JOURNAL OF VIROLOGY, May 2005, p. 5326–5336
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 9
RhoA Signaling Is Required for Respiratory Syncytial Virus-Induced
Syncytium Formation and Filamentous Virion Morphology
Tara L. Gower,1† Manoj K. Pastey,2† Mark E. Peeples,3Peter L. Collins,4Lewis H. McCurdy,2
Timothy K. Hart,5Alex Guth,1Teresa R. Johnson,2and Barney S. Graham2*
Departments of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee1; Vaccine
Research Center2and Laboratory of Infectious Diseases,4National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland; Department of Pediatrics, The Ohio State University College of
Medicine and Public Health, Columbus, Ohio3; and SmithKline Beecham
Pharmaceuticals, King of Prussia, Pennsylvania5
Received 7 June 2004/Accepted 21 December 2004
Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threat-
ening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2
cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important
for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an
enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation
and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein
expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase
inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed
reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated
infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology,
cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of
infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous
virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron
microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifi-
able peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than
those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV
infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play
a role in initiating cell-to-cell fusion.
Human Respiratory syncytial virus (RSV) belongs to the fam-
ily Paramyxoviridae and is the leading viral cause of severe
lower respiratory tract illness in infants and young children.
The fusion (F) glycoprotein is necessary for cell-to-cell fusion
and syncytium formation and is thought to be necessary for
virion entry into cells, but the exact mechanisms of virus-
induced membrane fusion have not been defined. RSV F1is
expressed on the virus envelope and on the surfaces of infected
cells as a trimer (9, 53), similar to human immunodeficiency
virus type 1 (HIV-1) gp41. Fusion proteins from several diverse
enveloped viruses such as paramyxoviruses and lentiviruses
have similar structural and functional domains and share sim-
ilar fusion properties (7, 14, 24). Paramyxoviruses, including
RSV, have a broad pH range for fusion and syncytium forma-
tion and directly fuse with the plasma membrane (41). Virus-
mediated membrane fusion and entry are multistep processes
that generally require attachment to the primary virus recep-
tor, and in some cases, coreceptor binding. The fusion peptide
is then inserted into the target cell membrane, followed by
hemifusion, full fusion, the production of a fusion pore, and
the release of the viral genome into the target cell cytoplasm
(50). While the importance of virus-to-cell fusion during entry
is clear, the teleological advantage to viruses of forming syn-
cytia through cell-to-cell fusion is more uncertain. Viruses may
use syncytium formation to spread quickly to neighboring cells
or to evade host defense mechanisms. Cell-to-cell fusion me-
diated by some viral envelope proteins involves the cellular
actin cytoskeleton and cell surface integrins (4, 12, 21, 23).
Therefore, host cellular proteins that maintain cell membrane
integrity, cell mobility, and adhesion might be expected to play
a role in virus-induced fusion and syncytium formation since
fusion involves direct cell-to-cell contact and the mixing of cell
membranes, although there is currently no direct evidence for
their involvement. Virus-induced membrane fusion mediated
by the virus receptor and the fusion protein may occur similarly
to intracellular vesicle fusion. Integral membrane proteins on
the vesicle and target membrane known as v-snares and t-
snares interact and undergo conformational changes which
bring the target membranes close together to facilitate fusion
(46, 47). Interestingly, a small GTPase, Rab5, is known to play
a role in v-snare- and t-snare-mediated vesicle fusion (15, 45).
Many enveloped viruses cause characteristic changes in the
surface morphology of infected cells. The surfaces of infected
cells are covered by large clumps of filamentous protrusions,
which can be visualized by light microscopy, immunofluores-
cence staining, and electron microscopy (2, 3, 35, 51). The
* Corresponding author. Mailing address: Vaccine Research Center,
Building 40, Room 2502, NIAID, NIH, 40 Convent Dr., MSC 3017,
Bethesda, MD 20892-3017. Phone: (301) 594-8468. Fax: (301) 480-
2771. E-mail: email@example.com.
† T.L.G. and M.K.P. contributed equally to this study.
morphology of budding virions depends on cellular determi-
nants such as polarized cell phenotype and the integrity of the
actin microfilament network (6, 39). The determinants of
RSV’s spherical and filamentous morphological forms and the
roles of such particles in virus transmission and pathogenicity
are not clearly defined. In RSV-infected cells, the filaments are
coated with the viral envelope proteins F and G, suggesting a
potential role for these proteins in forming cell-to-cell contacts
that might initiate syncytium formation.
We have previously demonstrated that RhoA and its down-
stream signaling cascades are activated during RSV infection
(16). RhoA is a small GTP binding protein in the Ras super-
family. RhoA is ubiquitously expressed in mammalian cells,
and activated RhoA influences a variety of essential biological
functions in eukaryotic cells, including gene transcription, cell
cycle, vesicular transport, adhesion, cell shape, fusion, and
motility, through its activation of signaling cascades (18, 30).
