JOURNAL OF VIROLOGY, Feb. 2007, p. 1230–1240
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 3
The ERK Mitogen-Activated Protein Kinase Pathway Contributes to
Ebola Virus Glycoprotein-Induced Cytotoxicity?
Carisa A. Zampieri,1Jean-Francois Fortin,2† Garry P. Nolan,2and Gary J. Nabel1*
Vaccine Research Center, NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-3005, 40 Convent Drive, Bethesda,
Maryland 20892-3005,1and Baxter Laboratory of Genetic Pharmacology, Dept. of Microbiology and Immunology,
Stanford University School of Medicine, 269 Campus Drive, CCSR, Rm. 3205, Stanford, California 94305-51752
Received 24 July 2006/Accepted 6 November 2006
Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates.
Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and
detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of
expression of a mutant form of GP lacking a mucin-like domain (GP?muc). GP-induced cell death is
nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways,
including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced
cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and
survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2,
was reduced in cells expressing GP compared to those expressing GP?muc as determined by flow cytometry,
in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and
kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption
of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown
potentiated the decrease in ?V integrin expression associated with toxicity. Conversely, activation of the
pathway through the expression of a constitutively active form of ERK2 significantly protected against this
effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a
role in pathogenicity induced by this gene product.
Ebola virus is an enveloped, negative-strand RNA virus in
the family Filoviridae, which is capable of inducing severe hem-
orrhagic fever syndrome in humans, nonhuman primates, and
other species (37). Four species of the virus have been identi-
fied to date: Zaire, Sudan, Ivory Coast, and Reston. Of these,
the highly pathogenic Zaire species produces mortality rates of
up to 90% (23). Though the pathogenic determinants of Ebola
virus remain incompletely defined, several lines of evidence
suggest that the viral glycoprotein (GP) is a key contributor to
the adverse events of infection. Expression of Ebola virus GP
from all four subtypes induces variable degrees of cytotoxicity
in cell lines and primary cells in vitro that is characterized by
cell rounding and detachment, followed by cell death (47).
Although there is some debate over the role of GP cytotoxicity
during live viral infection (2, 18, 35), differences in GP-induced
cytotoxicity are reflected in the mortality rates caused by the
different subtypes (40, 47), indicating the importance of this
gene product in the pathogenic course of the disease.
Membrane-associated Ebola virus GP is a heavily glycosyl-
ated type I transmembrane protein that is responsible for re-
ceptor binding and fusion of the virus with host cells (9, 37, 46).
During assembly, a GP precursor (GP0) is cleaved by a furin-
like protease into GP1 and GP2 subunits to form a het-
erodimer. Trimers of the GP1/GP2 heterodimer form the sole
structural protein on the surface of the virion (44). Transient
expression of Ebola virus GP induces loss of adherence from
the extracellular matrix in several different cell lines and pri-
mary cell types, including cell types infected by Ebola virus in
vivo (8, 40–42, 46). The morphological changes observed upon
GP expression in vitro correlate with the presence of a mucin-
like domain (46). Expression of wild-type GP, but not compa-
rable levels of GP lacking the mucin-like domain (GP?muc),
decreases the presence of cell surface molecules important for
cell adhesion, signaling, and immune evasion. These trans-
membrane glycoproteins include certain integrin subunits, epi-
dermal growth factor receptor (EGFR), and major histocom-
patibility complex class I (40–42). Since integrin-mediated
adhesion is important in cell-matrix interactions and intracel-
lular signaling, it is thought that the decreased surface expres-
sion of these important molecules may play a major role in the
induction of rounding and detachment by GP (40, 41).
In this study, we examined the effects of Ebola virus GP
expression on pivotal proteins in several cellular signal trans-
duction pathways and investigated whether they contribute to
GP-induced cytotoxicity. Using flow cytometric analysis of
phosphoprotein expression, we surveyed the activation of sev-
eral proteins important in cell growth and survival, including
the mitogen-responsive extracellular regulated kinases 1 and 2
(ERK1/2) and p38, the stress-activated c-Jun-NH(2) terminal
kinase (JNK), and the tumor suppressor protein p53. We
found that GP expression was associated with a mucin domain-
dependent reduction in active levels of mitogen-activated pro-
tein kinase (MAPK) effector ERK2, a kinase that is important
* Corresponding author. Mailing address: Vaccine Research Center,
NIAID, National Institutes of Health, Room 4502, Bldg. 40, MSC-
3005, 40 Convent Drive, Bethesda, MD 20892-3005. Phone: (301)
496-1852. Fax: (301) 480-0274. E-mail: firstname.lastname@example.org.
† Present address: Research and Development, Boehringer In-
gelheim (Canada) Ltd., 2100, Cunard St., Laval, Que ´bec, Canada
?Published ahead of print on 15 November 2006.
in mediating cellular responses such as proliferation, cell cycle
progression, and survival. Further analysis indicated a role for
this kinase in GP-mediated cell rounding and detachment.
