JOURNAL OF VIROLOGY, Oct. 2009, p. 10710–10718
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 20
Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated
Attachment Protein That Alters Its Infectivity and Dependence
Steven Kwilas,1,2Rachael M. Liesman,3Liqun Zhang,4Edward Walsh,5
Raymond J. Pickles,3,4and Mark E. Peeples2*
Division of Immunology, The Graduate College, Rush University, 1653 W. Congress Parkway, Chicago, Illinois 606121; Section of
Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Department of Pediatrics,
The Ohio State University College of Medicine, 700 Children’s Drive, Columbus, Ohio 432052; Department of
Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 275993;
Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 275994; and Department of Medicine, University of
Rochester School of Medicine and Dentistry, Rochester, New York 146215
Received 15 May 2009/Accepted 24 July 2009
Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein
(G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface
glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2
cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero
cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment
form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero
cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less
efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for
virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not
likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for
attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters.
Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in
animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection
or the efficacy of the vaccine.
Human respiratory syncytial virus (RSV) is a negative-sense,
single-stranded RNA virus in the family Paramyxoviridae, sub-
family Pneumovirinae. RSV causes mild respiratory disease in
all age groups, but the disease can be severe or fatal in infants
and the elderly (4, 9, 11). Initial attempts to produce a killed
vaccine were not successful, resulting instead in enhanced dis-
ease upon infection (26, 41). Efforts to produce a live attenu-
ated vaccine are ongoing (6, 7, 51).
RSV produces three glycoproteins which are important for
infection. The largest glycoprotein (G) is involved in attach-
ment to the host cell (35), the fusion (F) glycoprotein mediates
virion membrane fusion with the target cell membrane (2), and
the small hydrophobic (SH) glycoprotein may attenuate apop-
tosis (15). The F protein is the only glycoprotein that is abso-
lutely required for infection of cultured immortalized cells (27,
45) and syncytium formation, the most obvious cytopathic ef-
fect of RSV in immortalized cell culture. Although the G
protein is not absolutely required for infection, it enhances
infection and syncytium formation (45). The G protein at-
taches to cultured, immortalized cell lines (35) primarily via
glycosaminoglycans (GAGs) on the cell surface (13, 22, 23, 30).
GAGs are repeating disaccharide units of hexuronic acid and
hexosamine that form unbranched polysaccharide chains and
are found on the surface of most mammalian cells. The GAG
type that appears most important for RSV infection of HEp-2
cells is heparan sulfate (HS) (23, 30).
The G protein is a type II integral membrane protein with its
N terminus on the cytoplasmic side of the membrane and its C
terminus as the extracellular ectodomain (49). An unglycosy-
lated region in the center of the protein contains four cysteines
held together by disulfide bonds in a cysteine noose (19, 24,
33), followed, to the C-terminal side, by a predicted heparin-
binding domain (HBD) (12, 13). The 32-kDa G protein, while
in the endoplasmic reticulum (ER), is modified by the addition
of multiple N-linked carbohydrate chains, depending on the
strain. These N-linked additions would increase the molecular
mass of G to 45 to 60 kDa. Previous reports have found G
protein forms of this size in cells and in virions at low levels (5,
20, 21, 50). All of these reports suggest that these smaller forms
of the G protein are partially glycosylated processing interme-
Maturation of the N-linked carbohydrates of the G protein
occurs in the Golgi compartment, where a large number of
O-linked carbohydrate chains are added, resulting in an 84- to
92-kDa mature protein (14, 32, 35, 49). This size variation of
* Corresponding author. Mailing address: Section of Vaccines and
Immunity, The Research Institute at Nationwide Children’s Hospital,
Department of Pediatrics, The Ohio State University College of Med-
icine, 700 Children’s Drive, Columbus, OH 43205. Phone: (614) 722-
3696. Fax: (614) 722-3680. E-mail: mark.peeples@nationwidechildrens
?Published ahead of print on 5 August 2009.
the G protein is probably due, in part, to the difficulty in sizing
heavily glycosylated molecules and variations in molecular
The G protein shares no homology with the glycoproteins of
paramyxoviruses outside the Pneumovirinae subfamily. The
high serine and threonine content and the high O-linked gly-
cosylation levels are similar to those found in mucins. The
amount of O-linked glycosylation is partially dependent on the
cell type used to produce the virus (18).
In the present study, we examined virus produced in HEp-2
and Vero cells, which are both commonly used to grow RSV in
the laboratory, for dependence on GAGs by the ability to
infect cells expressing GAG or deficient in GAG expression.
We also examined the ability of the viruses to infect primary,
well-differentiated human airway epithelial (HAE) cell cul-
tures. In both systems, infectivity was greatly dependent upon
the cell line used to grow the virus. Biochemical characteriza-
tion of purified virus grown in these two cell lines revealed a
smaller form of the RSV G protein in virions from Vero cells.
Using C terminus-specific antibodies and a six-His tag at the C
terminus of the G protein, we determined that the smaller G
protein form was lacking its C terminus. These results highlight
the importance of the C-terminal portion of the G protein and
suggest that the cell line used to produce a virus can alter its
MATERIALS AND METHODS
Viruses and cells. The recombinant green fluorescent protein (GFP)-express-
ing RSV used in these experiments were rgRSV-SGF (strain A2) and mutants of
this virus lacking one or more of the glycoprotein genes designated by the
glycoproteins that they express, i.e., rgRSV-F, rgRSV-GF, and rgRSV-SF (45).
The HEp-2 (American Type Culture Collection, Manassas, VA), MRC-5, A549,
primary monkey kidney (PMK), and BSC-1 cell lines were grown in Opti-MEM
I containing 2% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA),
and Vero and World Health Organization (WHO)-approved Vero cells were
grown in RPMI 1640 medium containing 5% FBS. The Chinese hamster ovary
(CHO) K1 cell line and its mutant A745, which is severely deficient in the
production of all GAGs due to a defective xylosyl transferase (10), were grown
in F-12 containing 10% FBS. All media were purchased from Invitrogen (Carls-
bad, CA). Cells were incubated at 37°C in 5% CO2. All virus stocks and cells
tested negative for mycoplasma by PCR (Intronbio, Seongnam-Si, Korea).
GAG dependency index. The GAG dependency index was derived by dividing
the titer of the same virus sample determined on CHO K1 cells by its titer on
CHO A745 cells. A volume of 40 ?l of RSV whose titer had not been determined
was added to 160 ?l of medium as the first fivefold dilution. Additional serial
fivefold dilutions of RSV were used to inoculate both CHO K1 and CHO A745
cells in 96-well plates that were ?70% confluent. At 24 h, the infected cells were
fixed in 3% paraformaldehyde for 20 min, permeabilized with 0.1% Triton
X-100, stained with fluorescein isothiocyanate-labeled anti-RSV polyclonal an-
tibodies (Virostat, Portland, ME), and counted.