RhoA affects the cytoskeleton by inducing the organization of
actin stress fibers and the formation of focal adhesion plaques
(37). Stress fiber formation is known to require RhoA activa-
tion (34). RhoA signaling pathways can also induce the pro-
duction of interleukin-8, which is produced in abundance by
RSV-infected cells (22). RhoA activation also leads to the
formation of microvilli by the phosphorylation of moesin via
Rho kinase (34, 44). Interestingly, viral filaments which are
apparent during RSV infection resemble RhoA-induced mi-
crovilli. Based on these data, the goal of this study was to
define the role of RhoA signaling in RSV infection. We used
the following agents to determine if activated RhoA signaling
is important for RSV replication and syncytium formation: the
C3 exoenzyme from Clostridium botulinum, which specifically
inactivates RhoA by ADP ribosylation of Asn 41 (40, 43);
Y-27632, a Rho kinase inhibitor (49); and cytochalasin D,
which inhibits actin polymerization (10). In this paper, we
demonstrate that RhoA signaling is necessary for RSV-in-
duced cell-to-cell fusion and for the formation of microvilli
that promote the formation of filamentous virions. However,
RhoA signaling is not required for an efficient infection or for
the production of infectious but nonfilamentous virions. We
also demonstrate an association between RSV-induced syncy-
tium formation and the presence of RhoA-induced viral fila-
ments. The data indicate that the requirements for the pro-
duction of infectious RSV virions can be dissociated from the
process of cell-to-cell fusion and that virus-induced RhoA ac-
tivation and signaling are necessary for the filamentous virus
structure and syncytium formation.
MATERIALS AND METHODS
Viruses and cells. R. Chanock, National Institutes of Health, Bethesda, Md.,
provided the A2 strain of RSV. RSV stocks were prepared as previously de-
scribed (17). Recombinant, green fluorescent protein-expressing RSV (rgRSV)
was generated as previously described (19). The Long strain of RSV was ob-
tained from the American Type Culture Collection. All cells were maintained in
Eagle’s minimal essential medium or Dulbecco’s modified Eagle medium
(DMEM) supplemented with glutamine, gentamicin, penicillin G, and 10% fetal
Plaque assay. Two-day-old HEp-2 monolayers at 80% confluence in 12-well
plates (Costar, Cambridge, Mass.) were used for plaque assays. The assay was
performed as previously described (17).
RSV growth curves. HEp-2 monolayers at 80% confluence in 96-well plates
were pretreated with medium containing 30 ?g of C3 (CalBiochem, La Jolla,
Calif.)/ml or 20 ?M Y-27632 (a gift from Shuh Narumiya, Welfide Corporation,
Iruma, Japan) beginning 24 h before RSV infection and continuing throughout
the infection. Fifty microliters of RSV at a multiplicity of infection (MOI) of 0.1
was added to the cells and allowed to adsorb for 1 h at room temperature. After
the adsorption of RSV, medium containing C3 or Y-27632 was added and the
plates were incubated at 37°C. Untreated RSV-infected wells were used as
controls. The dose levels and treatment effects of C3 and Y-27632 have been
validated in previous publications (16, 27). Medium supernatants were collected
daily, and virus growth was measured by a plaque assay for 8 consecutive days
postinfection. Virus-induced syncytia and plaques were visualized at 4 days
Western blot detection of RSV F. HEp-2 cells were either left untreated or
pretreated with 30 ?g of C3/ml 24 h prior to RSV infection. At 72 h postinfec-
tion, the cells were harvested and 1-ml aliquots were centrifuged at 14,000 rpm
for 10 min at 4°C in a Sorvall Surespin 630 centrifuge. The pellet was resus-
pended in mammalian protein extraction reagent (Pierce, Rockford, Ill.). The
amount of protein was quantified by use of a BCA assay (Pierce). Equal amounts
of proteins from C3-treated or untreated RSV-infected cells were resolved by
sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis and transferred
to polyvinylidene difluoride membranes. The F protein was detected with an
anti-F monoclonal antibody followed by a horseradish peroxidase (HRP)-conju-
gated anti-mouse antibody (Amersham Pharmacia Biotech, Bucks, United King-
dom). Proteins were visualized by ECL (Amersham Pharmacia Biotech).
Analysis of F expression on C3-treated and untreated RSV-infected HEp-2
cells. Untreated HEp-2 cells or cells treated with 30 ?g of C3/ml for 24 h were
infected with RSV (MOI ? 0.5) and fixed in 4% formaldehyde 48 h after
infection. The fixed cells were stained with a 1:1,000 dilution of an anti-F
monoclonal antibody (Chemicon, Temecula, Calif.) in 5% nonfat dry milk for
1 h. After three washes with phosphate-buffered saline (PBS)-Tween 20, the cells
were stained with a 1:5,000 dilution of Alexa fluor 488 goat anti-mouse immu-
noglobulin G (IgG; Molecular Probes, Eugene, Oreg.) in 5% nonfat dry milk for
1 h followed by washing with PBS-Tween 20. The cells were analyzed with a
FACSCaliber (Becton Dickinson, San Jose, Calif.) argon ion laser at 15 mW and
488 nm. Data were analyzed with FlowJo, version 6.0 (Tree Star, San Carlos,
Immunofluorescence analysis. HEp-2 cells were grown on coverslips in six-
well plates and infected with 200 ?l of 103-PFU/ml rgRSV. These cells were
analyzed by immunofluorescence microscopy at 24 h post-RSV infection. For the
visualization of RSV-infected cells, cells were fixed on coverslips at room tem-
perature in 3.7% formaldehyde for 10 min. The cells were observed under a Zeiss
Axioplan fluorescence microscope, and photographs were taken with SPOT
image capture software and a Zeiss MC80 microscope camera.
