JOURNAL OF VIROLOGY, Dec. 2007, p. 12927–12935
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 23
Interaction of Decay-Accelerating Factor with Coxsackievirus B3?
Susan Hafenstein,1Valorie D. Bowman,1Paul R. Chipman,1Carol M. Bator Kelly,1Feng Lin,2
M. Edward Medof,2and Michael G. Rossmann1*
Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, Indiana 47907-2054,1and Institute of
Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 441062
Received 1 May 2007/Accepted 24 August 2007
Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral
canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an
alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor
(CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been
identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a
variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The
DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with
one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of
echoviruses, suggesting independent evolutionary processes.
Coxsackieviruses belong to the family Picornaviridae, which
contains some of the most common viral pathogens of verte-
brates (39, 45, 48, 54, 56, 62). Picornaviruses are small, icosa-
hedral, nonenveloped animal viruses. Capsids have 60 copies
of each of four viral proteins, VP1, VP2, and VP3 (Fig. 1) and
VP4, that form an icosahedral shell ?300 Å in diameter filled
with a positive-sense, single-stranded RNA genome. A prom-
inent feature of the capsid surface is a depression around the
fivefold axes of symmetry called the “canyon” (41). The results
of both genetic and structural studies have shown that the
canyon is the site of receptor binding for many of these viruses
(2, 9, 19, 32, 43), including coxsackieviruses which utilize the
coxsackievirus-adenovirus receptor (CAR) (13). Receptor
molecules that bind into the canyon have been found to belong
to the immunoglobulin superfamily (43). When these receptor
molecules bind into the canyon, they dislodge a “pocket factor”
within a pocket immediately below the surface of the canyon
whose shape and environment suggest that it might be a lipid
(19, 52). The flexible floor of the canyon is depressed into the
pocket when receptor binds. In contrast, the roof of the pocket
is pushed upwards when occupied by pocket factor. Hence, the
receptor molecule and pocket factor have overlapping binding
sites, thus competing with each other for binding to the virus.
Absence of the hydrophobic pocket factor destabilizes the vi-
rus and initiates transition to altered “A” particles (10), a likely
prelude to uncoating of the virion, possibly during passage
through an endosomal vesicle (31, 59).
Not all receptors of picornaviruses bind in the canyon. A
group of minor rhinoviruses bind to very-low-density lipopro-
teins (16, 30, 61), and some other picornaviruses, including
certain coxsackie- and echoviruses, utilize decay-accelerating
factor (DAF) (CD55) as a cellular receptor (7, 22, 36, 50). In
general, coxsackievirus serotype B2 (CVB2), CVB4, and CVB6
do not bind DAF, whereas CVB1, CVB3, and CVB5 do bind
DAF (49), although there are variations in DAF binding within
the CVB3 serotype. Passage of CVB3 through human rhabdo-
myosarcoma (RD) cells, a cell line that is normally resistant to
CVB3 infection, gives rise to a variant form of the virus, CVB3-
RD. This variant is able to grow well on RD cells while retain-
ing the ability to infect HeLa cells, albeit with less efficiency
and by forming smaller plaques. The binding site of DAF on
the surfaces of echovirus 7 (EV7) and EV12 crosses the ico-
sahedral twofold axes of symmetry (4, 13, 35). Mutational
analysis has shown that two amino acids, residues 2108 and
2151 (residues in VP1, VP2, VP3 and VP4 are numbered
sequentially starting with 1001, 2001, 3001, and 4001, respec-
tively), determine the CVB3-RD phenotype in some strains
(24). These residues are located near the twofold axes of sym-
metry (29). However, subsequent work showed that additional
surface residues, 1080, 3137, 3184, and 3234, might contribute
to defining the CVB3-RD phenotype (51).
DAF is a member of a family of proteins that regulate
complement activation by binding to and accelerating the de-
cay of convertases, the central amplification enzymes of the
complement cascade (5, 20, 21, 27). As DAF is expressed on
virtually all cell surfaces, the host’s cells are protected from
attack by the host’s own immune system. The functional region
of DAF consists of four short consensus repeats (SCR1, -2, -3,
and -4), each about 60 amino acids long. The four domains rise
about 180 Å above the plasma membrane on a serine- and
threonine-rich stalk of 94 amino acids, 11 of which are O
glycosylated and attached to the plasma membrane by a gly-
coslphosphatidylinositol anchor. The entire DAF molecule is
relatively rigid, forming an extended rod with dimensions of
160 by 50 by 30 Å (25). Each of the SCR domains is folded into
a ? structure stabilized by disulfide bridges. The solution struc-
tures of the SCR2 and -3 domains and the crystal structures of
the SCR3 and -4 domains as well as the full-length human
DAF have been determined (25, 60, 64).