Metabolic labeling of RSV. Virion proteins were metabolically labeled with
Tran35S-label (?70%35S-labeled L-methionine and ?15%35S-labeled L-cys-
teine; MPBiomedical, Solon, OH) by adding 20 ?Ci/ml to Opti-MEM I with 2%
FBS at 4 h postinoculation (p.i.). At 72 h p.i., virus was collected by scraping cells
from the tissue culture dish with a rubber policeman, pipetting, and vortexing.
Cells were removed by low-speed centrifugation (400 ? g for 5 min). The
supernatant was centrifuged at 20,000 ? g for 90 min in 50-ml high-speed conical
tubes (Sorvall) to pellet the virus in a Sorvall 600TC rotor. The pellet was
resuspended in Hanks balanced salt solution with Ca2?, Mg2?, and 25 mM
HEPES by repeated pipetting. This material was loaded onto a linear 25 to 55%
sucrose gradient in SW28 tubes and centrifuged for 18 h at 4°C in an SW28 rotor
at 125,000 ? g. The gradient was fractionated from the bottom with a capillary
tube connected to a peristaltic pump and a fraction collector. Samples of each
fraction were analyzed with a Tri-Carb 2100TR scintillation counter (Packard).
Fractions containing peak radioactivity were pooled, diluted, loaded onto a
second linear 25 to 55% sucrose gradient, and purified a second time under the
same conditions as the first spin. Fractions from the second gradient were
similarly analyzed. The fractions were also tested for infectivity, and peak infec-
tivity was found to coincide with peak radioactivity. The fractions containing
peak radioactivity were pooled for analysis.
Western blotting. Similar amounts of purified virions (15,000 cpm, metaboli-
cally labeled in parallel) were solubilized with lysis buffer that contained protease
inhibitors (Pierce) and 2 mg/ml N-ethylmaleimide, reduced in Laemmli sample
buffer with 5% ?-mercaptoethanol, and boiled for 3 to 5 min, and proteins were
separated by electrophoresis on a 10% polyacrylamide gel containing sodium
dodecyl sulfate (SDS-PAGE) (31). The proteins were transferred at 200 mA for
1 h to an Immobilon (Millipore) membrane, blocked overnight with 5% nonfat
milk–0.1% Tween 20, probed with primary antibody, washed twice with blocking
solution, and probed with a horseradish peroxidase (HRP)-labeled goat anti-
mouse antibody (Kirkegaard & Perry Laboratories). Lumi-Light Western blot-
ting substrate (Roche) was added, and the membranes were exposed to film for
5 s or less to generate the images in the figures and separately scanned with a
PhosphorImager (Typhoon; GE Healthcare). Quantification was done by aver-
aging the density of bands from three different membranes. Primary antibodies
used to stain the membranes were monoclonal antibody (MAb) L9 against the
conserved central region of the RSV G protein; MAb D14 against the RSV
nucleocapsid (N) protein (48); MAb 130-2G, which recognizes the C terminus of
the G protein (1, 43); and MAb 5His (Qiagen, Valencia, CA), which recognizes
the six-His tag.
Pulse-chase. A 70% confluent six-well plate of Vero and HEp-2 cells was
inoculated at a multiplicity of infection (MOI) of 3 with tipping for 1 h and then
rinsed with PBS before fresh medium was added. At 24 h p.i., wells were washed
three times with PBS before being pulsed with 20 ?Ci/ml Tran35S-label in
Met/Cys-free Dulbecco modified Eagle medium for 15 min at 37°C, after which
the medium was removed, the cells were rinsed with PBS, complete Dulbecco
modified Eagle medium–10% FBS supplemented with 10 ?M unlabeled methi-
onine was added, and the mixture was incubated at 37°C. At various times after
this pulse, the cells were solubilized with lysis buffer that contained protease
inhibitors (Pierce) and 2 mg/ml N-ethylmaleimide. As a control that lacks Golgi
compartment modifications, monensin (10 ?M) was added 1 h before the pulse
and maintained throughout the pulse and chase. Lysates were cleared of insol-
uble debris by centrifugation at 14,000 ? g for 10 min. The cleared lysate was
immunoprecipitated (IP) with MAb L9 and displayed by 10% SDS-PAGE. The
gel was soaked overnight in a 20% methanol–3% glycerol solution to prevent
cracking, followed by EN3HANCE (Perkin-Elmer) for 30 min, before drying and
exposure to film.
Endoglycosidase H (Endo H) treatment. Virions or IP samples were prepared
and digested with 500 U of Endo H at 37°C for 2 h according to the manufac-
turer’s instructions (New England BioLabs, Ipswich, MA).
Cell surface biotinylation. A 60% confluent plate of Vero cells in RPMI
medium and HEp-2 cells in Opti-MEM I was infected at an MOI of 0.1. At 72 h
p.i., cell monolayers were rinsed three times with PBS to remove medium and
FBS, incubated with 0.5 mg/ml Sulfo-NHS-Biotin (Pierce) at 4°C for 30 min,
rinsed three times with PBS containing 1% FBS, and covered with lysis buffer
that contained protease inhibitors (Pierce) and 2 mg/ml N-ethylmaleimide. The
lysates were cleared of insoluble debris by centrifugation at 14,000 ? g for 10
min, IP with MAb L9, separated by 10% SDS-PAGE, and transferred to an
Immobilon membrane. The membrane was blocked with 2% FBS in PBS, probed
with HRP-conjugated streptavidin (Kirkegaard & Perry Laboratories), treated
with Lumi-Light Western blotting substrate (Roche), and exposed to film.
His-tagged recombinant G protein. Six histidines were added to the C termi-
nus of the G protein immediately before the terminal glutamine. Mutagenesis
was performed by inverse PCR (3) with primers CATCACCATTAGTTACTT
AAAAACATATTATCACAAAAGGCCTTGACCAACCG and AGGTGGGT
TGTGTGGTGCGGTCGTAGTGGTA. The six-His sequence was inserted into
SD-2, a plasmid containing the G protein sequence from the A2 strain flanked by
the RSV gene start and gene stop transcription signals. Once modified, the G
gene was inserted into RW30, a full-length, RSV cDNA carrying the gene for
GFP in the first position (A. Kwilas and M. E. Peeples, unpublished data). Virus
was recovered from this plasmid by the standard recovery protocol for rgRSV
(45). The His-tagged G protein was detected by Western blotting and probing
with MAb 5His.
Infection of HAE cell cultures. Primary, well-differentiated HAE cell cultures
grown on Transwell filters (Costar) at the air-liquid interface (16) were rinsed
with PBS three times over a 30-min period to remove apical secretions and
supplied with fresh basolateral medium prior to inoculation. The virus inoculum
was diluted to 6.2 ? 106PFU in100 ?l of Hanks balanced salt solution (?MOI,
20) and applied to the apical surface of the HAE cell cultures for 2 h of
incubation at 37°C, after which the inoculum was removed by aspiration and
VOL. 83, 2009 TRUNCATION OF RSV G PROTEIN INHIBITS INFECTION10711
cultures were incubated at 37°C. At indicated times postinoculation, images were
obtained with a Leica DMIRB inverted fluorescence microscope equipped with
a cooled-color charge-coupled device digital camera (Retiga 1300; QImaging,
Burnaby, British Columbia, Canada). The proportion of the epithelium positive
for GFP was determined by pixilating a black-and-white image, inverting the
image, and calculating the percentage of black pixels by computer for five images
per culture and averaging the results.