Cell fusion assay using vaccinia virus-based expression of RSV envelope
glycoproteins. A cell-to-cell fusion assay was used to assess the requirement for
RhoA signaling in RSV-induced cell-to-cell fusion. One population of HEp-2
cells (effector population) was infected with recombinant vaccinia virus vTF7-3,
which encodes T7 polymerase, at a multiplicity of infection of 10 PFU per cell
and then transfected with plasmids encoding RSV glycoproteins F, G, and SH
under the control of the T7 promoter by the use of FuGene (Boehringer Mann-
heim, Indianapolis, Ind.). While F is the major determinant of cell-to-cell fusion,
syncytium formation is optimized when all three surface proteins are expressed
(20, 36). In addition, we have shown that coinfecting cells with recombinant
vaccinia viruses expressing individual RSV proteins that combine the expression
of F, G, and SH maximizes filament formation (unpublished observations). A
second population of HEp-2 cells (target population) was infected with a recom-
binant vaccinia virus expressing ?-galactosidase under the control of the T7
promoter (provided by E. A. Berger, National Institutes of Health, Bethesda,
Md.). Four hours after transfection, the cells were trypsinized and suspended in
DMEM containing 2.5% fetal bovine serum to a density of 2 ? 107cells per ml.
The cells were divided evenly into aliquots, RhoA inhibitors were added to either
effector or target cells, and the cells were incubated for 16 h at 37°C. The two sets
of cells were then washed and suspended in DMEM at a concentration of 106
cells per ml. The two cell populations were then mixed in triplicate by adding 100
?l of each cell population to 96-well tissue culture plates, which were then
incubated at 37°C for 4 h. The inhibitors were not present during the 4-h
incubation for fusion. After 4 h, the cells were fixed in 2% glutaraldehyde–20%
formaldehyde (Sigma, St. Louis, Mo.) in PBS for 10 min. One hundred fifty
microliters of X-Gal solution (5 mM potassium ferricyanide, 5 mM potassium
ferrocyanide, 2 mM magnesium chloride, 1 mg of X-Gal [5-bromo-4-chloro-3-
indolyl-?-D-galactopyranoside; Fisher, Springfield, N.J.]/ml, freshly diluted from
a 40-mg/ml stock solution in dimethyl formamide) was then added. After 8 h,
blue-stained fused cells were viewed with an inverted phase-contrast microscope.
Scanning electron microscopy (SEM). HEp-2 cells on 12-mm-wide coverslips
(Fisher, Pittsburgh, Pa.) were treated with 20 ?M Y-27632 or 30 ?g of C3/ml for
VOL. 79, 2005RSV MORPHOLOGY IS RhoA DEPENDENT5327
16 h and then infected with RSV (MOI ? 1). Medium containing C3 or Y-27632
was added after adsorption to maintain the treatment throughout the course of
infection. Untreated RSV-infected cells and uninfected cells treated with 20 ?M
lysophosphatidic acid (LPA) for 30 min were used as controls. LPA is a known
activator of RhoA signaling. Infected samples were fixed 24 h after infection in
4% glutaraldehyde (Sigma) for 1 h, treated with 1% osmium for 15 min, and
dehydrated through a series of 70 to 100% ethanol washes. Dehydrated cells
were critical point dried, sputter coated with gold, and visualized by use of a
Hitachi S4200 scanning electron microscope.
Transmission electron microscopy (TEM) and immunostaining. Vero cells or
HEp-2 cells (1.5 ? 105cells) were plated in 24-well plates 1 day prior to infection
with the Long strain of RSV at an MOI of 0.015 in medium containing 2% fetal
calf serum for 1 h. The cells were then washed and incubated in fresh medium for
For immuno-TEM, the medium was then replaced with fresh medium con-
taining 10 ?g of SB-209763, a humanized monoclonal antibody specific for the F
protein of RSV that prevents syncytium formation, per milliliter for 24 to 48 h.
The medium was aspirated and the cultures were fixed with 2.5% glutaraldehyde
in 0.1 M phosphate buffer overnight. The cultures were washed and incubated for
45 min with HRP-conjugated donkey anti-human IgG (Jackson Immunore-
search, West Chester, Pa.). After the wash step, the peroxidase reaction product
was developed with diaminobenzidene, and the cultures were postfixed with 1%
osmium for 1 h and dehydrated in a graded series of ethanol.
For all samples, the cells were lifted off the wells with propylene oxide and
embedded in EMBed 812 resin (Electron Microscopy Sciences, Ft. Washington,
Pa.). Thin sections were cut and stained with uranyl acetate and lead citrate. For
some sections, tannic acid staining was performed prior to the uranyl acetate
stain to accentuate the glycoprotein spikes. The specimens were examined by use
of a JEOL 100EX transmission electron microscope at 80 kV.
For evaluations of particles separated by velocity sedimentation, the bands
associated with the peak fractions were concentrated by centrifugation onto a
60% sucrose cushion. A sample from the concentrated band was applied to a
grid, stained with 0.5% uranyl acetate, and examined by use of a Hitachi H-7000
transmission electron microscope.
Sucrose gradient analysis of virus particles. HEp-2 cells in 25-cm2flasks were
either left untreated or treated with 30 ?g of C3/ml for 24 h and then infected
with RSV (MOI of 0.5). After a 72-h infection, the infected cells were harvested
and layered over a continuous 15 to 60% sucrose gradient. The virus was then
separated by velocity centrifugation at 14,000 rpm for 10 min at 4°C in a Sorvall
Sure Spin 630 centrifuge. Two-milliliter fractions were collected, and plaque
assays were performed with the fractions as described previously (17).
