JOURNAL OF VIROLOGY, Nov. 2004, p. 12677–12682
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 22
Novel Role for Decay-Accelerating Factor in Coxsackievirus
A21-Mediated Cell Infectivity
Nicole G. Newcombe,1Leone G. Beagley,1Dale Christiansen,2Bruce E. Loveland,2
E. Susanne Johansson,1Ken W. Beagley,3Richard D. Barry,1
and Darren R. Shafren1*
Picornaviral Research Unit1and Discipline of Immunology/Microbiology,3School of Biomedical Sciences,
Faculty of Health, The University of Newcastle, Newcastle, New South Wales 2300, and
The Austin Research Institute, Heidelberg, Victoria 3084,2Australia
Received 7 December 2003/Accepted 14 June 2004
Decay-accelerating factor (DAF) is involved in the cell membrane attachment of many human enteroviruses.
Presently, further specific active roles of DAF in mediating productive cell infection and in the pathogenesis
of natural enterovirus infection are poorly understood. In an attempt to more fully understand the role of DAF
in lytic cell infection we examined the specific interactions of the prototype strain of coxsackievirus A21
(CVA21) with surface-expressed DAF. Investigations into discrete DAF-CVA21 interactions focused on viral
binding; viral particle elution with respect to the parameters of time, temperature, and pH; and subsequent cell
infection. Radiolabeled-virus binding assays revealed that peak elution of CVA21 from DAF occurred within 15
min of initial attachment and that the DAF-eluted virus increased in a linear fashion with respect to
temperature and pH. CVA21 eluted from endogenous surface-expressed DAF was highly infectious, in contrast
to CVA21 eluted from intercellular adhesion molecule 1 (ICAM-1), which retained little to no infectivity. Using
an adenovirus transduction system, we demonstrate that CVA21 can remain infectious for up to 24 h after DAF
binding and is capable of initiating a multicycle lytic infection upon delayed ICAM-1 surface expression. Taken
together, the data suggest that a major role of DAF in cell infection by the prototype strain of CVA21 is to
provide membrane concentration of infectious virions, effectively increasing viral interactions with endogenous
or induced ICAM-1.
Coxsackievirus A21 (CVA21) is a human enterovirus and a
causative agent of upper respiratory tract infections (7). The
prototype strain of CVA21 (Kuykendall) utilizes two separate
host cell receptors, decay-accelerating factor (DAF) for cell
binding and intercellular adhesion molecule 1 (ICAM-1) for
both attachment and cell internalization (24). The use of two
receptors by CVA21 is thought to provide multiple cell attach-
ment sites and alternative mechanisms of viral cell entry,
thereby increasing the chances of a productive infection. Not
all cellular receptors that mediate enteroviral attachment to
the cell surface facilitate cell entry. Coxsackievirus B3 (CVB3)
can bind to DAF or the coxsackievirus-adenovirus receptor
(CAR), with only interaction with CAR permitting successful
infection (27). CVA9 recognizes the vitronectin receptor
?v?3and ?2-microglobulin but requires the heat shock protein
GRP78 and interactions with major histocompatibility complex
class I molecules for internalization (31).
A direct involvement for ICAM-1 in viral infection has been
established for the major group of human rhinoviruses (8) and
numerous group A coxsackieviruses (16, 24, 25). The ICAM-1
binding footprint on the CVA21 virion capsid maps to a deep
surface depression or “canyon” surrounding each fivefold ver-
tex (1, 5, 32). In contrast to ICAM-1, DAF is postulated to bind
in a more exposed region on the capsid surface (11). Many
human enteroviruses use DAF as a cellular attachment recep-
tor; these include echovirus 3 (EV3), -6, -7, -11, -12, -13, -19,
-20, -21, -24, -29, and -33; enterovirus 70; CVB1, -3, and -5; and
CVA21 (2, 12, 23). DAF appears to play a passive role during
enteroviral infection, as expression of DAF alone on rodent
cells is capable of facilitating cellular binding but not infection
by CVB1, CVB3, CVB5, EV7, and CVA21 (2, 23, 24).
Specific roles for DAF during virus-induced lytic infection,
other than passive viral attachment, have at present not been
established. The inability of DAF to facilitate enteroviral in-
fection is possibly due its failure to induce capsid conforma-
tional changes (22). More recently, to various degrees, low-
cell-culture-passage clinical isolates of CVA21 were shown to
be capable of lytically infecting DAF-expressing rhabdomyo-
sarcoma (RD) cells in the absence of ICAM-1, most probably
mediated by capsid cross-linking of DAF in a manner similar
to that of the anti-DAF monoclonal antibodies (17).