Structural and genetic studies have shown that closely re-
lated viruses have adapted to bind to DAF at different sites on
* Corresponding author. Mailing address: Department of Biological
Sciences, Purdue University, 915 W. State Street, West Lafayette, IN
47907-2054. Phone: (765) 494-4911. Fax: (765) 496-1189. E-mail: mr
?Published ahead of print on 5 September 2007.
the receptor surface (Table 1) (7, 18, 26, 37, 38, 50, 63). Fur-
thermore, different regions on the same SCR can be utilized by
different viruses for attachment (3, 35). Although DAF binding
is likely to facilitate viral adsorption and mediate tropism, the
availability of DAF receptor molecules on the host is normally
not sufficient for CVB3 or EV7 to be able to enter cells (37,
47). Presumably, viral adaptation to bind DAF provides ad-
ditional advantages. In order to investigate DAF interac-
tions with CVB3, a cryoelectron microscopy (cryoEM) re-
construction of DAF bound to CVB3-RD was made. The
binding site of DAF was found to cross the canyon on the
viral surface near the “puff” region on VP2 (41) that defines
the southern canyon rim.
MATERIALS AND METHODS
Protein and virus production. Human DAF was expressed in Pichia pastoris as
a C-terminally His6-tagged protein (14). The DAF construct consisted of the
full-length ectodomain, containing SCR1, -2, -3, and -4 (amino acids 1 to 254) but
lacking the S/T-rich linker domain and the glycoslphosphatidylinositol anchor.
The ectodomain of CAR protein (D1D2) used in the plaque reduction assays was
kindly provided by Paul Freimuth (Brookhaven National Laboratory) and was
produced and purified as described previously (13).
The CVB3 M strain (11) was propagated in HeLa cells and purified as de-
scribed previously (29). CVB3-RD was propagated in RD cells from an inoculum
kindly provided by Jeff Bergelson (University of Pennsylvania). RD cells were
brought to confluence in 150-mm-diameter plates at 37°C in Dulbecco minimal
Eagle medium (Invitrogen) with 10% fetal bovine serum. The cells were rinsed
with phosphate-buffered saline (PBS), followed by the addition of 5 ml of
CVB3-RD stock inoculum diluted in Dulbecco minimal Eagle medium per dish,
for a multiplicity of infection of 1 to 5. After incubation at 37° for 1 h, 7 ml of
fresh medium was added per dish, and infection was allowed to progress for 48 h.
Cells were harvested, pooled, and stored at ?80°C.
Virus was purified by freezing and thawing the cells three times before adding
NP-40 (1%). After homogenization, the preparation was centrifuged at 5,000
rpm for 10 min. MgCl2(to 5 mM), DNase (0.05 mg ml?1), and sodium dodecyl
sulfate (to 0.5%) were added to the supernatant and incubated for 30 min at
room temperature. Trypsin was added (0.5 mg ml?1) and incubated for 10 min
at 37°C. EDTA (10 mM) and sarcosine (1%) were added, and the pH was
adjusted to neutral with NH4OH. The virus was pelleted through 30% sucrose in
50 mM MES (morpholineethanesulfonic acid) (pH 6.0) by centrifugation at
48,000 rpm for 2 h using a 50.2 Ti rotor. The pellets were resuspended in 50 mM
MES, loaded onto 10 to 40% K-tartrate–50 mM MES gradients, and centrifuged
at 36,000 rpm for 90 min using an SW41 rotor. The virus bands were collected,
diluted in buffer, and pelleted by centrifugation at 48,000 rpm for 90 min using
a 50.2 Ti rotor. Pellets were resuspended in buffer to estimate the virus concen-
tration by measuring the absorbance at wavelengths of 260, 280, and 310 nm.