Fluorescence microscopy of viral particles. Viruses were concentrated from
peak individual fractions on a 60% sucrose cushion by ultracentrifugation at
14,000 rpm for 10 min at 4°C in a Sorvall Sure Spin 630 centrifuge. Virus particles
were collected from the layer just above the sucrose cushion. Specimens for
fluorescence microscopy were prepared on coverslips and stained. Briefly, 50 ?l
of the virus sample was smeared on a coverslip and allowed to air dry. The virus
particles were fixed with 3.7% formaldehyde in PBS for 30 min. The samples
were washed twice with PBS-Tween 20 and blocked with 5% nonfat dry milk in
PBS for 30 min. They were then stained with anti-RSV antibody (Maine Bio-
technology Services) in 1% nonfat dry milk for 1 h, followed by Alexafluor 488
anti-mouse IgG (Molecular Probes). After three washes with PBS-Tween 20, the
coverslips were mounted on microscope slides. Specimens were viewed with a
Zeiss AxioPlan 2 fluorescence microscope, and pictures were taken with a
Hamamatsu ORCA-ER digital camera.
Effect of C3 on RSV syncytium formation and replication.
The C3 protein from C. botulinum is known to specifically
ADP-ribosylate RhoA and irreversibly inactivates its ability to
initiate signaling pathways. We used C3 to determine whether
activated RhoA is required for RSV-induced syncytium for-
mation. HEp-2 cells were treated with C3 beginning 24 h prior
to virus infection. Untreated RSV-infected cells exhibited syn-
cytium and plaque formation (Fig. 1A). However, RSV-in-
fected cells that had been pretreated and incubated with C3
did not have virus-induced syncytium or plaque formation (Fig.
1B) and appeared similar to uninfected monolayers (Fig. 1C).
The response to C3 is dose dependent. HEp-2 cells were
treated with several doses of C3, up to 50 ?g/ml, beginning 24 h
prior to RSV infection (data not shown). RSV-induced syncy-
tium formation was completely inhibited by 30 ?g of C3/ml,
while lower doses resulted in partial inhibition. Next, we asked
whether RhoA activation is required for RSV infection and
replication. HEp-2 cells in 96-well plates were either left un-
treated or pretreated with C3 beginning 24 h prior to RSV
infection and throughout the course of infection. The contents
of individual RSV-infected wells were transferred to HEp-2
cell monolayers in 12-well plates for plaque assays on eight
consecutive days after RSV infection. Surprisingly, the number
of PFU produced by C3-treated cells was equal to that pro-
duced by untreated RSV-infected cells, indicating that the
production of infectious virions was not affected by the inacti-
vation of RhoA (Fig. 1D). In addition, RSV was produced
from the C3-treated cells without producing syncytia for more
than 8 days. To determine the potential effect of C3 treatment
on the level of F protein expression, we analyzed the F protein
by Western blotting and flow cytometry of RSV-infected cells,
using a monoclonal anti-F antibody. Analyses of the RSV F
protein in untreated or C3-treated RSV showed similar levels
of protein production by Western blotting and similar levels of
F expression on the cell surface by flow cytometry, indicating
that an altered magnitude of RSV F expression is not the
explanation for C3’s inhibition of syncytium formation (Fig. 2).
These data suggest that the presence of constitutive levels of
inactive RhoA associated with the membrane is sufficient for
virus entry and replication but that RhoA activation is required
for virus-induced cell-to-cell fusion to form syncytia.
Effect of Rho kinase inhibitor Y-27632 on RSV infection and
replication. The Rho kinase inhibitor Y-27632 was used to
further characterize the requirements for RhoA signaling in
RSV infection and replication. First, we asked whether
Y-27632 could interfere with single-cell infection by RSV.
HEp-2 cells were treated with 20 ?M Y-27632 beginning 24 h
prior to infection with rgRSV (MOI ? 0.1). The cells were
fixed in 3.7% formaldehyde at 24 h postinfection. RSV-in-
fected, green fluorescent protein-expressing cells were then
visualized by fluorescence microscopy. Y-27632 had no effect
on the number of RSV-infected cells (Fig. 3B) compared to
untreated controls (Fig. 3A).
Next, Y-27632 was used to create a virus growth curve to
determine its effect on RSV replication. HEp-2 cells in 96-well
plates were left untreated or treated with 20 ?M Y-27632
beginning 24 h prior to RSV infection (MOI ? 0.1). The
contents of individual wells were transferred to HEp-2 cell
monolayers in 12-well plates for plaque assays on eight con-
secutive days after RSV infection. The number of PFU of RSV
produced by Y-27632-treated cells was equal to that produced
by untreated RSV-infected cells, indicating that the production
of infectious virions was not affected by the inactivation of
RhoA signaling (Fig. 3C). These data suggest that downstream
signaling through Rho kinase is not required for virus entry or
RhoA activation and signaling are required for RSV-in-
duced cell-to-cell fusion. The role of RhoA in RSV-induced
cell-to-cell fusion was then evaluated by use of a cell-to-cell
fusion assay. As shown in Fig. 4, one population of HEp-2 cells
was infected with a recombinant vaccinia virus expressing T7
5328GOWER ET AL.J. VIROL.
polymerase and transfected with plasmids expressing RSV F,
G, and SH under the control of a T7 promoter (effectors).
Another population of HEp-2 cells was infected with a recom-
binant vaccinia virus expressing lacZ under the control of a T7
promoter (targets). At 4 h postinfection, the effectors (hatched
bars) or the targets (black bars) were treated with 20 ?M
Y-27632, 30 ?g of C3/ml, or 10 ?M cytochalasin D, an inhibitor
of actin polymerization. The cells were incubated separately
for 16 h and then mixed together. After 4 h, the cells were fixed
and stained with X-Gal. Fused blue cells were then counted.