Together these data provide insight into the signaling pathways
modulated by Ebola virus GP and suggest a new potential
therapeutic target against GP-mediated cytopathicity.
(This work is included in a dissertation presented to the
Genetics Program of the Institute for Biomedical Sciences at
the George Washington University, Washington, DC, by
Carisa Zampieri in partial fulfillment of the requirements for
the Ph.D. degree).
MATERIALS AND METHODS
Plasmids and cell culture. Expression vectors p1012, pGP(Z), and pGP?muc
contain a cytomegalovirus enhancer promoter as previously described (46). The
ERK1DN, ERK2-MEK1, ERK2-MEK1LA, and KRERK2-MEK1LA expres-
sion vectors were generously provided by Melanie H. Cobb (University of Texas
Southwestern Medical Center, Dallas, TX). Human embryonic kidney 293 cells
were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum, penicillin, and streptomycin (GIBCO). Cells were transfected
using either the Fugene 6 (Roche) or Lipofectamine 2000 (Invitrogen) transfec-
tion reagent according to the protocol supplied by the manufacturer.
Flow cytometric detection of intracellular phosphoproteins. Cells (1.2 ? 106)
were seeded in a 10-cm2plate 1 day prior to transfection. The cells were trans-
fected with 5 ?g of plasmid by using Fugene 6 transfection reagent. At 12, 18, 24,
and 36 h posttransfection, cells were harvested by pipetting up and down six
times to resuspend both adherent and nonadherent cells. They were centrifuged
for 5 min at 900 ? g and fixed by resuspension in cold phosphate-buffered saline
(PBS) containing 2% paraformaldehyde to have a concentration of no more than
1 ? 106cells/ml. Cells were incubated at room temperature for 10 min, centri-
fuged for 5 min at 900 ? g, and resuspended in cold PBS at a concentration of
10 ? 106cells/ml. Nine volumes of cold methanol were slowly added to the cell
suspension during vortexing to permeabilize the cells. This fixation decreased the
reactivity of antibodies with GP?muc relative to GP when equivalent levels were
detected by cell surface staining. The cells were then incubated on ice for 30 min
and stored at ?80°C. To rehydrate the cells prior to staining, cells in methanol
were diluted with 2 volumes of staining buffer, spun at 800 ? g for 10 min at 4°C,
and then resuspended in staining buffer at a concentration of 107per ml. Cells
were stained for 30 min on ice with the indicated antibodies (see below) and
washed once with staining buffer. Cells were analyzed on a FACSCalibur appa-
ratus. Cells were stained with antibodies to GP (biotin-conjugated mouse mono-
clonal antibody followed by streptavidin-phycoerythrin) and phosphorylated pro-
teins phospho-ERK1/2 (clone 20a, pT202/pY204), phospho-p38 MAPK (clone
36, pT180/pY182), phospho-p53 (polyclonal, pS46), and phospho-JNK1/2 (poly-
clonal, pT183/Y185) conjugated to either Alexa Fluor 647 or Alexa Fluor 488
Phospho-MAPK antibody arrays. 293 cells (1.2 ? 106) were transfected in
10-cm plates with 5 ?g of control vector, GP, or GP?muc plasmid by using
Fugene 6 transfection reagent. An analysis of the phosphorylation states of all
MAPKs was performed using a human phospho-MAPK array kit, equivalent to
immunoprecipitation and Western blot analysis (R&D Systems, Minneapolis,
MN). At 24 h after transfection, cells were rinsed with PBS and lysed with the
buffer provided. Arrays were incubated overnight at 4°C with 250 ?g of lysate
from control-, GP-, or GP?muc-transfected cells. The arrays were washed three
times with 20 ml of wash buffer provided and incubated for 2 h with the provided
detection antibody cocktail containing phospho-site-specific MAPK biotinylated
antibodies. The wash steps were repeated, after which the arrays were exposed to
chemiluminescent reagents and film. The data on the developed X-ray film were
scanned and quantitated using image analysis software (Quantity One).
ERK1 and ERK2 kinase assays. p44/42 kinase assays were performed using
nonradioactive kits from Cell Signaling Technology (Beverly, MA). Briefly, 293
cells were transfected with control, GP, or GP?muc expression vectors. The cells
were harvested and lysed with the provided 1? lysis buffer. The protein content
in the lysates was determined by the Bradford method (7). Either 25 ?g, 100 ?g,
500 ?g, or 1 mg of cell lysates was immunoprecipitated with polyclonal antibodies
against total ERK1 (Upstate Cell Signaling Solutions) or ERK2 (Upstate Cell
Signaling Solutions) by use of immobilized protein G (Invitrogen). The precip-
itated enzymes were then used for kinase assays with Elk-1 substrate followed by
Western blot analysis with antibodies that allow detection and quantitation of
siRNA-mediated knockdown. The ERK2-specific short interfering RNAs
(siRNAs) (siRNA-A [GGGUUCCUGACAGAAUAUGtt] and siRNA-B
[GGAAAAGCUCAAAGAACUAtt]) and the negative control siRNA (con-
trol siRNA, Silencer negative control 1) were obtained from Ambion (Austin,
TX). Lowercase letters indicate sequences not complementary to ERK2 but
necessary for SiRNA duplex function. 293 cells (1 ? 105) were transfected
with 25 ?M of each siRNA by using Lipofectamine transfection reagent.