To collect virus for the GAG dependency assay and Western blotting, 300 ?l
of serum-free medium was added to the apical surface of the HAE cell cultures.
After 30 min of incubation at 37°C, the medium with released virus was collected
and snap-frozen on dry ice. Because the sample volume in these cultures is too
small to process in sucrose gradients, we performed low-speed centrifugation at
400 ? g for 5 min to remove cells and debris. One-fifth of the virus sample was
then centrifuged at 15,000 ? g for 10 min at 4°C to pellet the virus for analysis.
Quantification of GAG dependency. We and others have
previously reported that RSV infection of HEp-2 and other
immortalized cell lines is largely dependent upon the presence
of GAGs on the cell surface (12, 22, 23, 30, 36, 46). This
conclusion was based in part on the much greater sensitivity to
RSV infection of CHO K1 cells than a mutant derivative, CHO
A745 (10). The CHO A745 cell line is defective in xylosyl
transferase, the enzyme that initiates GAG synthesis by linking
xylose to a serine or threonine in the proper context. The result
of this defect is a severe deficiency in total GAG expression
(10). To quantify GAG usage by RSV, we have titrated the
same virus sample on these two cell lines. The ratio of the
CHO K1 titer to the CHO A745 titer is the GAG dependency
index. A GAG dependency index of 1 means no preference for
GAG-expressing cells, whereas values of ?1 indicate an in-
creasing use of GAG for infection.
We used the GAG dependency index to compare the GAG
usage of recombinant GFP-expressing strain A2 RSV express-
ing all three of the viral glycoproteins, rgRSV-SGF, and mu-
tants of this virus that are missing the G or SH glycoprotein
gene or both. rgRSV-SGF and rgRSV-GF grown in HEp-2
cells displayed 17-fold and 14-fold GAG dependency, respec-
tively (Fig. 1A). The two viruses lacking the G protein,
rgRSV-F and rgRSV-SF, both displayed low GAG depen-
dency, fourfold and twofold, respectively. These results con-
firm previous findings that the G protein is the major GAG-
binding protein while the F protein plays a minor role in GAG
We also determined the GAG dependency index of virus
grown in Vero cells. Surprisingly, rgRSV-SGF and rgRSV-GF
grown in Vero cells showed 4-fold and 2-fold GAG depen-
dency, in contrast to the 17-fold and 14-fold GAG dependency
of the same viruses grown in HEp-2 cells (Fig. 1A). The GAG
dependency of the Vero cell-grown viruses expressing the G
protein was similar to that of viruses lacking the G protein,
suggesting that the G protein in Vero cell-grown RSV has
severely reduced function, at least with regard to the GAG
The G protein in virions. To compare the amount of G
protein in similar amounts of virions derived from HEp-2 and
Vero cells, we added Tran35S-label to the culture medium
during virus production, collected the supernatants after 72 h,
and purified these metabolically labeled virions twice by su-
crose density gradient. Similar amounts of virions based on the
amount of incorporated radioactivity were separated by SDS-
PAGE for Western blot analysis. The blot was probed with L9,
a MAb against the G protein. MAb D14, against the N protein,
was included as an internal virion control for loading variation.
As expected, the mature, 90-kDa form of the G protein was
readily detected in virions grown in HEp-2 cells. Minor
amounts of a 55-kDa and a 45-kDa form were also detected
(Fig. 1B, lane 1). The Vero cell-grown virions contained pri-
marily the 55-kDa form of the G protein, with a minor amount
FIG. 1. Importance of cell surface GAGs for infection and size of
the G protein in virions produced in HEp-2 and Vero cells. (A) GAG
dependency of recombinant RSVs from strain A2 expressing different
combinations of viral glycoproteins that were grown in HEp-2 or Vero
cells. Virus titers were determined on CHO K1 and CHO A745 cells
and compared as follows: CHO K1/CHO A745 ? GAG dependency
index. (B) Western blot assay of sucrose-purified virions (15,000 cpm)
probed with MAbs L9 (G protein) and D14 (N protein). All lanes are
from the same gel, from which irrelevant lanes were removed. (C) The
percentage of total virion G protein in the 90-kDa form was divided by
the amount of virion N protein to normalize the amount of G protein
in the virions produced by HEp-2 and Vero cells.
10712 KWILAS ET AL.J. VIROL.
of the 90-kDa and 45-kDa forms (Fig. 1B, lane 2). To deter-
mine whether the Vero cell line that we used was unique in this
regard, we examined virions produced in the WHO-approved
Vero cells used for vaccine production. Virions produced by
these Vero cells also contained predominantly the 55-kDa G
protein (Fig. 1B, lane 3).
If the virus from one cell line had more G protein than the
other, it might have an advantage in GAG binding. To examine
this possibility, we quantified the level of G protein in virions
from each cell line, normalized to the N protein (Fig. 1C).
There was minimal difference in the amounts of G protein in
virions from HEp-2 and Vero cells. However, the predominant
form of the G protein was very different. The 90-kDa form
represented 81% of the G protein in HEp-2 cell-grown virions
but only 12% of the G protein in Vero cell-grown virions, most
of the remainder in each case being the 55-kDa form. The low
abundance of the 90-kDa form of G protein in virions from
Vero cells correlates with the decreased GAG dependency of
Production and maturation of the G protein. G protein
forms ranging from 45 kDa to 60 kDa have previously been
described in infected cells and virions and suggested to be G
protein processing intermediates (5, 14, 20, 21, 50). To deter-
mine whether the G protein intermediates correspond to the
55-kDa form found in Vero cell-grown RSV, we performed a
pulse-chase experiment. At 24 h p.i., infected cells were pulsed
with Tran35S-label for 15 min, chased with excess unlabeled
methionine for the times indicated, and lysed and the G pro-
tein was IP with MAb L9. In HEp-2 cells, a 45-kDa processing
intermediate was present in the pulse (Fig. 2A, lane 3), while
the 90-kDa fully O-glycosylated, mature 90-kDa form of the G
protein first appeared in the 30-min chase (Fig. 2A, lane 5).
The 45-kDa G protein intermediate appeared similarly in Vero
cells (Fig. 2B, lane 3), but the 90-kDa form was less prominent.