Inhibiting RhoA signaling in effector cells (hatched bars) with
Y-27632 or C3 dramatically reduced cell-to-cell fusion. A cy-
tochalasin D treatment of effector cells also inhibited cell-to-
cell fusion. In contrast, the treatment of target cells with
Y-27632 or cytochalasin D did not have an effect on cell-to-cell
fusion, although a treatment with C3 partially inhibited cell-
to-cell fusion. These data indicate that RhoA signaling and
actin polymerization are important for the effector cells in
cell-to-cell fusion and that RhoA signaling through Rho kinase
and actin polymerization are not required in the target cells for
cell-to-cell fusion to occur. The inhibition of another RhoA
signaling pathway or altered RhoA membrane localization may
explain the partial effect on cell-to-cell fusion in C3-treated
To verify that the inhibition of cell-to-cell fusion was not due
to an inhibition of vaccinia virus replication or the expression
of T7 polymerase and lacZ, we infected HEp-2 cells with both
the vaccinia virus expressing T7 polymerase and the vaccinia
virus expressing lacZ, and after 4 h, added various inhibitors.
Sixteen hours later, the cells were fixed and stained with X-Gal.
All of the cells turned blue within 30 min, regardless of the
treatment received, indicating that the inhibitors did not sig-
nificantly affect vaccinia virus infection or the expression of T7
polymerase or lacZ (data not shown). Also, as indicated by
trypan blue exclusion, none of the inhibitors caused toxicity to
the cells during the 16 h of treatment compared to untreated
cells (data not shown).
Taken together, as shown in Fig. 4, there were distinct re-
quirements for cell-to-cell fusion for the effector cells, which
were transfected with plasmids encoding RSV envelope pro-
teins, and for the target cells, which lacked RSV envelope
proteins. RhoA signaling and actin polymerization were re-
FIG. 1. C3 prevents RSV-induced syncytium formation in HEp-2 cells. (A) RSV-infected cells showed extensive syncytium formation in HEp-2
cells at 3 days postinfection. (B) Treatment with 30 ?g of C3/ml blocked RSV-induced syncytium formation. (C) Uninfected control HEp-2 cells
treated with 30 ?g of C3/ml showed that C3 has no visible effect on cell morphology or viability. (D) HEp-2 cell monolayers grown in 96-well plates
were either treated with 30 ?g of C3/ml for 24 h or mock treated and then were infected with RSV at an MOI of 0.1. Virus titers were measured
for eight consecutive days after RSV infection by harvesting the entire contents of each well and performing plaque assays in triplicate. RSV growth
curves were created for untreated cells (squares) and cells treated with C3 (diamonds). The data shown are representative of three separate
experiments. Error bars represent standard deviations.
VOL. 79, 2005 RSV MORPHOLOGY IS RhoA DEPENDENT5329
quired for the infected cells to fuse with target, uninfected
cells. On the other hand, RhoA signaling through Rho kinase
and actin polymerization were not required in the uninfected
RhoA signaling is required for RSV-induced filament for-
mation. We next asked whether RhoA signaling and actin
polymerization affected RSV-induced microvillus formation.
We previously reported that the treatment of HEp-2 cells with
30 ?g of C3/ml or 20 ?M Y-27632, a Rho kinase inhibitor,
beginning 24 h prior to infection and throughout RSV infec-
tion alters the pattern of F protein localization in infected cells
(16). Untreated RSV-infected cells have punctate staining for
F in the cytoplasm, and late in infection, have filamentous
structures extending from the cell, which stain with an anti-F
antibody (16). In C3- and Y-27632-treated cells, there were no
F-staining filaments, and the cytoplasmic staining for F was
more diffuse (16).
Therefore, we next determined the effect of RhoA activation
and signaling on viral filament formation by SEM (Fig. 5).
Untreated cells or cells treated with 30 ?g of C3/ml for 24 h
were infected with RSV and fixed in 4% glutaraldehyde 24 or
48 h after infection. When visualized by SEM, RSV-infected
cells had large clumps of long filaments that protruded from
several places across the cell surface (Fig. 5A and B) compared
to uninfected HEp-2 cells (Fig. 5D). These filaments also
bridged across cell junctions to neighboring cells. By 48 h
postinfection, the filaments covered the entire surface of the
infected cell (Fig. 5B). Interestingly, the viral filaments pro-
duced by C3-treated, RSV-infected cells were attenuated (as
shown at 24 h in Fig. 5C). These cells produced blunted fila-
ments that were not in clumps but were sparsely distributed
and more disorganized across the surfaces of the cells. The
filaments induced by RSV in the absence of C3 closely resem-
bled the microvilli in uninfected cells induced by LPA, a known
inducer of RhoA signaling (Fig. 5E) (34, 44). These data sug-
gest that RhoA-induced microvilli may play an important role
in RSV filament formation and virus-induced cell-to-cell fu-
sion, possibly by initiating cell-to-cell contacts.
Viral filaments are budding virus particles. RSV-induced
filament formation was next evaluated by immunoperoxidase
staining and TEM to confirm the presence of the F glycopro-
tein on the membranes of the filamentous particles. In Vero
cells infected with the Long strain of RSV and processed for
TEM, both viral particles and viral filaments were produced by
infected cells (Fig. 6A). At a higher magnification, viral glyco-
protein spikes were evident on the surfaces of both viral par-
ticles and filaments (Fig. 6B and C), and nucleocapsid struc-
tures were evident within these structures. Immunoelectron
microscopic localization of the anti-F antibody revealed in-
tense staining of both viral particles and viral filaments (Fig.