After a 24-h incubation, cells were transfected in the same manner with
siRNA as well as 250 ng of plasmids expressing the control or GP. At 2, 3, and
4 days after the initial siRNA transfection, cells were harvested and analyzed
for both GP cytotoxicity and ERK2-specific knockdown by using flow cytom-
etry and Western blot analysis followed by quantitation (Quantity One),
respectively, as described below.
Western blot analysis. Cell lysis for Western blotting was performed in cell
lysis buffer (Cell Signaling Technology). Antibodies against GP (rabbit necropsy
serum), ERK1/2 (Cell Signaling Technology), ERK2 (Cell Signaling Technol-
ogy), and ?-actin (Sigma) were used in immunoblotting in accordance with the
GP cytotoxicity assay. To quantitate GP-induced cytotoxicity, we measured the
amount of ?V integrin downregulation in GP-expressing cells as previously
described (41). Briefly, 293 cells were transfected with the indicated plasmid
vectors and analyzed by fluorescence-activated cell sorting (FACS) for cell sur-
face expression of both Ebola virus GP and ?V integrin surface molecules at 24
and 48 h after transfection. Both adherent and detached cells were collected and
incubated with antibodies to Ebola virus GP and integrin ?V for 30 min on ice.
The cells were washed with 1 ml ice-cold PBS containing 2% fetal bovine serum
and incubated with allophycocyanin (Jackson ImmunoResearch Laboratories)-
or phycoerythrin (Sigma)-conjugated secondary antibodies for 30 min on ice.
Cells were washed as described above and resuspended in PBS containing 1%
paraformaldehyde. Analysis was conducted using a Becton Dickinson four-color
Calibur flow cytometer and FlowJo analysis software.
Flow cytometric analysis of kinase phosphorylation in cells
expressing Ebola virus GP. Ebola virus GP modulates the
surface expression of cellular proteins, downregulating cell sur-
face integrins, major histocompatibility complex class I, and
other cell surface molecules (40–42). Since these proteins have
been implicated in cellular signaling pathways, we analyzed the
activation of several kinases and molecules involved in cell
growth and survival to determine if perturbation in one or
more signaling pathways correlated with GP cytotoxicity. Phos-
phoprotein flow cytometry was used to analyze the phosphor-
ylation states of the signaling molecules ERK1/2, JNK, p38,
and p53 in cells expressing Ebola virus GP. Human embryonic
kidney 293 cells were transfected with plasmids expressing
full-length GP, GP?muc, or a no-insert vector control, and
phosphorylation states were analyzed at 12, 18, 24, and 36 h
At 18 h after transfection, the expression of Ebola virus GP
reduced the levels of phosphorylated ERK1/2 and, to a lesser
extent, p38 but not those of JNK or p53 (Fig. 1A). At this time
point, the ratio of high- to low-phosphorylated ERK1/2 among
the GP-expressing cells was approximately 1 to 3 (Fig. 1A, top
middle panel [15.3 versus 43.9%, right upper versus lower
quadrant]). In contrast, this ratio was slightly greater than 1 in
GP?muc-transfected cells (Fig. 1A, top right panel [23.6 versus
18.9%, right upper versus lower quadrant]). This effect was
also apparent at the earliest, 12-h time point, when the ratio of
high- versus low-phosphorylated ERK1/2 in GP-transfected
cells was approximately 1 to 2 (Fig. 1B, middle panel [14.3 to
25.2%, right upper versus lower quadrant]). The reduction in
ERK1/2 phosphorylation remained constant and did not
change significantly beyond the 18-h time point when exam-
VOL. 81, 2007ROLE OF ERK2 IN EBOLA VIRUS GP TOXICITY1231
FIG. 1. Expression of Ebola virus GP decreases phospho-ERK1/2, the active form of this kinase. (A) 293 cells were transfected with a plasmid
encoding a vector control, Ebola virus GP, or GP?muc. Both adherent and nonadherent cells were collected at 18 h after transfection. The cells
were fixed, permeabilized, and stained with antibodies to Ebola virus GP and the phosphorylated forms of the signaling kinases ERK1/2, JNK, p38,
and p53. (B) 293 cells were transfected and stained as described for panel A. Cells stained with antibodies against Ebola virus GP and
phospho-ERK1/2 are shown at 12 h.