Endo H digestion of these pulse-chase samples (Fig. 2A and
B, lanes 4, 6, 8, and 10) revealed that the 45-kDa G protein was
Endo H sensitive while the 90-kDa form was not. Endo H
sensitivity indicates the presence of immature N-linked sugars,
while resistance indicates that the N-linked sugars have been
matured in the Golgi compartment. This result confirms that
the 45-kDa G protein intermediate contains immature
N-linked carbohydrate chains, as expected for glycoproteins in
the ER, prior to maturation in the Golgi compartment. The
Endo H resistance of the 90-kDa form indicates that it has
migrated through the Golgi compartment. The size increase in
the G protein, from 45 kDa to 90 kDa, is consistent with the
addition of many O-linked carbohydrates, a process that also
takes place in the Golgi compartment.
To confirm that the increase in size requires transport to the
Golgi compartment, infected HEp-2 cells were treated with
monensin, a drug that blocks the transport of glycoproteins
from the ER to the Golgi compartment. Monensin treatment
prevented the appearance of the 90-kDa G protein (Fig. 2A
and B, lanes 9), and the 45-kDa form remained Endo H sen-
sitive (Fig. 2A and B, lanes 10), as expected. The 90-kDa
species of the G protein in these cell lysates is similar to the
size of the predominant form of the G protein in virions pro-
duced by HEp-2 cells. The 45-kDa species of the G protein in
these cell lysates appears to be somewhat smaller than the
55-kDa form that predominates in virions produced by Vero
cells. The lack of a 55-kDa species following a 1-h pulse indi-
cates that it is generated by a process that occurs more than 1 h
Maturation state of the G protein in virions. Purified virions
from both HEp-2 and Vero cells were treated with Endo H to
determine whether their 45- to 55-kDa forms were processing
intermediates. The minor 45-kDa species from both HEp-2
and Vero cell-grown viruses was sensitive to Endo H digestion,
increasing in migration to 36 kDa (Fig. 3B, lanes 2 and 4), the
size of unglycosylated G protein. This minor 45-kDa G protein
species is therefore an intermediate that has not been pro-
cessed in the Golgi compartment. In contrast, the 90-kDa and
55-kDa species were resistant to Endo H, indicating the pres-
ence of mature N-linked sugars on these forms, the result of
processing in the Golgi compartment. Since the 55-kDa form
has passed through the Golgi compartment, this form could be
the result of inefficient O-linked glycosylation or of cleavage of
the 90-kDa G protein with the loss of its C terminus.
Analysis of the C terminus of the G protein. To determine
whether the C terminus of the G protein is present in the
55-kDa form, we analyzed HEp-2 and Vero cell-purified viri-
ons by Western blot assay. The blot was probed with MAb L9,
which binds within the central, conserved cysteine region of the
G protein (Fig. 3A) (47). MAb L9 detected the 90-kDa, 55-
kDa, and 45-kDa forms, indicating that they are all present in
these virions (Fig. 3C, lanes 1 and 2). The blot was stripped and
reprobed with MAb 130-2G, which recognizes the C terminus
of the G protein (Fig. 3A) (43). This MAb stained the 90-kDa
and 45-kDa forms of the G protein but not the 55-kDa form
(Fig. 3C, lanes 3 and 4), indicating that the C terminus is
missing. A second C terminus-specific MAb, 232-1F (43), had
the same reactivity pattern (data not shown).
To confirm the loss of the C terminus of the G protein in the
55-kDa form, we constructed and rescued a virus with a six-His
tag at the C terminus of the G protein, rgRSV-G-6His (Fig.
3A). This virus was grown in HEp-2 and Vero cells, and puri-
FIG. 2. Pulse-chase of G protein in RSV-infected cells. (A) HEp-2
and (B) Vero cells were pulsed with Tran35S-label for 15 min. Cells
were lysed and then IP with MAb L9 at the times indicated (in min-
utes). After IP, the samples were treated with 500 U of Endo H for 2 h
at 37°C (?) or left untreated (?). After SDS-PAGE separation, the gel
was fixed and dried. The gel was exposed to film at ?80°C for 1 week
before film development. Mon, monensin.
VOL. 83, 2009 TRUNCATION OF RSV G PROTEIN INHIBITS INFECTION10713
fied virions were subjected to Western blot analysis with MAbs
L9 and 5His. MAb L9 reacted with both the 55-kDa and
90-kDa forms (Fig. 3D, lanes 1 and 2) from both virus prepa-
rations, whereas MAb 5His only reacted with the 90-kDa form
(Fig. 3D, lanes 3 and 4). These results confirm that the 55-kDa
G protein found in virions from both cell lines is missing its C
Cell and cell surface forms of the G protein. As a first step
in determining the location of the 55-kDa form within cells, we
compared infected total cell lysate with cell surface proteins
identified by biotinylation. The samples were electrophoresed
and probed with MAb L9 in order to visualize the forms and
relative proportions of the G protein in each sample. Infected
HEp-2 cell lysates and cell surface and purified virions (Fig. 4)
all displayed the 90-kDa form as the major form, with minor
amounts of the 55-kDa and 45-kDa forms. Cell lysates from
infected Vero cells (Fig. 4) also displayed more of the 90-kDa
form than of the 55-kDa form. In striking contrast, most of the
G protein on the cell surface was the 55-kDa form, with a
minor amount of the 90-kDa form, similar to the virions. The
difference between the Vero cell lysate and cell surface sug-
gests that the G protein is processed to the 55-kDa form at the
cell surface or in the Golgi compartment just before arriving at
the cell surface. Therefore, virions do not selectively incorpo-
FIG. 3. Glycan maturity and presence of the C terminus in virion G protein. (A) Schematic of the RSV G protein, an N-terminally anchored
type II glycoprotein. The soluble form of the G protein (sG) starts at Met-48 and is released from the cell surface by cleavage. A central conserved
domain with four conserved cysteines (CCCC) is followed by a predicted HBD. The G protein is modified by N-linked glycans (N) and many
O-linked glycans (potential sites are indicated by stalks with filled circles). The binding areas for MAbs L9, 130-2G, and 232-1F are designated by
arrows. The six-His tag at the COOH terminus is also shown. TM, transmembrane domain. (B) Western blot assay of viruses treated with Endo
H and probed with MAb L9. Samples containing each virus (15,000 cpm) were treated with 500 U of Endo H for 2 h at 37°C. (C) HEp-2 and Vero
cell-grown viruses were probed with MAb L9. The same blot was stripped, blocked, and reprobed with MAb 130-2G. (D) Western blot assay of
rgRSV-G-6His purified virions (15,000 cpm) from HEp-2 and Vero cells probed with MAb L9 or 5His.
FIG. 4. Comparison of G proteins from total cell lysates, the cell surface, and purified virions. Lanes 1, 4, 5, and 8 were stained with MAb L9.
Lanes 2, 3, 6, and 7 were processed separately to detect the biotinylated cell surface proteins that had been IP with MAb L9 and developed with
streptavidin-HRP and a chemiluminescent substrate.
10714 KWILAS ET AL.J. VIROL.
rate the 55-kDa form instead of the 90-kDa form as they are
budding from infected Vero cells. Instead, they incorporate the
cell surface G protein, which is predominantly the 55-kDa
Phenotype of virions from other cell lines. To determine
whether Vero cells are unique in the production of virions with
major amounts of a cleaved G protein, we analyzed rgRSV-G-
6His virions produced in other cell types used to grow RSV.