6D, E, and F) overlying the locations of the glycoprotein
spikes. These observations, along with those for Fig. 5, suggest
that the microvilli induced by RSV infection and RhoA acti-
vation are budding filamentous virus particles.
C3 treatment shifts virion morphology from filamentous to
more spherical. A scanning electron microscopic examination
of C3-treated infected cells showed reduced quantities of sin-
gle blunted filaments compared to the large clumps of long
filaments in untreated infected cells (Fig. 5). Next, we devel-
oped a sucrose gradient velocity sedimentation technique to
separate spherical and filamentous viruses and to attempt a
semiquantitative measurement of the influence of C3 treat-
ment on virus morphology. HEp-2 cells were either left un-
treated or treated with 30 ?g of C3/ml for 24 h and then
FIG. 2. Level of F expression in C3-treated or untreated infected cells. (A) HEp-2 cells were either left untreated or pretreated with 30 ?g of
C3/ml 24 h prior to RSV infection. At 72 h postinfection, the cells were harvested and a 1-ml aliquot was centrifuged at 14,000 rpm for 10 min
at 4°C. The pellet was resuspended in mammalian protein extraction reagent. Equal amounts of proteins from C3-treated or untreated
RSV-infected cells were resolved by sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride
membranes. The F protein was detected with an anti-F monoclonal antibody followed by an HRP-conjugated anti-mouse antibody. Proteins were
visualized by ECL. ?, C3 treatment; ?, no treatment. (B) RSV F expression. C3-treated cells infected with RSV for 24 h were evaluated by flow
5330 GOWER ET AL.J. VIROL.
infected with RSV. After a 72-h infection, the infected cells
and media were harvested and layered over a continuous 15 to
60% sucrose gradient. Viruses were then separated by velocity
centrifugation in a Sorvall centrifuge. Two-milliliter fractions
were collected and plaque assays were performed on fractions
as described previously (17). RSV titers from sucrose gradient
fractions showed a predominance of spherical forms for C3-
treated RSV compared to equal amounts of filamentous and
spherical virions for untreated RSV (Fig. 7A). The peak frac-
tions (2 and 6) were examined by fluorescence microscopy to
confirm the separation of two morphological forms of the virus
into two identifiable peaks. The virus particles were fixed on
coverslips and stained with a rhodamine-conjugated anti-RSV
antibody. The specimens were viewed with a Zeiss AxioPlan 2
fluorescence microscope. The peak fractions 2 and 6 showed
spherical and filamentous morphologies, respectively (Fig. 7B
and C). To confirm that the RSV F-expressing particles seen by
fluorescence microscopy were virus particles and not mem-
brane fragments, we performed TEM on the peak fractions.
This confirmed that peak 1 consisted primarily of pleomorphic,
somewhat spherical virus particles and peak 2 consisted of
primarily filamentous particles.
We have previously shown that RhoA is activated during
RSV infection (16). We now report data that suggest that
RhoA-mediated signaling and actin polymerization are associ-
ated with the filamentous virion morphology and the syncyti-
um-inducing phenotype. RhoA is an essential host cell protein
with GTPase activity and is known to influence a variety of
signaling pathways and basic cell functions (18, 30). The role of
RhoA in virus-induced fusion and subsequent signaling events
may have significance in virus infection to ensure the coordi-
nated control of cellular activities required for virus replica-
tion, such as the stage of the cell cycle and reorganization of
the actin cytoskeleton. There are many examples of viruses
using the host cell machinery to complete their life cycles, so it
is not surprising that RSV has adapted to utilize an essential
GTPase to modify its morphological properties.
In C3-treated RSV-infected cells, there was no formation of
syncytia (Fig. 1B). Interestingly, blocking RhoA signaling with
C3 or Y-27632 did not affect the efficiency with which RSV
initiated infection or the production of infectious virions in cell
culture (Fig. 1D and 3C). Therefore, we have shown a distinc-
tion in the requirement for RhoA signaling events between the
entry of cell-free virions (independent of RhoA signaling) and
cell-to-cell fusion and syncytium formation (dependent on
RhoA signaling). This distinction is not related to changes in
the overall expression of RSV F, since Western blotting and
flow cytometric analysis of C3-treated and untreated RSV-
infected cells showed equivalent levels of F expression (Fig. 2).
The fusion assays whose results are shown in Fig. 4 showed
that there were distinct requirements for cell-to-cell fusion for
the effector cells, which expressed the RSV envelope glyco-
proteins, and for the target cells, which did not express RSV
envelope glycoproteins. This suggests that RhoA signaling and
actin polymerization are required for an infected cell to fuse
with an uninfected target cell. On the other hand, the data
FIG. 3. Effect of Y-27632 on RSV replication. rgRSV-infected cells can be visualized by immunofluorescence microscopy. (A) At 24 h
postinfection, rgRSV-infected cultures had many green infected single cells. (B) Pretreatment with 20 ?M Y-27632 for 24 h did not affect the
single-cell infection by rgRSV. (C) HEp-2 cell monolayers grown in 96-well plates were either treated with 20 ?M Y-27632 or left untreated and
infected with RSV at an MOI of 0.1. Virus titers were measured for eight consecutive days after RSV infection by harvesting the entire contents
of each well and performing plaque assays in triplicate. RSV growth curves were created for untreated cells (squares) and cells treated with 20 ?M
Y-27632 (circles). The data shown are representative of three separate experiments. Error bars represent standard deviations.