The study herein focuses on investigations addressing the
active role(s) of DAF during infection by the CVA21 proto-
type strain. Characterization of DAF binding and the relative
rates of CVA21 elution from DAF with respect to time, tem-
perature, and pH were investigated. Furthermore, a virion
concentration role for DAF in enteroviral infection was inves-
tigated by examining the capacity of DAF-bound CVA21 to
maintain infectivity prior to and following elution and evalu-
ating its subsequent potential in facilitating productive cell
infection via interactions with delayed ICAM-1 expression.
Binding of CVA21 and its elution from DAF. Picornaviral
cell attachment and subsequent cell entry are characterized by
* Corresponding author. Mailing address: The Picornaviral Re-
search Unit, Discipline of Immunology and Microbiology, Faculty of
Health, The University of Newcastle, Level 3, David Maddison Clinical
Sciences Building, Royal Newcastle Hospital, Newcastle, New South
Wales 2300, Australia. Phone: 61 2 4923 6158. Fax: 61 2 4923 6814.
elution of high levels of viral particles from their specific cell
surface receptors following initial attachment (6). Similar to
what is found for many picornaviruses, when the prototype
strain of CVA21 is eluted from its natural internalizing recep-
tor, ICAM-1, it possesses a significantly reduced infectivity,
hence minimizing its capacity to initiate subsequent infections
(25). A focused characterization of the relative kinetics and
infectivity of prototype CVA21 particles eluted directly from
surface-expressed DAF has not previously been undertaken.
We concentrated our investigations on the effects of time,
temperature, and pH on elution of CVA21 from DAF, with
radiolabeled-virus binding assays performed using DAF-ex-
pressing CHO cells as the CVA21 binding substrate. The pro-
totype strain of CVA21 used in these investigations had been
passaged approximately 10 times in HeLa and/or human lung
fibroblasts and three or four times in ICAM-1-expressing RD
cells. Elution of CVA21 from DAF reached maximal levels at
15 min, with no further significant elution increase after this
time (Fig. 1A). The amount of CVA21 eluted from DAF was
increased by gradually elevating the temperature from 4 to
42°C while maintaining a constant elution time of 30 min (Fig.
1B). In this environment, maximal levels of CVA21 were eluted
at 42°C. Not surprisingly, the infectivity of virus eluted at 42°C
was significantly less than that of virus eluted at 37°C (data not
shown). Temperatures above 37°C may have an adverse effect
on the integrity of the virion capsid, resulting in reduced re-
ceptor binding and hence diminished infective capacity. The
significant increases in CVA21 elution levels occurring at tem-
peratures in excess of ambient levels may be representative of
temperature-dependent A-particle formation, as is the case for
DAF is expressed on the surfaces of cells in the gastrointes-
tinal tract (acidic pH environment) and cells in the respiratory
tract (neutral pH environment) (14, 19). Cells from both of
these areas are potential targets for CVA21 membrane attach-
ment, and we assessed whether changes in environmental pH
impacted CVA21 DAF elution. Increasing the pH of the elu-
tion environment from 5.5 to 8.0 while maintaining an elution
FIG. 1. CVA21 elution from DAF-expressing CHO cells in re-
sponse to time, temperature, and pH of the incubation media. (A) Ra-
diolabeled CVA21 (5 ? 105cpm) was incubated with DAF-expressing
CHO cells (approximately 2 ? 107) for 2 h at 4°C; following three
washes with phosphate-buffered saline (PBS) to remove unbound viri-
ons, cell aliquots were resuspended in 200 ?l of PBS and incubated at
37°C for 0, 1, 5, 15, 30, and 60 min to induce viral-particle elution.
Levels of CVA21 eluted were determined by liquid scintillation count-
ing (23). Approximately 10 to 15% of input radiolabeled CVA21
bound to DAF-expressing CHO cells (data not shown). (B) Radiola-
beled CVA21 was bound to cell surface-expressed DAF at 4°C for 2 h
and then eluted by incubation at the appropriate temperature for a
further 30 min. (C) Radiolabeled CVA21 was bound to cell surface-
expressed DAF at 4°C for 2 h and then eluted by incubation in media
of the appropriate pH at 37°C for a further 30 min.