The MRC-5 human diploid fibroblast cell line is also approved
for vaccine production. PMK cells are often used in clinical
virology laboratories to isolate virus from patients for identi-
fication, and similar cells were used to grow the original for-
malin-inactivated RSV vaccine (26). BSC-1 cells were used in
early studies to identify RSV proteins, but the 90-kDa protein
was difficult to detect (52). Virions produced from these cell
lines incorporated less 90-kDa protein and more 55-kDa pro-
tein than did the HEp-2-grown virus (Fig. 5A and B), and all of
these virions displayed low GAG dependency (Fig. 5C).
HEp-2 and Vero cell-grown rgRSV-G-6His had a GAG
dependency similar to that of rgRSV-SGF (Fig. 1A), indicating
that addition of the His tag does not affect GAG binding. The
ratios of total G protein to N protein were also generally
similar (Fig. 5B). The 90-kDa G protein of virus grown in
MRC-5 cells represents 72% of the total G protein, compared
to 89% for HEp-2 cells, but its GAG dependency was reduced
by more than 3.5-fold (from 18-fold to 5-fold). These results
suggest that a threshold amount of 90-kDa G protein is needed
to determine GAG dependency. It is possible that the G pro-
tein is oligomeric and that it does not function if one or more
of the monomers is the 55-kDa form.
Phenotype of virions produced in HAE cell cultures. As
shown above, the cleavage and function of the virion G protein
depend on the cell line producing the virion. We have previ-
ously used primary well-differentiated HAE cell cultures as a
model to study RSV pathogenesis (54). The apical surface of
HAE cell cultures includes ciliated cells and mucus-producing
goblet cells. HAE cell cultures were inoculated with rgRSV-
SGF grown in HEp-2 cells, and virus was harvested from the
apical surface at 3 days p.i. The HAE cell-grown virus was
highly GAG dependent (Fig. 6A), similar to HEp-2 cell-grown
virus rather than to Vero cell-grown virus. To assess the size of
the G protein in HAE cell culture-grown virus, we examined
virions released from the apical surface of HAE cell cultures.
Because of the small sample size, these virions could not be
sucrose purified; however, virions were partially purified by
removing cell debris by low-speed centrifugation and by pel-
leting virions at a higher speed. The virus pellet was reduced
prior to SDS-PAGE. Western blotting identified a predomi-
nant, novel form of the G protein migrating at approximately
180 kDa (Fig. 6B, lanes 2 and 3) in addition to a small amount
of the 90-kDa form and a minor amount of a smaller G protein
form or breakdown product. The inoculum virus (Fig. 6B, lane
1) was grown in HEp-2 cells and displays primarily the 90-kDa
We also examined rgRSV-G-6His virus from HAE cell cul-
tures to determine if this 180-kDa form contains full-length G
protein. MAb L9 (Fig. 6C, lanes 1 and 2) and MAb 5His (Fig.
6C, lanes 3 and 4) both reacted with the 180-kDa form from
HAE cells, as well as the 90-kDa HEp-2 cell-grown virus con-
trol. This result clearly demonstrates that the C terminus of the
G protein is present in the 180-kDa form and therefore that
the G protein is full length. This conclusion is consistent with
the results above (Fig. 6A) demonstrating that the HAE cell
culture-grown virus retains high GAG dependency. We hy-
pothesize that this “supersized” G protein is either a dimer of
the 90-kDa G protein or the 90-kDa form with additional or
more extensive O-linked carbohydrate chains. In the absence
of reducing agent or boiling, the protein also migrates at 180
kDa (data not shown). These results imply that the protein is
not an aggregated form of the G protein.
Infection of HAE cell cultures with Vero cell-grown virus.
We have demonstrated that RSV grown in Vero cells does not
use GAGs efficiently for infection of immortalized cells and
that the cause of this reduced infection is loss of the C terminus
of the G protein. The loss of GAG binding should not affect
infection of HAE cell cultures because these cells do not ex-
FIG. 5. Forms of the G protein in virions produced by other cell
lines. (A) Western blot assay of purified rgRSV-G-6His virions probed
with MAbs L9 and D14. (B) Ratio of the total G protein versus N
protein and the percent 90-kDa G protein. Ratios were determined
from triplicate blots. (C) GAG dependency of rgRSV-G-6His grown in
different cells. Virus titers on CHO K1/CHO A745 ? GAG depen-
VOL. 83, 2009 TRUNCATION OF RSV G PROTEIN INHIBITS INFECTION10715
press HS on their apical surface (53) and RSV infects only via
the apical surface (54). Nevertheless, loss of the G protein C
terminus may affect its function, attachment to respiratory
epithelial cells. We tested this possibility by inoculating HAE
cell cultures with rgRSV-SGF produced in HEp-2 or Vero
cells. Equivalent numbers of infective units of each virus were
used to inoculate the HAE cell cultures. The virus titers used
to calculate the infectivity of the stock virus was determined on
GAG-deficient CHO A745 cells. In this way, the titer advan-
tage that GAG binding provides HEp-2 cell virus when titrated
on a GAG expressing cell line was eliminated.
At 1 day p.i. with rgRSV-SGF grown in Vero cells, the level
of GFP expression was 600- to 1,800-fold less than that of HAE
cell cultures inoculated with virus from HEp-2 cells (Fig. 7).
The finding that Vero cell-grown RSV is extremely inefficient
at infecting HAE cell cultures compared to HEp-2 cell-grown
RSV indicates that the complete 90-kDa G protein is impor-
tant for infection of primary respiratory cells. In the same
experiment, we inoculated HAE cell cultures with rgRSV-F, a
virus completely lacking the G and SH genes, grown in HEp-2
cells. This virus also initiated infection of HAE cell cultures
inefficiently, 9- to 13-fold less than rgRSV-SGF grown in
At 2 days p.i., all of the viruses had begun to spread, includ-
ing Vero cell-grown rgRSV-SGF. The form of the G protein in
the virions produced by HAE cells would be determined by the
HAE cell cultures at this point, not by Vero cells. rgRSV-SGF
spread more rapidly than rgRSV-F. This result is consistent
with progeny virions from HAE cell cultures that contain the
full-size (or supersized) G protein which enables subsequent
infection and spread in HAE cell cultures.
In early biochemical studies, the large RSV glycoprotein,
eventually named the G protein, was found in virions produced
from infected HeLa cells (34, 40) but not in virions from BSC-1
cells (52). BSC-1 cells were derived from African green mon-
key kidneys, as are Vero cells. A study to resolve this dramatic
difference between American- and Scottish-grown RSVs found
that both cell lines produced the 85-kDa form, though the
BSC-1 cells produced less (42). In the present report, we have
identified an associated phenotype: RSV produced in HEp-2
cells (or HeLa cells [data not shown]) are highly dependent
on GAGs for virus entry, while RSV grown in Vero, PMK,
MRC-5, A549, or BSC-1 cells is much less dependent on
GAGs for infection.