1232 ZAMPIERI ET AL.J. VIROL.
ined at 24 and 36 h posttransfection (data not shown). An
increase in the phosphorylation of the tumor suppressor gene
p53 at residue S46 was also observed (Fig. 1A, lower row), but
it occurred both in cells expressing GP and in those expressing
GP?muc and therefore was deemed unlikely to be related to
GP-induced cytotoxicity. GP expression had little effect on the
activation state of the JNK kinase (Fig. 1A). Although a mod-
est difference in the staining of intracellular GP and GP?muc
was observed (Fig. 1A and B), this effect likely does not reflect
real differences in the levels of these proteins in cells. The GP
monoclonal antibody detects similar levels of GP and GP?muc
cell surface staining (one- to twofold differences) in transfected
cells. However, in order to measure intracellular kinase levels,
the cells were fixed and permeabilized prior to staining. This
procedure affected antibody binding to GP?muc more than
that to GP (data not shown). Taken together, these results
suggest that GP expression correlates with a specific reduction
in the phosphorylation of ERK1/2 and that the perturbation of
this kinase requires the mucin-like domain of GP.
A survey of the MAPK family reveals reduced ERK2 phos-
phorylation in cells expressing GP. ERK1 and -2 are members
of the mitogen-activated protein kinase family. To determine
whether GP expression modulated phosphorylation of other
kinases in this family, we examined the activities of several
related proteins. At 24 h after transfection, the levels of phos-
phorylated MAPK proteins in the cell lysates were analyzed
using a human phospho-MAPK array. GP expression signifi-
cantly reduced the amount of phosphorylated ERK2 compared
to that with a vector control (Fig. 2A) (P ? 0.01) and reduced
ERK1 (P ? 0.1) and p38 (P ? 0.02) expression to lesser extents
that were either not statistically significant (ERK1) or less
substantial (p38). The effect on ERK2 was dependent on the
mucin domain: cells expressing GP?muc had significantly
higher levels of phosphorylated ERK2 than cells expressing
full-length GP (P ? 0.007). GP had little effect on other ki-
nases, including JNK (Fig. 2A) and Akt and GSK-3? (data not
shown). However, there were also no significant changes in the
activation state of Rsk-1, a downstream substrate of ERK1 and
ERK2. This may be due to the relatively low levels of Rsk-1 in
unstimulated 293 cells, making changes in phosphorylation
difficult to observe. In addition, GP expression did not alter
phosphorylation of the unrelated heat shock protein 27 (Fig.
2A). Expression levels of the transfected plasmids and total
ERK1 and ERK2 protein levels determined by Western blot-
ting were the same in all lysates (Fig. 2B), indicating that the
reduction in ERK2 phosphorylation was not the result of re-
duced kinase levels in the cells. The decreased intensity of
GP?muc relative to GP on the Western blot is likely due to the
presence of fewer antigenic determinants on the protein, be-
cause a significant portion is removed by the deletion muta-
tion. These data demonstrate that GP exerts a more pro-
FIG. 2. Ebola virus GP specifically decreases the phosphorylation of the mitogen-activated protein kinase ERK2. (A) 293 cells were transfected
with a plasmid encoding a vector control, Ebola virus GP, or Ebola virus GP?muc. After 24 h, both adherent and nonadherent cells were harvested,
lysed, and analyzed for MAPK activity as described in Materials and Methods. The results are representative of two separate experiments done
in duplicate. Error bars indicate standard deviations. (B) The cell lysates obtained from the experiment described for panel A were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gels were immunoblotted using antibodies specific for Ebola virus GP, total
ERK1/2, and total ERK2. The two bands in the GP immunoblot correspond to the cleaved and uncleaved versions of the GP and GP?muc proteins
VOL. 81, 2007ROLE OF ERK2 IN EBOLA VIRUS GP TOXICITY1233
nounced effect on the activation state of a specific MAPK,
ERK2, which is mediated by the mucin-like domain.
ERK activity is inhibited by GP expression. ERK1 and
ERK2 are closely related enzymes. In order to confirm a spe-
cific effect of GP on the activity of ERK2, we performed kinase
assays with both ERK1 and ERK2 from control-, GP-, or
GP?muc-transfected cells. Cells were harvested at 24 h after
transfection, and various amounts of lysates were immunopre-
cipitated using antibodies against either ERK1 or ERK2. The
immunoprecipitated kinases were incubated with the ERK1/2
substrate Elk-1. Elk-1 phosphorylation was then detected by
Western blotting using an anti-phospho-Elk-1 antibody. Ex-
pression of GP reduced Elk-1 phosphorylation of both ERK1
and ERK2, but the reduction in ERK2 activity was more sub-
stantial and significant (Fig. 3A). Quantitation of band inten-
sities from 500 ?g of lysate revealed a 34% reduction in ERK1
activity versus a 72% reduction in ERK2 activity compared to
control-transfected cells (Fig. 3B). The expression of GP?muc
stimulated both ERK1 and ERK2 activities. This phenomenon
was also observed in the MAPK array analysis of ERK1 phos-
phorylation (Fig. 2A). Taking these results together, we sug-
gest that ERK1 may play a contributory role in GP toxicity,
though it is likely to be necessary and not sufficient for this
effect. Western blotting of cell lysates indicated that the alter-
ations in kinase activities were not due to differences in the
amounts of ERK1 and ERK2 proteins within the cells (Fig.