Data collection and cryoEM reconstruction. Full-length DAF molecules were
incubated with CVB3-RD at room temperature for 1 h at a ratio of four DAF
molecules per potential binding site on the virus (240:1). Small aliquots of this
mixture were applied to carbon-coated grids and vitrified in liquid ethane. Elec-
tron micrographs were recorded on Kodak SO-163 film by using a Phillips
CM300 FEG microscope. Micrographs were digitized with a Zeiss PHODIS
microdensitometer at 7-?m intervals. The scans were averaged in boxes of 2 by
2 pixels. The final averaged pixel size was 3.11 Å. Approximately 3,500 particles
were selected and corrected for contrast transfer function of the microscope
using the program RobEM (http://cryoem.ucsd.edu/programs.shtm). The defo-
cus distance ranged from 1.1 to 4.6 ?m. The EM reconstruction processes were
FIG. 1. Diagrammatic view of a picornavirus (top left). A thick black line outlines a protomer, an assembly intermediate. Blue, green, and red
correspond to VP1, VP2, and VP3, respectively. One icosahedral asymmetric unit is enlarged (center) to show the location of the canyon and the
entrance (white disc) to the hydrophobic binding pocket (white dashed lines) containing the lipid pocket factor. On the right is shown a side view
of the pocket, showing the entrance to the pocket from the canyon.
TABLE 1. Dominant DAF SCR interactions with different
DAF interaction at site:
12928 HAFENSTEIN ET AL.J. VIROL.
performed using icosahedral averaging with the programs EMPFT and EM3DR
(1). The cryoEM density of native CVB3 was used as an initial starting structure.
The final resolution was estimated by using maps of the reconstructed cryoEM
density representing the viral capsid between radii 75 and 160 Å and then
determining where the Fourier shell correlation fell below 0.5 using the
CUTPIFMAP, FFT, and EMRESOL programs written by Chuan Xiao (http:
//bilbo.bio.purdue.edu/?viruswww/Rossmann_home/river_programs/). A similar
cryoEM reconstruction procedure was used for a reconstruction of the native
virus (Table 2).
Difference map and fitting of the DAF structure into the cryoEM densities.
The program EMfit (42) was used to calibrate the exact magnification of the
cryoEM native CVB3 map by comparing it with a map derived from the X-ray
crystallographically determined coordinates of CVB3 (Protein Data Bank [PDB]
accession number 1COV). A difference map was calculated by subtracting the
native map from the cryoEM map of the CVB3-RD and DAF complex. The
SCR2 and -3 fragment was fitted into the cryoEM difference density and refined
by using the program EMfit (42) (PDB accession codes for X ray, 1OJV, 1OJW,
and 1OJY; PDB accession code for nuclear magnetic resonance, 1NWV). The
fitted SCR2 and -3 fragment was then removed from the cryoEM difference
density by setting to zero the density within a radius of 4.5 Å of each C?atom,
prior to the independent fitting of SCR1 and SCR4. Residues in the virus/
receptor interface were identified as those in CVB3 that had any atoms less than
3.6 Å from any atom in the fitted DAF structure. The buried surface area was
calculated using CCP4 programs Areaimol and Surface (Table 3) (6, 8, 23).
Plaque reduction assays. Monolayers of HeLa or RD cells were grown to
confluence in six-well 35-mm culture dishes. Virus inoculum was diluted in PBS
plus 0.1% (wt/vol) bovine serum albumin to a titer that would allow formation of
50 to 100 plaques. Soluble receptor or an equal volume of PBS plus 0.1% bovine
serum albumin was added to the diluted virus and incubated for 1 h at room
temperature before being applied to cells. After incubation for 45 min at room
temperature to allow virus to attach to cells, the cultures were fed with medium
containing 0.8% agar and incubated for 24 to 48 h. The agar overlay was
removed, and cells were stained with crystal violet to visualize plaques.
Accession numbers. Accession numbers for data bank deposition are as fol-
lows: EMD-1412, cryoEM reconstruction of CVB3 RD strain complexed with
DAF; 2QZD, 2QZF, and 2QZH, structures of the ectodomain of the DAF fitted
into a cryoEM reconstruction of CVB3 RD strain and DAF complex; EMD-
1411, cryoEM reconstruction of CVB3 M strain.
RESULTS AND DISCUSSION
Fitting the DAF structure into the cryoEM complex map.
The cryoEM reconstruction of CVB3-RD complexed with full-
length DAF (Table 2; Fig. 2) showed that the receptor does not
bind into the canyon as does CAR but rather lies across the
surface of the virus, crossing over the canyon to interact with
the southern rim of the canyon at the “puff” region of VP2
(loop EF, residues 2129 to 2180) (Fig. 2). The cryoEM density
corresponding to DAF was approximately equal in magnitude
to that of the virion capsid, indicating that nearly all of the 60
possible DAF binding sites were fully occupied. The length and
shape of the DAF density corresponded to an extended DAF
molecule consisting of four SCR domains. This contrasts with
the binding of DAF to the surfaces of EV7 and EV12, which
showed only partial occupancy because of low affinity of re-
ceptor for virus and steric clashes at the twofold symmetry axes
(4, 13, 35).