We have found that while virions from Vero cells have low
levels of the 90-kDa G protein, they contain much more of a
smaller, 55-kDa version of the G protein. Using our GAG
dependency assay, we have determined that this Vero cell-
grown RSV infects GAG-expressing CHO cells fourfold less
efficiently than virus produced in HEp-2 cells and containing
primarily the 90-kDa full-length G protein. It appears that the
90-kDa form of the G protein uses GAGs on the surface of
immortalized cells to initiate infection much more efficiently
than the 55-kDa form.
Estimating the cleavage position in the 90-kDa G protein
that would generate a 55-kDa G protein is challenging. Iden-
tifying the site is helped by knowing that the cleavage site is
conserved between the A2 and Long strains of RSV because
virions from Long also contain primarily the 55-kDa G protein
when produced in Vero cells (data not shown). The real diffi-
culty in estimating the position is due to the large amount of
carbohydrates attached to this protein. The G protein contains
FIG. 6. (A) GAG dependency indexes of RSVs produced in
HEp-2, Vero, and HAE cells. Virus titers were determined on CHO
K1 and CHO A745 cells and compared as follows: CHO K1/CHO
A745 ? GAG dependency index. (B) Western blot assay of crude virus
released from HAE cell cultures, probed with MAb L9. HEp-2 virus
initial inoculum bound to HAE cell culture at 0 days p.i. (lane 1), HAE
cell virus culture no. 1 (lane 2), HAE cell virus culture no. 2 (lane 3).
(C) Western blot assay of rgRSV-G-6His virions from HEp-2 cells and
HAE cell cultures probed with MAb L9 or 5His.
FIG. 7. Infection of primary HAE cell cultures inoculated with
rgRSV-SGF grown in HEp-2 and Vero cells and rgRSV-F grown in
HEp-2 cells (6.2 ? 106PFU in 100 ?l [?MOI, 0.05]). The percent GFP
was calculated from five random pictures obtained from each HAE cell
culture. Each time point represents three HAE cell cultures.
10716 KWILAS ET AL.J. VIROL.
seven potential N-linked glycosylation sites, but the difference
of 9 kDa between the 45-kDa intermediate and its size follow-
ing Endo H treatment, 36 kDa, suggests that only three or four
of these sites are used. Approximately half of the final 90-kDa
molecular mass is due to O-linked glycosylation. We used the
program NetNGlyc 1.0 to predict the three or four N-linked
sites most likely to be used and the NetOGlyc 3.1 (25) program
to predict the most likely O-glycosylation sites (ExPASy Pro-
teomics Server, http://ca.expasy.org/tools/#ptm). Using these
programs to predict molecular weight, we estimate that the
cleavage site would be C terminal to the central disulfide-
bonded region (13), within or just C terminal to the HBD (Fig.
4A). HBDs are defined by a high concentration of basic amino
acids. Cleavage in or near the HBD might well destroy the
ability of the G protein to bind GAGs. One or more of the
many lysines in this area could be a target for a plasmin-like
enzyme, or the lone arginine could be the substrate for a
The original, formalin-inactivated RSV vaccine that led to
increased pathology rather than protection upon challenge was
produced in cells derived from the same source as Vero cells,
African green monkey kidneys (17, 26, 29). Vero cells have
been used to produce similar vaccines for many of the animal
studies that have focused on the cause of the increased pathol-
ogy. Along with the increased pathology, the vaccine induced
nonfunctional antibodies. Deletions from the G protein C ter-
minus result in a loss of recognition by human, rabbit, and
murine antibodies (32, 38, 39), suggesting that the C terminus
of the G protein contains its major antigenic determinants. The
reduced amount of the C-terminal antigenic determinants and
the presence of a possibly misfolded, truncated G protein may
have also contributed to the decrease in neutralizing antibod-
ies and the increase in nonfunctional antibodies in children
who received the formalin-inactivated RSV vaccine (37).
While GAG interactions are important for infection of im-
mortalized cells in culture, it is unlikely that they are critical for
infection of airway cells in vivo. The well-differentiated HAE
cell cultures that appear and function like normal airway epi-
thelium do not express HS on their apical surface (53). We
anticipate, therefore, that the ability of the RSV G protein to
bind to GAGs will not be important for the initiation of RSV
infection of HAE cell cultures or of airway cells in vivo. How-
ever, the loss of the C-terminal region of the G protein might
well lead to a loss of attachment function. Indeed, we found
that the infectivity of Vero cell-grown virus for HAE cell cul-
tures is much lower (600- to 1,800-fold) than that of HEp-2
cell-grown virus (Fig. 7, 1 day p.i.).
Since HAE cell cultures closely model the human airway
epithelium, attenuated vaccines grown in Vero cells would also
be expected to infect humans inefficiently. In trials of attenu-
ated virus vaccines, escalating doses are used to identify the
lowest dose that induces an adequate antibody response. Vero
cell-grown attenuated vaccine candidates do induce neutraliz-
ing antibodies in infants. However, these vaccine candidates
have caused respiratory symptoms in a small number of infants
and these symptoms have been deemed unacceptable (28).
We predict that if such vaccines were produced in a different
cell line, less inoculum would be needed to induce the same
antibody response. In addition to increased efficiency of vac-
cine production, a lower inoculum would also reduce exposure
to nonreplicating virus antigens, cell culture-derived cytokines,
chemokines, and other possible contaminants in the inoculum.
Exposure to local high levels of these proteins could contribute
to inflammation and symptoms in the minority of infants who
have been vaccinated. After the initial infection of HAE cell
cultures, the source of the virus would not play a role, as
progeny virus from the infected HAE cells would contain pri-
marily intact G protein (Fig. 6B). This virus spreads at a rate
similar to infection initiated by HEp-2 cell-grown RSV (Fig. 7),
after a 3-day lag. The same pattern would likely be true in
The G protein is important for infection in vivo, since RSV
lacking its G gene replicates poorly in rodents and nonhuman
primates (8). In model animal experiments, as in vaccine trials,
Vero cell-grown virus could reduce the efficiency of the initial
infection. In fact, Vero cell-grown virus has been shown to
infect mice poorly compared to HeLa cell-grown virus (44),
consistent with our data and predictions.
In summary, efficient RSV infection requires the intact G
protein, particularly infection of primary respiratory epithelial
target cells. Most of the G protein in virions produced from
Vero cells is truncated by cleavage. This truncated form of the
G protein does not appear to be functional in either GAG
attachment to immortalized cells or attachment to primary
HAE cell cultures, since the initiation of infection is reduced in
both cases. This loss of attachment function would most likely
also result in poor infection initiation in vivo, negatively im-
pacting on both animal experiments and attenuated-vaccine
studies with volunteers.