3C). These results suggest that GP expression decreases ERK2
and, to a lesser extent, ERK1 kinase activities.
Expression of a dominant negative ERK potentiates GP
cytotoxicity. To investigate further the role of ERK activation
on GP-induced cytotoxicity, the effects of a dominant negative
ERK1 K71R (ERK1 DN) mutant protein were examined di-
rectly by counting the percentage of rounded and detached
cells and also by flow cytometry to quantitate ?V integrin
FIG. 3. Effect of GP expression on the kinase activities of ERKs. (A) 293 cells were transfected with a control plasmid lacking an insert, GP,
or GP?muc expression vectors. Cells were harvested and lysed at 24 h after transfection. Total ERK1 and ERK2 were immunoprecipitated from
increasing amounts of cell lysates, and the effects of GP expression on ERK1 and ERK2 kinase activities were determined as measured by
phosphorylation of the substrate Elk-1. (B) The band intensities (500 ?g lysate input) were quantitated to obtain percent kinase activities. (C) The
cell lysates obtained from the experiment described for panel A were analyzed by Western blotting using an antibody specific for total ERK1/2.
1234 ZAMPIERI ET AL.J. VIROL.
surface expression by comparison. The FACS-based assay was
used to quantitate GP cytotoxicity because cell surface down-
regulation of ?V integrin has been shown to correlate with
rounding and detachment in previous studies (40–42). Briefly,
293 cells were transfected with an empty vector control or a
vector containing the dominant negative ERK1 DN. This mu-
tant is deficient in kinase activity and binds to substrate, thus
inhibiting the substrate’s access to catalytically active ERK1
and ERK2 (25). Cells were cotransfected with increasing
amounts of either an empty vector control or a GP or GP?muc
expression vector. After 24 h, cells were harvested and dually
stained for GP and ?V integrin. Expression of GP decreased
FIG. 4. A dominant negative form of ERK1/2 increases the cytotoxicity of Ebola virus GP. 293 cells were cotransfected with 50 ng, 100 ng, or
250 ng of Ebola virus GP or an empty vector control and 2 ?g of ERK1 DN as indicated. Cells were incubated for 24 h, and both floating and
adherent cells were counted to calculate percent rounding and then collected for staining with ?V integrin and GP antibodies followed by FACS
analysis. FACS results are shown for events in the live cell gate and are representative of at least three independent experiments. Error bars
indicate standard deviations.
VOL. 81, 2007 ROLE OF ERK2 IN EBOLA VIRUS GP TOXICITY1235
surface ?V integrin in a dose-dependent manner with a con-
comitant decrease in GP expression, giving a “comma” appear-
ance (Fig. 4A, top row), possibly due to the surface removal of
both ?V integrin and GP, as noted previously (41). Deletion of
the mucin-like domain, which eliminates toxicity, completely
reversed the decrease in ?V integrin levels (Fig. 4A, bottom
row). In contrast, cells cotransfected with dominant negative
ERK and GP showed a substantial increase in ?V integrin
downregulation. The number of cells with surface ?V integrin
increased substantially when cells were transfected with 50 ng
of GP as well as the ERK1 DN construct (Fig. 4B, third panel
versus fourth panel [9.69% versus 18.8%, lower right quad-
rant]). The expression of ERK1 DN alone had no effect on ?V
surface expression (Fig. 4B, second panel). To directly mea-
sure cytotoxicity, both adherent and rounding cells in Fig. 4B
were counted prior to staining, and the percent rounding was
calculated (Fig. 4C). At the lowest GP dose, GP expression
increased the percentage of rounded cells, compared to less
than 5% in control-transfected cells (Fig. 4C) (P ? 0.05). The
addition of ERK1 DN further increased cell rounding to ap-
proximately 20% (Fig. 4C) (P ? 0.03). The percent rounding
corresponded well to the quantitation of cytotoxicity by using
the FACS-based approach and indicate that this indirect ap-
proach provides consistent and accurate measurements of GP-
induced cytotoxicity. Together, these results suggest that inhi-
bition of active ERK1/2 through the expression of a dominant
negative ERK potentiates GP-induced toxicity as measured by
both downregulation of ?V integrin surface molecules and cell
siRNA-mediated knockdown of ERK2 increases the GP-in-