Three different crystal structures of DAF (25) generated
eight different models with variations of twist and tilt between
SCR1 and -2 and between SCR3 and -4. Each of these eight
models of full-length DAF was fitted into the DAF difference
density using all nonhydrogen atoms (see Materials and Meth-
ods for the difference map calculation). The general shape of
the DAF molecule, in particular the ?30 to 40o“kink” be-
tween SCR1 and SCR2, readily determined the overall orien-
tation. Although the DAF molecule is generally long and cy-
lindrical in shape, the kink or elbow angle modified its shape to
permit an accurate orientation of the DAF molecule about its
FIG. 2. Surface-rendered cryoEM reconstruction of CVB3-RD
complexed with full-length DAF molecules. Density further than a
160-Å radius from the center of the virus is shown in gray. An asym-
metric unit is outlined in black. Density corresponding to a full-length
DAF molecule with a His tag lies across the asymmetric unit stretching
from a threefold axes of symmetry, across and partially blocking the
canyon, to the neighboring protomer, rising towards a fivefold vertex.
The figure was produced using the program DINO (DINO: Visualizing
Structural Biology , http://www.dino3d.org).
FIG. 3. DAF fitted into the difference density map. The SCR do-
mains are labeled, and the C?backbone of DAF is shown in red. The
figure was produced using the program O (17).
TABLE 2. CryoEM data collection
9 994 5922.8–4.8
TABLE 3. Areas of contact between DAF subunits and the
DAF subunitResidue no.Area (A ˚2)
VOL. 81, 2007 INTERACTION OF DAF WITH COXSACKIEVIRUS B312929
long axis. The asymmetry of the DAF molecule determined the
placement of SCR1 closer to a fivefold axis and SCR4 closer to
a threefold axis (Fig. 3). However, none of the eight different
crystal structures used in their entirety exactly filled the DAF
density, indicating that the structure of DAF bound to the
surface of the virus had a significantly different tilt and twist
between SCR1 and -2 and between SCR3 and -4 than observed
in any of the crystal structures. Consequently, a stepwise fitting
and refinement operation was used, which first fitted the struc-
ture of the central part of the DAF molecule, SCR2 and -3,
and subsequently fitted SCR1 and SCR4 separately into the
DAF density, by using the EMfit program (40) (Table 4). The
best fit of SCR2 and -3 was obtained by using the nuclear
magnetic resonance structure (60). The density corresponding
to SCR2 and -3 was subtracted from the map, and the full-
length structure of DAF (25) was used to initiate the place-
ment of SCR1 and SCR4 into corresponding densities. The
position and orientation of SCR1 were refined while restrain-
ing the distance between the C terminus of SCR1 and the N
terminus of the fitted SCR2 and -3 to be less than 8 Å. Simi-
larly, the SCR4 position and orientation were refined while
restraining the N-terminal end of SCR4 to be no more than 8
Å from the C-terminal end of SCR2 and -3. Throughout the
stepwise fitting procedure, the full-length DAF structure was
used to check that the individual SCRs retained the correct
spatial and angular relationships to each other.
Interactions of CVB3-RD and DAF. The crystal structure of
CVB3 (CVB3 M strain, PDB accession code 1COV) was
placed into the cryoEM density of the CVB3-RD and DAF
complex by superimposing the icosahedral symmetry elements.
The fitted DAF structure was also placed into the map, and
atoms located on the virus surface, as well as atoms in the DAF
molecule, were identified if they were within 3.6Å of the two
fitted structures (Table 5; Fig. 4). Two loops in the flexible puff
region of the CVB3 virus, residues 2134 to 2145 and 2160 to
2168, clashed with the SCR2 of the fitted DAF molecule.