FIG. 2. Infectivity of the prototype strain of CVA21 following elu-
tion from DAF and ICAM-1. (A) CHO cells (approximately 107)
expressing either ICAM-1 or DAF were incubated with 200 ?l of
[35S]methionine-labeled CVA21 (5 ? 105cpm) in serum-free Dulbec-
co’s modified Eagle medium for 2 h at 4°C. Following removal of
unbound virions, the cells were incubated at 37°C for 1 h. Levels of
[35S]methionine-labeled virus bound and eluted were measured by
liquid scintillation counting on a 1450 Microbeta TRILUX (Wallac,
Turku, Finland) (23). Results are expressed as means of triplicate
samples plus standard deviations. (B) The infectivity of virions eluted
from both cell lines was determined by lytic-end point dilution on
96-well monolayers of ICAM-1-expressing RD cells. Cell survival was
quantitated from quadruplicate wells by staining with a crystal violet-
methanol solution, and the relative absorbance of stained cell mono-
layers was read on a multiscan enzyme-linked immunosorbent assay
plate reader (Flow Laboratories, McLean, Va.) at 540 nm. Fifty per-
cent end point titers were calculated by the method of Reed and
Muench (21), where a well was scored as positive if the absorbance was
less than that of the no-virus control minus three standard deviations.
Results are expressed as log10TCID50of CVA21 per 102cpm.
FIG. 3. CVA21 induced lytic infection of RD cells following delayed induction of ICAM-1 expression. (A) Time course of ICAM-1 expression
following adenovirus transduction. RD cells were induced to express human ICAM-1 by transduction with 2.5 ? 106TCID50of a recombinant
adenovirus containing human ICAM-1 cDNA. Cells were assessed by flow cytometry for ICAM-1 expression at various times after adenovirus
inoculation with the anti-ICAM-1 domain monoclonal antibody (WEHI). (B) Flow-cytometric analysis of RD cells showing surface expression of
DAF, ICAM-1, and CD36 24 h following mock transduction or transduction with recombinant adenoviruses containing human ICAM-1 or CD36
cDNA. The solid histograms represent DAF expression, while the pink histograms represent ICAM-1 expression and the blue histograms represent
CD36 expression. The recombinant adenoviruses containing ICAM-1 cDNA or CD36 cDNA were constructed with an Adeno-quest kit (Quantum
Biotechnologies Inc.) in accordance with the manufacturer’s instructions. (C) CVA21 lytic infection of RD cells via the delayed expression of
ICAM-1. Monolayers of DAF-expressing RD cells in six-well culture plates were infected with the prototype strain of CVA21 (multiplicity of
infection ? 1.0 TCID50) for 1 h at 37°C. Following removal of non-DAF-bound CVA21 virions by four washes with serum-free Dulbecco’s modified
Eagle medium (DMEM), cell monolayers were transduced with 2.5 ? 106TCID50of a recombinant adenovirus containing ICAM-1 or CD36 cDNA
immediately or at 6 and 24 h after CVA21 inoculation. At 6 and 24 h after CVA21 inoculation, cell monolayers were washed with serum-free DMEM
prior to adenovirus transduction. Following adenovirus transduction, cell monolayers were incubated at 37°C for 24 h, at which time cell supernatants
were harvested. RD cells that were inoculated with CVA21 but mock transduced served as background cell controls. Levels of progeny CVA21
in the cell supernatants were determined by lytic-end point dilution on 96-well monolayers of ICAM-1-expressing RD cells. Cell survival from quadrup-
licate wells was quantitated by staining with a crystal violet-methanol solution, and the relative absorbance of stained cell monolayers was read on a
multiscan enzyme-linked immunosorbent assay plate reader (Flow Laboratories) at 540 nm. Fifty percent end point titers were calculated by the method
of Reed and Muench (21), where a well was scored as positive if the absorbance was less than the no-virus control minus three standard deviations.
VOL. 78, 2004NOTES 12679
time of 30 min and a temperature of 37°C resulted in a con-
tinual increase in the level of CVA21 eluted from DAF (Fig. 1C).