duced phenotype. Since ERK1 and ERK2 sequences are highly
homologous within their catalytic cores, the dominant negative
ERK1 K71R protein can inhibit both protein kinase activities
(10, 25). To determine whether the reduction in endogenous
ERK2 function alone was sufficient to enhance GP-induced
cell rounding and detachment, two ERK2-specific siRNA du-
plexes, ERK2 siRNA-A and ERK2 siRNA-B, directed against
different regions of ERK2 mRNA were utilized to specifically
decrease endogenous ERK2 protein expression. These two
siRNA constructs were transfected in parallel with a negative
control siRNA into 293 cells. After 24 h, cells were transfected
again with the same siRNA duplexes and with plasmids ex-
pressing GP or a control empty vector plasmid. Cells were
collected at 48, 72, and 96 h after the initial siRNA transfec-
tion, and at each time point the amount of GP-induced ?V
integrin downregulation was quantified by flow cytometry. The
specificity of the ERK2 siRNAs was confirmed by Western blot
analysis (Fig. 5A). At 96 h, ERK2 protein levels were reduced
to 8% and 3% of normal levels (compared to control siRNA)
with ERK2 siRNA-A and ERK2 siRNA-B, respectively. At
this time, cells expressing GP and treated with the siRNAs
demonstrated greater ?V integrin downregulation than that
induced by GP with control siRNA. Indeed, there was approx-
imately threefold and twofold more downregulation of ?V
integrin in cells treated with ERK2 siRNA-A (Fig. 5B, top left
versus top middle panel [8.25 versus 26%, right lower quad-
rant]) and ERK2 siRNA-B (Fig. 5B, top left versus top right
panel [8.25 versus 19.9%, right lower quadrant]), respectively.
ERK2 knockdown had minimal effects on ?V integrin surface
levels when transfected with vector control (Fig. 5B, lower
panel). The results are representative of two independent ex-
periments using three different amounts of GP. Taken to-
gether, these results suggest that the reduction in cellular
ERK2 levels enhances the cytotoxicity induced by GP.
Expression of a constitutively active ERK2 reduces GP-
induced cytotoxicity. Since an increase in GP-induced cytotox-
icity was observed when ERK2 activity was diminished, we
examined whether constitutive activation of ERK2 signaling
would decrease this effect. Robinson et al. previously demon-
strated that the fusion of wild-type ERK2 to its upstream
regulator MAPK, MEK1 (ERK2-MEK1), produces a consti-
tutively active form of ERK2 (32). A nuclear form of this
fusion protein (ERK2-MEK1LA) is active in the absence of
extracellular signals and does not activate endogenous ERK1
or ERK2. The expression of this fusion protein is sufficient to
cause several transcriptional and phenotypic responses in
mammalian cells. Expression of inactive ERK2 fused to the
upstream regulator (KRERK2-MEK1LA) eliminates the cat-
alytic activity of the fusion protein and acts as a negative
control for the constitutively active fusion protein (32). We
examined the effects of the expression of these constructs on
GP cytotoxicity by cotransfecting 293 cells with expression vec-
tors encoding GP and either ERK2-MEK1, ERK2-MEK1LA
(active ERK2), or KRERK2-MEK1LA (inactive ERK2). Cells
were incubated for 24 h, after which the amount of cytotoxicity
was quantified with the flow cytometric assay for ?V integrin
downmodulation. None of the three fusion proteins, ERK2-
MEK1, ERK2-MEK1LA, and KRERK2-MEK1LA, had an
effect on basal ?V integrin surface expression in the absence of
GP (Fig. 6, upper row). Cells cotransfected with constitutively
active ERK2 and GP showed 50% less surface downregulation
of ?V integrin than cells transfected with GP alone (Fig. 6, first
versus third lower panel [23.2 versus 56.6%, right lower quad-
rant]). In contrast, expression of the kinase-inactive KRERK2-
MEK1LA fusion protein had no effect on ?V integrin down-
modulation by GP (Fig. 6, lower row, first versus fourth panel
[56.6 versus 52.9%, right lower quadrant]). In addition, cells
cotransfected with GP and the cytoplasmic form of the fusion
protein (ERK2-MEK1) had a more modest decrease in ?V
integrin surface expression than cells transfected with GP
alone (Fig. 6, lower row, first versus second panel [56.6 versus
34.8%, right lower quadrant]). These data demonstrate that
GP-mediated ?V integrin downmodulation and cytotoxicity
are diminished by expression of constitutively active ERK2.