TABLE 4. Results of fitting the structural components of DAF into the cryoEM difference density map
Center (A ˚)f
Sumf as a function of SCR
thet1 thet2 thet3centxcenty centzSCR1 SCR2 SCR3SCR4
aLukacik et al. (25) provide eight different X-ray crystallography structures of DAF from three different crystal forms: two structures of 10JC, designated a and b;
two structures from 10JW, designated a and b; and four structures from 10JY, designated a, b, c, and d. XR corresponds to the X-ray crystallographic coordinates for
SCR2 and -3 from 10JVa. NMR23 is the first of 42 conformers submitted by Uhrinova et al. (60).
bSumf is defined as the average value of density for all nonhydrogen atomic positions normalized by setting the highest density in the map to 100.
cClash represents the percentage of atoms in the model that have steric clashes with symmetry-related subunits.
d?Den provides the percentage of atoms that are positioned in negative density.
eOrientation Eulerian angles that rotate the standard orientation of the model into the cryoEM density are given in degrees as described by Rossmann (40).
fx, y, and z are the three translational positions of the mass center for the fitted model.
TABLE 5. Fitted DAF residues within 3.6 A ˚of virus
Gln254......................................................Lys76, Gln77, Tyr79, Glu92, Arg101
Glu256......................................................Lys76, Glu94, Arg101
Gln264......................................................Glu94, Cys95, Arg101
Pro265 ......................................................Arg101, Ser104, Leu105
Ser266.......................................................Ser104, Leu105, Pro107
Gly267 ......................................................Leu105, Ser106, Pro107
Ala135a.....................................................Arg100, Arg101, Lys127b
Thr136a.....................................................Gly98, Arg100, Lys127b
Asn138a....................................................Pro97, Gly98, Ser176, Val177
Asn139......................................................Pro130, Ser176, Val177
Val160 ......................................................Arg100, Arg101, Pro103
Ser162a.....................................................Gly98, Arg100, Arg101
Gly163a.....................................................Gly98, Arg100, Lys127b, Ser128b
Ser164a.....................................................Lys127b, Ser128b, Pro130
Asn165a....................................................Lys127b, Ser128b, Pro130
Asn63........................................................Ser104, Leu105, Ser106
Ser232.......................................................Glu77, Tyr79, Glu92
Gln233......................................................Gln77, Pro78, Tyr79
Glu234......................................................Lys76, Glu77, Pro78, Tyr79, Ile80,
Asp152......................................................Tyr79, Ile80, Thr81
aCVB3 loops 2161–166 and 2135–138 clash with fitted DAF.
bFitted DAF loop 126–128 clashes with CVB3,
12930HAFENSTEIN ET AL.J. VIROL.
Presumably these flexible loops alter their structure when
forming the complex. Whereas the SCR1 domain of DAF has
no interaction with the virus, DAF residues located in SCR2
(residues 76 to 81, 92 to 98, and 101 to 107) were within the
contact region. Residues in the SCR2 and -3 junction make a
few interactions with the puff, but the main part of SCR3
crosses over the canyon, without entering into the canyon and
without making contact with the viral surface. The C terminus
of each DAF molecule interacts with two other DAF mole-
cules related by icosahedral threefold symmetry. The site of
interaction corresponds to a Ca2?ion, coordinated by
Glu3200, as observed in the crystal structure of CVB3 (29). In
the complex of CVB3-RD and DAF, this Ca2?ion would also
be associated with the carboxy-terminal His6nickel binding tag
from each of three symmetry-related DAF molecules. Thus,
the DAF molecules are effectively tethered to the virus at two
sites, namely, at SCR2 and at the carboxy end of SCR4, causing
SCR3 to block access to the canyon (Fig. 4).
In agreement with previous affinity measurements and mu-
tational analyses (3, 38), most of the interactions between the
receptor and virus occur between SCR2 of DAF and the puff
region of the virus. Although the DAF footprints onto the EV7
and EV12 surfaces are similar and involve the puff region on
the south side of the canyon, they differ greatly from the foot-
print of DAF onto CVB3 (Fig. 5 and 6). Furthermore, there is
little overlap between the DAF residues involved in binding
CVB3 and those that bind EV12 (4) (Fig. 7, left). Most of the
DAF residues in the CVB3 contact region are located on the
opposite side of DAF than are the residues that interact with
convertases, consistent with binding and immunological inves-
tigations (12, 20, 21, 53) (Fig. 7, right).
