed using FlowJo v7.5 (http://www.flowjo.com). Gates
were set to exclude cells that were not infected and not expressing
the tagged protein, as determined in control experiments. These
were done for normal cells infected by virus but not stained, cells
not infected by virus but stained with VP40 specific antibody and
cells expressing the tagged protein and not infected (stained with
antibody against the tagged protein when used). To quantitate the
infection dependency of ZEBOV on expression of each construct,
the proportion of cells that were expressing each tagged protein
construct and infected by ZEBOV was calculated as a fraction of
the total cell population. While not used here, this analytical
approach can be extended further by setting gates for low,
moderate and high levels of ectopic gene expression and then
correlating the outcome on infection.
To measure the effect of DN-Cav1 on 10A1 MLV infection,
HEK293 cells were transfected with plasmid encoding GFP or
GFP tagged DN-Cav1 protein. Thirty six hours after transfection
cells were infected with 10A1 MLV pseudotype encoding
truncated CD4 receptor (Miltenyi, Germany) as a marker for
infection. 36 h after infection the cells were stained for CD4
expression with PE-labeled mouse anti-human CD4 antibody (BD
Pharmingen Cat#555347). After 1 h the cells were washed in PBS
and fixed in 4% paraformaldehyde. Cells were stained with DAPI
to identify cell nuclei and were imaged by a Nikon TE eclipse
microscope with an automated motorized stage. To analyze the
effect of DN-Cav1 on infection, images were analyzed using Cell
Profiler software (Broad Institute, Cambridge, MA) to detect total
cells, cells expressing the expression construct and those infected
by detection of CD4. Analysis was then performed as above.
Acknowledgments
Technical support was provided by Shramika Adhikary and the work was
aided by discussions with other members of the laboratory, including
Zeming Chen, Jia Wang and Mary Miller.
Author Contributions
Conceived and designed the experiments: MFS AAK RAD. Performed the
experiments: MFS AAK TA. Analyzed the data: MFS AAK RAD.
Contributed reagents/materials/analysis tools: TA RAD. Wrote the paper:
MFS RAD.
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