The envelope protein of Ebola virus has been shown to
mediate cell rounding and detachment in vitro and in vivo and
has been implicated in the pathogenicity of Ebola virus infec-
tion (41, 47). Ebola virus GP cytotoxicity correlates with the
downmodulation of several surface proteins important in ad-
hesion and signaling, including certain integrins and EGFR
(40–42). It is also dependent on recycling and endocytosis
mediated by the cytoskeletal modulator dynamin (41). Al-
though GP expression induces changes in cell surface protein
levels, the mechanisms of surface protein downmodulation and
cytotoxicity, as well as the cellular signaling events that mediate
these effects, have not been defined. This study has analyzed
the activation of several proteins that are pivotal to normal cell
1236 ZAMPIERI ET AL.J. VIROL.
signaling. Ebola virus GP significantly decreased the activation
of ERK2, and this effect was dependent on the mucin-like
domain of the glycoprotein, which is also required for cellular
cytotoxicity (40, 42, 47). Inhibition of ERK2 activity, either
through the overexpression of a catalytically inactive dominant
negative form of ERK1/2 or through specific knockdown of
endogenous ERK2, enhanced GP-induced ?V integrin down-
regulation. Conversely, expression of a constitutively active
ERK2 significantly decreased ?V integrin downmodulation.
Together, these data demonstrate that GP mediates its effect
on cellular viability and integrin expression at least in part
through ERK2 activity.
Cell adhesion is essential for proper cellular gene expres-
sion, growth, differentiation, and survival (19, 22, 38, 39). In-
tegrins are pivotal to cell-cell and cell-matrix adhesion. Previ-
ously appreciated primarily for their role in the maintenance of
adhesion, they are now well recognized as signaling receptors
that regulate the cellular response to mitogens and intersect
other signaling networks such as the ERK/MAPK cascade (22,
39). Several studies have shown that there is strong and sus-
FIG. 5. Ebola virus GP cytotoxicity is increased by siRNA-mediated knockdown of ERK2. 293 cells were transfected with 25 ?mol of control
siRNA, ERK2 siRNA-A, or ERK2 siRNA-B, and 24 h later cells were transfected again with 250 ng of plasmid encoding vector control or Ebola
virus GP and another 25 ?mol of control siRNA, ERK2 siRNA-A, or ERK2 siRNA-B. The cells were incubated for another 72 h, after which both
floating and adherent cells were collected and either lysed for Western blot analysis of ERK2 knockdown (A) or double stained with GP and ?V
integrin antibodies followed by flow cytometric analysis (B). Results are representative of two independent experiments.
VOL. 81, 2007 ROLE OF ERK2 IN EBOLA VIRUS GP TOXICITY1237
tained ERK activity in adherent cells (reviewed in references 4
and 5). In contrast, endothelial cells treated with integrin
?V?3 agonists in vivo show diminished long-term ERK acti-
vation (15). GP expression substantially reduced surface ?V
integrin levels as well as a loss of cell adherence. It may be that
the observed GP-induced reduction in ERK activity is an up-
stream signaling event which subsequently leads to surface
integrin downmodulation. However, given the importance of
cell adhesion and integrins in the regulation of the ERK path-
way, it is likely that the diminished ERK signaling is down-
stream of GP-induced cell rounding and detachment. This
effect was most pronounced in ERK2 and was less obvious in
other mitogen-activated kinases such as JNK and p38, which
are not regulated by integrin-mediated adhesion (3).
Our results suggest that GP affects the catalytic activity of
the ERK2 protein. Given the 80% sequence homology be-
tween ERK1 and ERK2 (10) and their similar modes of reg-
ulation, the preferential effect on ERK2 was not anticipated;
however, several studies have shown more recently that despite
their sequence homology, these two proteins have unique
properties (16, 36, 43). For instance, although the proteins are
coexpressed in virtually all tissues, their relative abundances
vary widely. In addition, ERK1-deficient mice have a different,
significantly milder phenotype than ERK2-deficient mice,
which die early in development (20, 29). These recent findings
suggest that ERK1 and ERK2 are not redundant proteins and
that they have individual functions in cell signaling. In addi-
tion, the results presented here show a similar effect, to a lesser
degree, on ERK1, Thus, the finding that GP expression has a
more pronounced effect on ERK2 activity is consistent with the
current understanding of ERK1 and ERK2 function.
While this study demonstrates that GP expression reduces
ERK2 phosphorylation and activation, the data also provide
evidence that increasing or decreasing active ERK2 levels in-
versely affects GP-induced rounding and detachment. While
the events that ensue to induce cell death are unknown, ERK2
is a pivotal protein within the ERK/MAPK signaling cascade,
and it is likely that downstream substrates of ERK2 mediate
GP cytotoxicity. Once activated, the ubiquitously expressed
ERK1 and ERK2 MAPKs modify a diverse array of substrates
that relay signals mediating critical cellular responses (re-
viewed in reference 48). They phosphorylate approximately
160 proteins with substantial regulatory functions, including
other protein kinases, transcription factors, cytoskeletal pro-
teins, and other enzymes (10, 48). Reduced ERK2 activity
during GP expression may lead to altered activity of one of
these diverse substrates, thereby increasing cell rounding and
detachment. The results from experiments utilizing constitu-
tively active ERK2 indicate that at least one important sub-
strate most likely localizes to the nucleus. The expression of a
nuclear form of constitutively activated ERK2 decreased cell
rounding and detachment, an effect not observed with a cyto-
plasmic version of active ERK2. A study by Robinson et al.