We thank Barb Newton for excellent technical help, Peter Collins
for the recombinant RSV system, Larry Anderson for the C-terminal
antibodies for the G protein, Russell Durbin for helpful discussions,
Cliff Beall for the WHO-approved Vero cells, and Jeff Esko for the
This work was supported by NIH grants AI047213 and HL051818.
1. Anderson, L. J., P. Bingham, and J. C. Hierholzer. 1988. Neutralization of
respiratory syncytial virus by individual and mixtures of F and G protein
monoclonal antibodies. J. Virol. 62:4232–4238.
2. Barretto, N., L. K. Hallak, and M. E. Peeples. 2003. Neuraminidase treat-
ment of respiratory syncytial virus-infected cells or virions, but not target
cells, enhances cell-cell fusion and infection. Virology 313:33–43.
3. Byrappa, S., D. K. Gavin, and K. C. Gupta. 1995. A highly efficient proce-
dure for site-specific mutagenesis of full-length plasmids using Vent DNA
polymerase. Genome Res. 5:404–407.
4. Chanock, R. M., and L. Finberg. 1957. Recovery from infants with respira-
tory illness of a virus related to chimpanzee coryza agent (CCA). II. Epide-
miological aspects of infection in infants and young children. Am. J. Hyg.
5. Collins, P. L., and G. Mottet. 1992. Oligomerization and post-translational
processing of glycoprotein G of human respiratory syncytial virus: altered
O-glycosylation in the presence of brefeldin A. J. Gen. Virol. 73:849–863.
6. Collins, P. L., and B. R. Murphy. 2005. New generation live vaccines against
human respiratory syncytial virus designed by reverse genetics. Proc. Am.
Thorac. Soc. 2:166–173.
7. Collins, P. L., and B. R. Murphy. 2002. Respiratory syncytial virus: reverse
genetics and vaccine strategies. Virology 296:204–211.
8. Crowe, J. E., Jr., P. T. Bui, C. Y. Firestone, M. Connors, W. R. Elkins, R. M.
Chanock, and B. R. Murphy. 1996. Live subgroup B respiratory syncytial
virus vaccines that are attenuated, genetically stable, and immunogenic in
rodents and nonhuman primates. J. Infect. Dis. 173:829–839.
9. Dowell, S. F., L. J. Anderson, H. E. Gary, Jr., D. D. Erdman, J. F. Plouffe,
T. M. File, Jr., B. J. Marston, and R. F. Breiman. 1996. Respiratory syncytial
virus is an important cause of community-acquired lower respiratory infec-
tion among hospitalized adults. J. Infect. Dis. 174:456–462.
10. Esko, J. D., T. E. Stewart, and W. H. Taylor. 1985. Animal cell mutants
VOL. 83, 2009TRUNCATION OF RSV G PROTEIN INHIBITS INFECTION10717
defective in glycosaminoglycan biosynthesis. Proc. Natl. Acad. Sci. USA Download full-text
11. Falsey, A. R., and E. E. Walsh. 2000. Respiratory syncytial virus infection in
adults. Clin. Microbiol. Rev. 13:371–384.
12. Feldman, S. A., S. Audet, and J. A. Beeler. 2000. The fusion glycoprotein of
human respiratory syncytial virus facilitates virus attachment and infectivity
via an interaction with cellular heparan sulfate. J. Virol. 74:6442–6447.
13. Feldman, S. A., R. M. Hendry, and J. A. Beeler. 1999. Identification of a
linear heparin binding domain for human respiratory syncytial virus attach-
ment glycoprotein G. J. Virol. 73:6610–6617.
14. Fernie, B. F., G. Dapolito, P. J. Cote, Jr., and J. L. Gerin. 1985. Kinetics of
synthesis of respiratory syncytial virus glycoproteins. J. Gen. Virol. 66(Pt.
15. Fuentes, S., K. C. Tran, P. Luthra, M. N. Teng, and B. He. 2007. Function
of the respiratory syncytial virus small hydrophobic protein. J. Virol. 81:
16. Fulcher, M. L., S. Gabriel, K. A. Burns, J. R. Yankaskas, and S. H. Randell.
2005. Well-differentiated human airway epithelial cell cultures. Methods
Mol. Med. 107:183–206.
17. Fulginiti, V. A., J. J. Eller, O. F. Sieber, J. W. Joyner, M. Minamitani, and
G. Meiklejohn. 1969. Respiratory virus immunization. I. A field trial of two
inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza
virus vaccine and an alum-precipitated respiratory syncytial virus vaccine.
Am. J. Epidemiol. 89:435–448.
18. García-Beato, R., I. Martinez, C. Franci, F. X. Real, B. Garcia-Barreno, and
J. A. Melero. 1996. Host cell effect upon glycosylation and antigenicity of
human respiratory syncytial virus G glycoprotein. Virology 221:301–309.
19. Gorman, J. J., B. L. Ferguson, D. Speelman, and J. Mills. 1997. Determi-
nation of the disulfide bond arrangement of human respiratory syncytial
virus attachment (G) protein by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry. Protein Sci. 6:1308–1315.
20. Gruber, C., and S. Levine. 1985. Respiratory syncytial virus polypeptides. IV.
The oligosaccharides of the glycoproteins. J. Gen. Virol. 66:417–432.
21. Gruber, C., and S. Levine. 1985. Respiratory syncytial virus polypeptides. V.
The kinetics of glycoprotein synthesis. J. Gen. Virol. 66:1241–1247.
22. Hallak, L., D. Spillman, P. L. Collins, and M. E. Peeples. 2000. Glycosami-
noglycan sulfation requirements for respiratory syncytial virus infection.
J. Virol. 74:10508–10513.
23. Hallak, L. K., P. L. Collins, W. Knudson, and M. E. Peeples. 2000. Iduronic
acid-containing glycosaminoglycans on target cells are required for efficient
respiratory syncytial virus infection. Virology 271:264–275.
24. Johnson, P. R., M. K. Spriggs, R. A. Olmsted, and P. L. Collins. 1987. The
G glycoprotein of human respiratory syncytial viruses of subgroups A and B:
extensive sequence divergence between antigenically related proteins. Proc.
Natl. Acad. Sci. USA 84:5625–5629.
25. Julenius, K., A. Molgaard, R. Gupta, and S. Brunak. 2005. Prediction,
conservation analysis, and structural characterization of mammalian mucin-
type O-glycosylation sites. Glycobiology 15:153–164.
26. Kapikian, A. Z., R. H. Mitchell, R. M. Chanock, R. A. Shvedoff, and C. E.
Stewart. 1969. An epidemiologic study of altered clinical reactivity to respi-
ratory syncytial (RS) virus infection in children previously vaccinated with an
inactivated RS virus vaccine. Am. J. Epidemiol. 89:405–421.
27. Karron, R. A., D. A. Buonagurio, A. F. Georgiu, S. S. Whitehead, J. E.
Adamus, M. L. Clements-Mann, D. O. Harris, V. B. Randolph, S. A. Udem,
B. R. Murphy, and M. S. Sidhu. 1997. Respiratory syncytial virus (RSV) SH
and G proteins are not essential for viral replication in vitro: clinical evalu-
ation and molecular characterization of a cold-passaged, attenuated RSV
subgroup B mutant. Proc. Natl. Acad. Sci. USA 94:13961–13966.