VOL. 79, 2005 RSV MORPHOLOGY IS RhoA DEPENDENT5331
suggest that RhoA signaling through Rho kinase and actin
polymerization are not required in uninfected cells for fusion
In Fig. 4, we show that C3 partially inhibited cell-to-cell
fusion in treated targets. A recent study showed that a C3
treatment does not remove RhoA from the membrane but may
cause it to be redistributed into detergent-soluble regions of
the membrane (29), thereby reducing its density within choles-
terol-rich membrane microdomains (lipid rafts). Specific pro-
teins, including RhoA and CD44, localize to these membrane
microdomains (29, 31). It has been shown by our group and
others that RSV proteins colocalize with cellular proteins as-
sociated with lipid microdomains, including caveolin-1 (5) and
CD44, as well as with RhoA (27). The ADP-ribosylation of
RhoA by C3 inactivates RhoA signaling, results in an overall
decrease in both the number and length of viral filaments, and
shifts the localization of F to nonlipid microdomain regions of
the membrane (27). This suggests that the selective incorpo-
ration of RSV proteins into lipid microdomains may result in
virus assembly in microvilli providing the filamentous viral
morphology associated with syncytium formation. Viruses such
as HIV-1, influenza virus, and simian virus 40 have also been
shown to enter cells or to bud from lipid rafts (1, 8, 32, 52). The
redistribution of RhoA into non-lipid-raft membrane domains
of uninfected target cells by C3 does not influence replication
or infection with individual virions, but it may diminish the
ability of RSV-induced filaments to mediate cell-to-cell fusion.
It is possible that there is a greater requirement for membrane
order and the coalescence of factors within lipid rafts to sup-
port cell-to-cell fusion than for virus-to-cell membrane fusion.
This may be related either to a required mixture of proteins
and heparin binding domains which are needed to mediate
cell-to-cell fusion or to the need for a threshold concentration.
RSV-infected cells form long filamentous protrusions ex-
pressing the F glycoprotein within 24 h after infection (16).
Several viruses, including influenza virus and Ebola viruses,
can also form long filamentous particles during infection, but it
is unclear what role these filaments play in the spread of virus
FIG. 4. Role of RhoA-induced signaling in RSV-induced cell-to-
cell fusion. Fusion was measured by combining effector cells (HEp-2
cells infected with a vaccinia virus expressing T7 polymerase and trans-
fected with plasmids encoding RSV F, G, and SH) with target cells
(HEp-2 cells infected with a vaccinia virus expressing lacZ under the
control of a T7 promoter). The cells were fixed 4 h after mixing and
stained with X-Gal for the visualization of blue fused cells. Prior to
mixing, either target cells (black bars) or effector cells (hatched bars)
were treated with C3, Y-27632, or cytochalasin D for 16 h beginning
4 h after vaccinia virus infection or transfection and then washed.
FIG. 5. Inactivating RhoA causes blunted viral filaments. Viral filaments were visualized by scanning electron microscopy. Cells infected with
RSV for 24 h (A) or 48 h (B) had large clumps of filaments protruding from the cells. HEp-2 cells treated with 30 ?g of C3/ml and infected with
RSV (C) had reduced quantities of single blunted filaments at 24 h, while uninfected HEp-2 cells (D) had no filamentous structures. The filaments
in RSV-infected cells resembled RhoA-induced microvilli in uninfected cells treated with 20 ?M LPA for 30 min (E). Magnification, ?6,300.
5332 GOWER ET AL.J. VIROL.
infection and in syncytium formation (11, 13). We were able to
visualize these filamentous structures in RSV-infected cells by
TEM (Fig. 6), SEM (Fig. 5), and F-specific indirect immuno-
fluorescence (16) (Fig. 7). The treatment of cells with C3 or
Y-27632 beginning 24 h before virus infection caused an al-
tered pattern of F staining in infected cells by immunofluores-
cence (16). C3- and Y-27632-treated cells had diffuse staining
for F throughout the cell, and there were no distinct filamen-
tous structures present on or around the infected cells (16). By
using SEM, we observed that viral filaments can bridge from
the infected cell to a neighboring cell (Fig. 5A). The viral
filaments were blunted and more diffusely expressed on the
surfaces of HEp-2 cells treated with C3 (Fig. 5C), suggesting
that filaments are needed to bridge cell junctions in order to
initiate the cell-to-cell fusion process.
Rho kinase can cause the production of microvilli which colocal-
ize with CD44, a lipid raft protein, and resemble the RSV fila-
ments shown by immunofluorescence (16) and SEM (Fig. 5) (34,
44). These microvilli are important for cell-to-cell adhesion in
epithelial cells (48). The inhibition of RhoA signaling by C3
inhibits microvillus formation (Fig. 5C) (44), suggesting that
RhoA-induced microvilli may play an important role in the nor-
mal assembly of virus particles and the formation of filamentous
virus structures. These data indicate a strong association between
the ability of RSV to form filaments and its ability to undergo
cell-to-cell fusion, further suggesting that viral filaments may be
important for cell-to-cell fusion and syncytium formation. Im-
muno-TEM of microvilli and filamentous virions showed that the
microvilli are coated with the RSV F glycoprotein (Fig. 6). These
data suggest that the microvilli are sites of viral assembly and
represent budding viral particles.