Residues defining the DAF binding sites. Previously, two
residues in VP2, Val108 and Ser151, had been identified as
necessary for CVB3 infection of RD cells (24). Although
Ser2151 maps to the viral surface near the twofold axes of
symmetry, Val2108 is located internally on the ?D strand of
VP2, suggesting that the RD phenotype may not be solely a
function of direct receptor interaction. Binding of DAF to the
Nancy-new strain of CVB3 has been observed, even though
this particular strain had a Ser at position 2151 (RD genotype)
and an Asp at position 2108 (wild-type genotype) (51). Fur-
thermore, the amino acid differences reported between Nancy,
Nancy-new, and Nancy-RD indicate that there might be some
uncertainty as to a common background between different
laboratory strains of CVB3 and CVB3-RD, making it difficult
to predict which amino acid differences confer DAF binding
ability. In contrast to the situation for EV7 and EV12, the
structure presented here shows no interaction between DAF
and the 2151 surface residue thought to be responsible for the
RD phenotype. Possibly the binding site of DAF on CVB3-RD
FIG. 4. The surface of CVB3 around a fivefold vertex, with VP1, VP,2, and VP3 in blue, green, and red, respectively. One DAF and one CAR
receptor are shown as C?backbones in yellow and cyan, respectively, where they would be bound to the viral surface. The DAF contact area is
shown in white in each of the five protomers. CAR is seen to clash with SCR3 of DAF if both receptors were to bind at the same time to the same
icosahedral asymmetric unit of the virus. The stereo figure was produced using Chimera (34, 46).
FIG. 5. Echovirus surface residues predicted to interact with DAF, given as equivalent CVB3 residues based on multiple-sequence alignment
(CLUSTALW) (57). Virus contact residues are color coded to identify specific DAF SCR interactions, with SCR2 shown in orange, SCR3 in
purple, and SCR4 in green. Residues marked with ? are located in symmetry-related proteins. EV7 and DAF contacts are according to fit (a) in
Table 2 in reference 14. EV12 and DAF contacts are from Fig. 6D and E in reference 4.
VOL. 81, 2007 INTERACTION OF DAF WITH COXSACKIEVIRUS B3 12931
has greater affinity to DAF than the binding site observed in
the cryoEM studies of echoviruses. This is supported by the
much higher occupancy of DAF on CVB3-RD compared to
the cryoEM studies of DAF binding to EV7 and EV12. As the
two receptor binding sites are mutually exclusive, it would be
expected that the site reported here, once established, would
Plaque reduction assays. Plaque reduction assays were per-
formed with the uncomplexed wild-type CVB3 (11) and the
mutant CVB3-RD, as well as with viruses preincubated with
either soluble CAR or DAF (Fig. 8). Reduction of plaques
occurred only when wild-type virus was preincubated with sol-
uble CAR and applied to HeLa cells or when mutant virus was
preincubated with soluble DAF and applied to RD cells
(?90% plaque reduction). In all other combinations, plaque
reduction was insignificant. These results can be interpreted by
noting that HeLa but not RD cells express CAR, that both
types of cells express DAF, and that the structural results show
that DAF blocks access to the canyon (Fig. 4). In order to
FIG. 6. The viral surface is shown as a stereographic projection where the polar angles ? and ? represent latitude and longitude, respectively
(65). The virus surface is represented as a quilt of amino acids (44). The icosahedral asymmetric unit of the virus is indicated by the triangular
boundary. The footprint of DAF for echoviruses was plotted using the equivalent CVB3 residues based on multiple-sequence alignment
(CLUSTALW) (57). The three viruses have sequence identities of 61% for VP1, 70% for VP2, and 67% for VP3. DAF contact residues for EV7
(14) and EV12 (4) are green and blue, respectively. The overlap between DAF footprints for the two echoviruses is shown in cyan. The DAF and
CAR (14) footprints on CVB3 are outlined in black and red, respectively.
FIG. 7. A surface rendering of the full-length fitted DAF structure,
in stereo. (Left) The CVB3 footprint onto DAF (blue) compared to
the EV12 footprint onto DAF (green) shows no overlap. (Right) The
DAF area predicted to interact with convertases (from reference 20)
(magenta) shows no overlap with the echovirus footprint on DAF and
a slight overlap of five residues with the CVB3-RD footprint.
12932 HAFENSTEIN ET AL.J. VIROL.
FIG. 8. Interactionofwild-typeCVB3virus(lightblue)ormutantCVB3-RDvirus(green)withHeLacells(darkblue)orRDcells(red).HeLacellsexpress
contains a pocket factor (pink), which can be displaced by binding of a receptor into the canyon, shown as a U-shaped depression at the top of the virus.