demonstrated that certain functions of ERK2 are dependent
on the nuclear localization of the active enzyme (32). In addi-
tion, it has been shown that adhesion to the extracellular ma-
trix is required for efficient accumulation of activated ERK in
the nucleus (4, 11). This finding suggests that the loss of ad-
herence in GP-expressing cells decreases active and nuclear
ERK2 levels. Substrates of nuclear ERK2 include several tran-
scription factors which regulate the expression of a large array
of genes involved in diverse cellular processes and could affect
cell viability. At the same time, we cannot exclude the possi-
bility that a cytoplasmic protein, such as a cytoskeletal sub-
strate, may be the target of phosphorylation. Indeed, one pos-
sible cytoplasmic target of ERK2 is dynamin, an important
FIG. 6. Expression of a constitutively active ERK2 decreases Ebola virus GP-induced cytotoxicity. 293 cells were cotransfected with 250 ng of
plasmid encoding a vector control (upper row) or Ebola virus GP (lower row) and 1 ?g of either vector control, ERK2-MEK1, ERK2-MEK1LA,
or KRERK2-MEK1LA as indicated and were incubated for 24 h. Supernatant cells were collected and pooled with adherent cells, double stained
with antibodies specific for Ebola virus GP and ?V integrin, and analyzed by flow cytometry. Results are shown for events in the live cell gate and
are representative of at least three independent experiments. WT, wild type.
1238ZAMPIERI ET AL. J. VIROL.
mediator of membrane internalization and integrin receptor
endocytosis (12, 14, 21). Previous results have shown that a
dominant negative version of this protein reduces GP-induced
integrin downmodulation (41). However, we were unable to
demonstrate an interaction between these two proteins (data
not shown), suggesting that the observed effects of ERK activ-
ity on GP cytotoxicity are not mediated by dynamin.
Several viruses have been shown to modulate the ERK/
MAPK signaling cascade, including influenza virus (31), borna
disease virus (30), coxsackievirus (26), visna virus (6), human
immunodeficiency virus (HIV) (28), vaccinia virus (13),
Epstein-Barr virus (17), cytomegalovirus (33), and human
herpesvirus 8 (1). These viruses stimulate the activation of
this pathway, which results in efficient cell cycle promotion
as well as high cellular and viral gene production. Fewer
studies have demonstrated a negative correlation between
ERK activity and viral protein expression such as we have
described. One such study demonstrated that the hepatitis C
virus nonstructural NS5A protein inhibits EGF-stimulated
activation of the ERK pathway by inhibiting EGFR complex
formation (27). A recent study by Yoshizuka et al. reported
that the expression of the HIV type 1 (HIV-1) Vpr protein
is associated with the downregulation of genes in the ERK/
MAPK pathway and with decreased phosphorylation of
ERK1/2 (49). Vpr is associated with the induction of a G2/M
cell cycle arrest. That study suggests that an alternative
mechanism of HIV-1 Vpr-induced cell cycle arrest is the
Vpr-associated decrease in ERK1/2 activation, and the au-
thors speculate that this function is important for HIV rep-
lication and pathogenesis. Currently, little is known about
the effects of Ebola virus GP expression on cell cycle pro-
gression. The findings with HIV-1 Vpr, along with the
known regulatory effects of adhesion and ERK activity on
the activation of cyclin-dependent kinases, which regulate
the cell cycle (5, 24, 34), raise this possibility.
The results of this study demonstrate that Ebola virus GP
reduces the phosphorylation and activation of the signaling
molecule ERK2 and that reduction of ERK2 activity further
enhances GP-induced ?V integrin downmodulation, a corre-
late of cytopathicity. The observation that both GP-mediated
cytotoxicity and ERK2 dephosphorylation are dependent on
the mucin-like domain of GP further suggests that these pro-
cesses may be intimately related. This is the first evidence of a
GP-induced perturbation on a specific cellular signaling cas-
cade. Future analysis of the activity of this signaling cascade
during live viral infection may indicate an important role for
this pathway not only in GP-mediated cytotoxicity but also in
Ebola virus pathogenesis as well. A more complete under-
standing of the mechanisms involved in this phenomenon may
facilitate the development of therapies for Ebola virus infec-
tion, which are currently unavailable.
We thank Ati Tislerics for assistance with manuscript preparation,
Toni Garrison and Brenda Hartman for figure preparation, and mem-
bers of the G. J. Nabel and N. J. Sullivan labs for helpful discussions
This research was supported in part by the Intramural Research
Program of the NIH, Vaccine Research Center, NIAID. J.-F.F. was
supported by a fellowship from the Damon Runyon Cancer Research
Foundation and by NIH grant N01 HV28183.
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