CVA21 eluted from DAF retains infectivity. A large propor-
tion of eluted picornaviral particles are generally recognized to
be noninfectious because cell-bound virions undergo specific
receptor-induced capsid conformational changes (9). To com-
pare the relative effects that binding to and elution from either
surface-expressed DAF or ICAM-1 exert on CVA21 infectiv-
ity and to determine whether DAF interactions induced tem-
perature-dependent A-particle formation, radiolabeled-virus
binding and cell lytic assays were performed using CHO DAF-
and CHO-ICAM-1-expressing cells as the CVA21 binding sub-
strates. Flow-cytometric analysis revealed high-level expres-
sion of DAF and ICAM-1 on the appropriate transfected-
CHO-cell surface (datanot
radiolabeled CVA21 bound to and eluted from both DAF and
ICAM-1 on the surfaces of the transfected cells (Fig. 2A). In
contrast to virus eluted from ICAM-1, only CVA21 eluted
from DAF displayed a significant retention of infectivity (?105
50% tissue culture infective doses [TCID50]/100 cpm) (Fig.
CVA21 can bind to DAF, retain infectivity, and initiate pro-
ductive infection following delayed expression of ICAM-1.
Once it was established that CVA21 eluted from surface-ex-
pressed DAF retains a high level of infectivity (Fig. 2B), in-
vestigations focused on whether DAF-eluted virus could play
an active role in the pathogenesis of natural CVA21 infections.
The specific question to be addressed was to determine wheth-
er virus bound by surface-expressed DAF could initiate a pro-
ductive infection utilizing a delayed induction of cell surface
ICAM-1. In an attempt to simulate such an environment,
DAF-expressing RD cells (ICAM-1 negative), normally refrac-
tive to CVA21 lytic infection, were transduced to express
ICAM-1 or CD36 (using recombinant adenovirus vectors) at 0,
6, and 24 h following initial CVA21 binding to RD cell surface
DAF. Flow-cytometric analysis revealed significant levels of
surface ICAM-1 expression at 4 h posttransduction, with ex-
pression increasing to maximal levels approximately 16 h after
adenovirus inoculation (Fig. 3A). Additional flow-cytometric
analysis (Fig. 3B) and Western blot assays (data not shown)
confirmed high-level expression of both ICAM-1 and CD36 at
24 h after transduction of the RD cells by the appropriate
receptor-bearing recombinant adenovirus, while levels of en-
dogenous DAF expression in all cells were comparable. Viral
infectivity assays were performed to compare the levels of
progeny CVA21 propagated in the presence of transduced
ICAM-1 and CD36 receptor expression at 0, 6, and 24 h fol-
lowing viral binding to endogenous RD cell DAF. The RD
cells transduced to express ICAM-1 at 0, 6, or 24 h after initial
inoculation with CVA21 produced significantly higher viral
yields (approximately 200-fold) than cells induced to express a
mock receptor (CD36) or nontransduced RD cells (Fig. 3C).
Multicycle replication of CVA21 in RD cells transduced to
express ICAM-1 at 0, 6, or 24 h after DAF binding resulted in
complete lytic destruction of the cell monolayers, whereas no
cell lysis was observed in cells expressing CD36 or in nontrans-
duced cells (Fig. 4; data not shown). No cell lysis of non-
CVA21-infected RD cells transduced with recombinant adeno-
viruses containing ICAM-1 and CD36 cDNAs was observed
Prototype and clinical isolates appear to vary with respect to
enteroviral interactions with DAF (3, 15, 17, 29). In the ab-
sence of ICAM-1 expression and antibody-cross-linked DAF,
clinical isolates of CVA21, to various degrees, achieve host cell
lytic infection, possibly by cross-linking DAF via specific viral
capsid interactions (17). Despite detailed descriptions of DAF
interactions for numerous clinical enterovirus isolates, actively
defined roles for DAF during lytic infection of prototype en-
teroviral strains have not been forthcoming. The general con-
sensus from many studies investigating enterovirus-DAF inter-
actions is that DAF functions as a viral concentration receptor,
simply accumulating virions on the cell surface for interaction
with additional functional internalizing receptors (26, 28). It
has been suggested that the nature of DAF binding to entero-
viral capsids is of low affinity because of a very fast dissociation
rate constant (13). In contrast, interactions of ICAM-1 with
viral capsids of similar architecture show comparable affinities
but with significantly slower kinetics, consistent with binding to
a relatively inaccessible site, the capsid canyon (4).
Studies addressing the impact of biophysical parameters,
such as time, temperature, and pH, on the elution of CVA21
from DAF highlight that CVA21 particles are eluted relatively
rapidly from DAF, and this elution is susceptible to increases
in temperature and pH (Fig. 1). Elution of CVA21 from
ICAM-1 is characterized by a dramatic reduction in viral in-
fectivity compared to that of virions eluted from DAF (Fig. 2).