VOL. 81, 2007 INTERACTION OF DAF WITH COXSACKIEVIRUS B312933
further explain the plaque assay results, it is also necessary to
make the following assumptions: adaptation by the mutant
virus to bind DAF allows DAF to compete successfully with
CAR, and either (i) a canyon-binding receptor molecule dis-
places the pocket factor or (ii) DAF binding alone is sufficient
for the virus to enter and infect cells.
Viral plaque reduction assays indicated that wild-type virus
was able to infect HeLa cells but not RD cells (Fig. 8). This is
consistent with wild-type virus being able to bind CAR but not
DAF. Preincubation of wild-type virus with soluble CAR re-
duced plaques on HeLa cells, presumably by blocking the CAR
binding site. The mutant virus was capable of infecting both
HeLa and RD cells. However, compared to wild type, the
mutant plaques in HeLa cells were smaller and fewer, suggest-
ing either the mutant virus no longer binds CAR as well as the
wild type or that the mutant virus is entering cells in a different,
less efficient way. Furthermore, the gained ability of the mutant
virus to bind to DAF may be demonstrated by plaque forma-
tion in RD cells, although it is unclear whether the virus uses
only DAF or an unknown canyon-binding receptor to enter
RD cells. Preincubation of the mutant virus with DAF inhibits
plaque formation on RD cells but not that on HeLa cells.
Either the mutant virus relies on an interaction with DAF to
infect RD cells or access to the canyon for a hypothetical
unknown canyon-binding receptor was blocked by bound
DAF. However, it is possible that CAR molecules on HeLa
cells successfully competed with the DAF bound to the surface
of the mutant virus, allowing infection. Incubating the mutant
virus with CAR caused no reduction in plaque formation on
either HeLa cells or RD cells. This lack of inhibition suggests
that preincubation of the mutant virus with CAR could have
expelled the pocket factor, making the virus susceptible for
uncoating. Subsequently, the mutant virus would be able to
infect HeLa or RD cells by interacting with DAF.
The above assumption (i) that there might be another mol-
ecule on the surface of RD cells that can act as receptor for the
mutant CVB3-RD virus would be consistent with the hypoth-
esis that the pocket factor needs to be displaced by a canyon-
binding molecule, such as CAR, to initiate uncoating of the
virus. It is also consistent with observations that indicate that
when CVB3 adapts to use DAF, it retains the ability to bind
CAR and remains dependent on a CAR interaction for un-
coating (12, 36, 47) and that DAF alone is insufficient to
initiate infection (28). Furthermore, CVB3 can bind to at least
five different cellular receptors distinct from either DAF or
CAR on the surface of human cardiac cells (33). However, the
alternative assumption (ii), namely, that DAF alone is suffi-
cient for a successful infection, without the assistance of a
canyon-binding molecule to displace the pocket factor, cannot
be ruled out. For instance, the minor-group rhinoviruses,
which use very-low-density lipoproteins for attachment and
infection (16, 61), bind the receptor on an exposed surface
outside the canyon (15). Similarly, it has been shown that a
DAF-binding strain of echovirus can enter cells by using DAF
as a receptor, apparently without forming A particles (55).
Closely related viruses bind different surfaces of DAF to
different locations on the virus, suggesting a convergent evo-
lution in the use of DAF that offers some advantage to the
virion. Such an advantage may be due to the ubiquitous nature
of DAF, which is found on most cell surfaces, including serum-
exposed cells that line the lumen through which enteroviruses
travel. For instance, DAF is necessary to enter polarized epi-
thelial cells (3), because the initial binding of CVB3 to DAF
sets off a cell-signaling cascade which causes transport of the
bound virus to the tight junction between cells and allows
interaction with CAR molecules that are sequestered there.
Similarly, coxsackievirus binding to DAF expressed on pancre-
atic cells causes recruitment of CAR to the site of the inter-
action (58), allowing subsequent binding of the CAR and suc-
cessful infection of the cells.
We thank Sheryl Kelly and Cheryl Towell for help in preparation of
the manuscript and Rodney McPhail for assistance with Fig. 1.
Figures 4 and 7 were produced using the UCSF Chimera package
from the Computer Graphics Laboratory, University of California, San
Francisco (supported by NIH grant P41 RR-01081). The work was
supported by National Institutes of Health grants to M.G.R. (AI
11219), to D.E.M. (AI 23598), and to F.L. (HL 077319). S.H. was
supported by a National Institutes of Health postdoctoral fellowship
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