28. Karron, R. A., P. F. Wright, R. B. Belshe, B. Thumar, R. Casey, F. Newman,
F. P. Polack, V. B. Randolph, A. Deatly, J. Hackell, W. Gruber, B. R.
Murphy, and P. L. Collins. 2005. Identification of a recombinant live atten-
uated respiratory syncytial virus vaccine candidate that is highly attenuated
in infants. J. Infect. Dis. 191:1093–1104.
29. Kim, H. W., J. G. Canchola, C. D. Brandt, G. Pyles, R. M. Chanock, K.
Jensen, and R. H. Parrott. 1969. Respiratory syncytial virus disease in infants
despite prior administration of antigenic inactivated vaccine. Am. J. Epide-
30. Krusat, T., and H. J. Streckert. 1997. Heparin-dependent attachment of
respiratory syncytial virus (RSV) to host cells. Arch. Virol. 142:1247–1254.
31. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
32. Lambert, D. M. 1988. Role of oligosaccharides in the structure and function
of respiratory syncytial virus glycoproteins. Virology 164:458–466.
33. Langedijk, J. P., W. M. Schaaper, R. H. Meloen, and J. T. van Oirschot.
1996. Proposed three-dimensional model for the attachment protein G of
respiratory syncytial virus. J. Gen. Virol. 77(Pt. 6):1249–1257.
34. Levine, S. 1977. Polypeptides of respiratory syncytial virus. J. Virol. 21:427–
35. Levine, S., R. Klaiber-Franco, and P. R. Paradiso. 1987. Demonstration that
glycoprotein G is the attachment protein of respiratory syncytial virus.
J. Gen. Virol. 68:2521–2524.
36. Martínez, I., and J. A. Melero. 2000. Binding of human respiratory syncytial
virus to cells: implication of sulfated cell surface proteoglycans. J. Gen. Virol.
37. Murphy, B. R., G. A. Prince, E. E. Walsh, H. W. Kim, R. H. Parrott, V. G.
Hemming, W. J. Rodriguez, and R. M. Chanock. 1986. Dissociation between
serum neutralizing and glycoprotein antibody responses of infants and chil-
dren who received inactivated respiratory syncytial virus vaccine. J. Clin.
38. Palomo, C., P. A. Cane, and J. A. Melero. 2000. Evaluation of the antibody
specificities of human convalescent-phase sera against the attachment (G)
protein of human respiratory syncytial virus: influence of strain variation and
carbohydrate side chains. J. Med. Virol. 60:468–474.
39. Palomo, C., B. Garcia-Barreno, C. Penas, and J. A. Melero. 1991. The G
protein of human respiratory syncytial virus: significance of carbohydrate
side-chains and the C-terminal end to its antigenicity. J. Gen. Virol. 72(Pt.
40. Peeples, M., and S. Levine. 1979. Respiratory syncytial virus polypeptides:
their location in the virion. Virology 95:137–145.
41. Prince, G. A., S. J. Curtis, K. C. Yim, and D. D. Porter. 2001. Vaccine-
enhanced respiratory syncytial virus disease in cotton rats following immu-
nization with Lot 100 or a newly prepared reference vaccine. J. Gen. Virol.
42. Pringle, C. R., P. V. Shirodaria, H. B. Gimenez, and S. Levine. 1981. Antigen
and polypeptide synthesis by temperature-sensitive mutants of respiratory
syncytial virus. J. Gen. Virol. 54:173–183.
43. Sullender, W. 1995. Antigenic analysis of chimeric and truncated G proteins
of respiratory syncytial virus. Virology 209:70–79.
44. Taylor, G., E. J. Stott, M. Hughes, and A. P. Collins. 1984. Respiratory
syncytial virus infection in mice. Infect. Immun. 43:649–655.
45. Techaarpornkul, S., N. Barretto, and M. E. Peeples. 2001. Functional anal-
ysis of recombinant respiratory syncytial virus deletion mutants lacking the
small hydrophobic and/or attachment glycoprotein gene. J. Virol. 75:6825–
46. Techaarpornkul, S., P. L. Collins, and M. E. Peeples. 2002. Respiratory
syncytial virus with the fusion protein as its only viral glycoprotein is less
dependent on cellular glycosaminoglycans for attachment than complete
virus. Virology 294:296–304.
47. Walsh, E. E., A. R. Falsey, and W. M. Sullender. 1998. Monoclonal antibody
neutralization escape mutants of respiratory syncytial virus with unique al-
terations in the attachment (G) protein. J. Gen. Virol. 79(Pt. 3):479–487.
48. Walsh, E. E., C. B. Hall, J. J. Schlesinger, M. W. Brandriss, S. Hildreth, and
P. Paradiso. 1989. Comparison of antigenic sites of subtype-specific respi-
ratory syncytial virus attachment proteins. J. Gen. Virol. 70(Pt. 11):2953–
49. Wertz, G. W., P. L. Collins, Y. Huang, C. Gruber, S. Levine, and L. A. Ball.
1985. Nucleotide sequence of the G protein gene of human respiratory
syncytial virus reveals an unusual type of viral membrane protein. Proc. Natl.
Acad. Sci. USA 82:4075–4079.
50. Wertz, G. W., M. Krieger, and L. A. Ball. 1989. Structure and cell surface
maturation of the attachment glycoprotein of human respiratory syncytial
virus in a cell line deficient in O glycosylation. J. Virol. 63:4767–4776.
51. Wright, P. F., R. A. Karron, R. B. Belshe, J. Thompson, J. E. Crowe, Jr., T. G.
Boyce, L. L. Halburnt, G. W. Reed, S. S. Whitehead, E. L. Anderson, A. E.
Wittek, R. Casey, M. Eichelberger, B. Thumar, V. B. Randolph, S. A. Udem,
R. M. Chanock, and B. R. Murphy. 2000. Evaluation of a live, cold-passaged,
temperature-sensitive, respiratory syncytial virus vaccine candidate in in-
fancy. J. Infect. Dis. 182:1331–1342.
52. Wunner, W. H., and C. R. Pringle. 1976. Respiratory syncytial virus proteins.
53. Zhang, L., A. Bukreyev, C. I. Thompson, B. Watson, M. E. Peeples, P. L.
Collins, and R. J. Pickles. 2005. Infection of ciliated cells by human parain-
fluenza virus type 3 in an in vitro model of human airway epithelium. J. Virol.
54. Zhang, L., M. E. Peeples, R. C. Boucher, P. L. Collins, and R. J. Pickles.
2002. Respiratory syncytial virus infection of human airway epithelial cells is
polarized, specific to ciliated cells, and without obvious cytopathology. J. Vi-
10718KWILAS ET AL. J. VIROL.