CVA21 virions eluted from ICAM-1 undergo irreversible cap-
sid conformational changes as a result of receptor binding,
leaving them incapable of binding and initiating lytic infection
(22, 25). The capacity of DAF-eluted particles to remain in-
fectious is most probably a result of the inability of DAF to
FIG. 4. CVA21-induced lytic infection of RD cells transduced with
ICAM-1- or CD36-expressing adenovirus. DAF-expressing RD cells
were infected with the prototype strain of CVA21 (multiplicity of in-
fection [moi] ? 1.0 TCID50) for 1 h at 37°C. Following removal of non-
DAF-bound CVA21 virions by four washes with serum-free Dulbec-
co’s modified Eagle medium, cell monolayers were transduced with
2.5 ? 106TCID50of a recombinant adenovirus (Ad) containing ICAM-1
or CD36 cDNA. Following incubation for 24 h at 37°C, cell monolayers
were microscopically examined for cell lysis. Photomicrographs were tak-
en at a magnification of ?200. Time (T) zero represents cell monolayers
immediately after adenovirus transduction, while time 24 represents cell
monolayers 24 h following adenovirus transduction.
induce CVA21 capsid conformational changes (22). CVA21
particles eluted from DAF-expressing CHO cells possessed a
sedimentation coefficient in sucrose gradients similar to that of
infectious 160S particles, whereas CVA21 particles eluted
from ICAM-1-expressing CHO cells exhibited a reduced sed-
imentation coefficient, closer to that of 135S altered particles
(data not shown).
The reversible nature of the CVA21 interaction with DAF
was highlighted by the capacity of CVA21 to bind to DAF (on
ICAM-1-negative cells) and remain in an infectious state for
up to 24 h. The retention of infectivity allowed DAF-bound
virions to undergo cell entry and subsequent lytic infection
when presented with delayed ICAM-1 expression (Fig. 3 and
4). In the absence of detectable changes in cell cytopathology,
relatively high levels of infectious CVA21 on monolayers of
RD cells and RD cells transduced with adenovirus expressing
CD36 (Fig. 3C) persisted throughout the course of investiga-
tions, most likely because the residual viral inoculum bound
to DAF retained infectivity (Fig. 2). Alternatively, it may be
due to the presence of a subpopulation of virions within the
CVA21 prototype preparation that possess an enhanced DAF
usage phenotype, allowing cross-linking of DAF and initiating
a subsequent slow infection, a finding previously described for
clinical isolates of CVA21 (17).
The capacity of the prototype strain of CVA21 to use
DAF as an attachment receptor and retain a highly infec-
tious capacity is an extremely advantageous mechanism
given the widespread distribution of DAF throughout the
mammalian body, particularly on erythrocytes (18). In this
environment, DAF-expressing erythrocytes provide the vi-
rus with a ready transport vehicle through the body; bound
infectious virus can leave the erythrocyte surface and inter-
act with ICAM-1-expressing cells for lytic infection. It is
generally accepted that surface expression of endogenous
ICAM-1 throughout the human body is relatively low, await-
ing induction by the action of inflammatory cytokines such
as tumor necrosis factor alpha and interleukin-1? (30). Dur-
ing natural human rhinovirus infections, infected cells re-
lease such cytokines (20), which mediate enhanced ICAM-1
expression on surrounding cells. As some low-cell-culture-
passage clinical isolates of CVA21 are able to infect cells via
DAF alone, the question arises as to whether multiple cell
passages have led to the usage of ICAM-1 as an internaliz-
ing receptor. The finding that low-passage clinical CVA21
isolates exhibit more rapid and dramatic lytic infection of
ICAM-1-bearing cells than those expressing DAF alone (17)
suggests that ICAM-1 usage is most likely not a phenotype
acquired through in vitro propagation.
The data presented herein confirm two major roles of DAF
during CVA21 lytic infection. First the virus can bind to DAF
and elute while still retaining receptor binding capacity and,
hence, cell infectivity. Second, CVA21 can bind to DAF and
wait for the availability of significant levels of ICAM-1 expres-
sion on the same cell or proximal cells to allow viral internal-
ization and subsequent lytic infection. In aspects of both viral
evolution and pathogenesis, the capacity to bind to DAF must
be viewed as most advantageous for the prototype strain of
CVA21 and other DAF-binding enteroviruses in maximizing
cell infectivity, a phenotype that is retained and even enhanced
in virulent clinical CVA2I isolates.
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