FIG. 6. Ultrastructural localization of RSV F protein on viral particles and filaments. Vero cells infected with the Long strain of RSV were fixed
at 48 h postinfection and processed for immunoelectron microscopic localization of the F protein. The cells in panels A to C were control
immunostained cultures grown in the absence of the F-specific monoclonal antibody SB-209763, while the cells in panels D to F were grown in
the presence of 10 ?g of SB-209763/ml, which was localized with HRP-conjugated donkey anti-human IgG. Viral particles (B and E) and filaments
(C and F) had viral glycoprotein spikes on their surfaces (B and C) that stained positively with the anti-F antibody (E and F). Nucleocapsid
structures are evident within both viral particles and filaments. Bar ? 1 ?m (A and D) or 100 nm (B, C, E, and F).
VOL. 79, 2005 RSV MORPHOLOGY IS RhoA DEPENDENT 5333
In order to confirm that C3 affects filamentous virus produc-
tion, we developed a sucrose gradient velocity sedimentation
technique to separate spherical and filamentous viruses. There
were two peaks of RSV PFU in both C3-treated and untreated
RSV-infected groups (Fig. 7A). The relative separation of
spherical and filamentous morphological forms into two iden-
tifiable peaks was further supported by immunofluorescence
staining of virus particles with an anti-F monoclonal antibody
and TEM (Fig. 7B and C). The results showed that the C3
treatment shifted the relative production of viral morphologies
to more spherical particles than those obtained with untreated
RSV (Fig. 7A). A previous report has shown that cytochalasin
D treatment enhances spherical influenza virion release and
reduces the formation of filamentous influenza virus particles
(39). These data are consistent with our findings and suggest
that the assembly of viral filaments requires an intact actin
It has been reported that filamentous structures similar to
those described for wild-type RSV formed at the cell surface,
even when all three envelope glycoproteins were replaced by a
single foreign viral glycoprotein (vesicular stomatitis virus G
protein) carrying the RSV F cytoplasmic domain (33). The
FIG. 7. Sucrose gradient analysis of virus particles. Velocity sedimentation was performed on RSV grown for 72 h and treated with 30 ?g of
C3/ml or left untreated. In each of three independent experiments, C3-treated cells produced more infectious RSV in the first peak and less in
the second peak of the gradient. (A) Representative graph of the PFU in each fraction. Evaluating the results of three separate experiments, we
found the ratio of untreated to C3-treated PFU for peak 1 to be 1.80 ? 0.18 and that for peak 2 to be 0.58 ? 0.001 (two-tailed t test; P ? 0.01).
Using this technique, we examined the morphology of virions from peaks 1 and 2 by fluorescence microscopy using anti-F monoclonal antibody,
followed by Alexafluor 488 anti-mouse IgG. The images shown represent untreated RSV, but the morphologies of the two peaks for C3-treated
RSV were similar. The first peak contained predominantly spherical or pleomorphic virus particles (B), and the second peak contained primarily
filamentous particles (C). The identities of the morphologically distinct virus particles were confirmed by electron microscopy (insets). These data
indicate that a C3 treatment shifts the virion morphology to a more spherical and less filamentous shape.
5334 GOWER ET AL. J. VIROL.
engineered recombinant virus induces filaments at the cell
surface and causes cell-cell membrane fusion at pH 5.0 but not
at pH 7.0. The requirement of viral genes for particle mor-
phology differs among several viruses. For efficient particle
assembly, vesicular stomatitis virus and rabies virus require the
M protein (26, 28), influenza virus requires the M1 and M2
proteins (38), and simian virus 5 (another paramyxovirus) re-
quires the coexpression of the N protein, the M protein, and
one of the homologous transmembrane glycoproteins (42). For
RSV, the interactions of the F protein cytoplasmic domain and
the M protein with cellular proteins, RhoA, and the actin
cytoskeleton may play a role in filamentous virus formation.
Our findings indicate that RhoA signaling is associated with
RSV-induced filament formation and that the production of
infectious virions in vitro does not require a syncytium-induc-
ing phenotype. In addition, we report an association between
RhoA-induced viral filaments and RSV-induced syncytium for-
mation. A temperature-sensitive (ts) strain of RSV with a non-
syncytium-inducing phenotype in cell culture has been re-
ported (25). This virus produces equivalent numbers of
progeny viruses as normal syncytium-inducing strains of RSV,
but the virus is severely attenuated in vivo (25). In addition, the
non-syncytium-inducing phenotype correlates with the lack of
viral filaments seen by SEM (25). Thus, RSV infection in the
presence of C3 and Y-27632 treatment may be analogous to ts
RSV strains, which replicate with equal efficiencies in vitro as
their counterpart syncytium-inducing strains, but without sig-
nificant syncytium formation (25). However, the ts strains are
attenuated in vivo, suggesting that viral filament formation and
cell-to-cell fusion may represent virulence determinants.
Learning how to produce non-syncytium-inducing viruses
would be valuable for the development of live attenuated vac-
cines for two reasons. First, the virus may be less virulent, and
second, if the virion morphology were more homogeneous,
then the virus might be easier to purify and concentrate. More
work is needed to define the precise steps at which RhoA
activation is required for syncytium formation and to deter-
mine whether RhoA-induced signaling events are involved in
We thank James Wittig and Gary Olsen for their assistance with
SEM (Vanderbilt University, Nashville, Tenn.), Beverly Maleeff and
Sandra Griego for technical support with immuno-TEM (SmithKline
Beecham, King of Prussia, Pa.), and Shuh Narumiya for the Y-27632
reagent (Welfide Corporation, Iruma, Japan).
This work was supported in part by grant RO1-AI-33933.
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