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Journal of Virology, June 2001, p. 5375-5380, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5375-5380.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Structural Analysis of a Fiber-Pseudotyped
Adenovirus with Ocular Tropism Suggests Differential Modes of Cell
Receptor Interactions
Charles Y.
Chiu,1
Eugene
Wu,2
Swati L.
Brown,2
Dan J.
Von
Seggern,2
Glen R.
Nemerow,2,* and
Phoebe L.
Stewart1,*
Department of Molecular and Medical
Pharmacology and Crump Institute for Molecular Imaging, UCLA School
of Medicine, Los Angeles, California 90095,1 and
Department of Immunology, The Scripps Research Institute,
La Jolla, California 920372
Received 1 November 2000/Accepted 26 February 2001
 |
ABSTRACT |
Adenovirus (Ad) entry into cells is initiated by the binding of the
fiber knob to a cell surface receptor. The coxsackie- and adenovirus
receptor (CAR) functions as the attachment receptor for many, but not
all, Ad serotypes. Ad type 37 (Ad37), a subgroup D virus that causes
keratoconjunctivitis in humans, does not infect cells via CAR despite
demonstrated binding of the Ad37 knob to CAR. We have pseudotyped a
fiber deletion Ad5 vector with the Ad37 fiber (Ad37f), and this vector
retains the ocular tropism of Ad37. Here we present a cryo-electron
microscopy reconstruction of Ad37f that shows the entire Ad37 fiber,
including the shaft and knob domains. We have previously proposed that
Ad37 may not utilize CAR for cell entry because of the geometric
constraints imposed by a rigid fiber (E. Wu, J. Fernandez, S. K. Fleck, D. Von Seggern, S. Huang, and G. R. Nemerow, Virology
279:78-89, 2001). Consistent with this hypothesis, our structural
results show that the Ad37 fiber is straight and rigid. Modeling of the interaction between Ad37f and host cell receptors indicates that fiber
flexibility or rigidity, as well as length, can affect receptor usage
and cellular tropism.
| |
TEXT |
Viral cell entry is a complex
process that is initiated by the specific interaction of capsid
proteins with cell surface receptors. Efficient infection of cells by
adenovirus (Ad) involves binding to two different receptors
(14). The Ad fiber protein recognizes the primary
receptor, allowing high-affinity binding of virus particles to the cell
surface (10, 15), and an Arg-Gly-Asp (RGD) sequence motif
in the Ad penton base promotes association with secondary receptors,
v
3 or
v5 integrin,
triggering virus internalization (32). The coxsackievirus
and Ad receptor (CAR) protein was initially identified as the fiber
receptor for Ad2 and Ad5 (5, 26) and functions as a
receptor for many, but not all, Ad serotypes (17). There
are 51 Ad serotypes classified into subgroups A to F, encompassing a
broad spectrum of tissue tropisms and disease. Members of Ad subgroups
B, C, and E, including Ad type 2 (Ad2) and Ad5, cause respiratory
infections; those of subgroups A and F, including Ad12 and Ad40/41, are
responsible for gastrointestinal infections; and those of subgroup D,
including Ad37, Ad8, and Ad19a, are associated with epidemic
keratoconjunctivitis, a highly contagious ocular infection with the
potential to impair visual function (4, 9, 16).
Understanding the molecular and cellular basis for these differences
would assist in the development of novel antiviral drugs as well as
improved gene delivery vectors.
The Ad fiber protein is a homotrimeric molecule extending from each of
the 12 vertices of the icosahedral capsid. The fiber consists of an
N-terminal tail domain that interacts with the Ad penton base, a
central shaft domain of variable length, and a C-terminal knob domain
that contains the receptor binding site. Sequence analysis indicates
that the fiber shaft is composed of 6 to 23 -repeats in various Ad
serotypes (8). The Ad37 fiber has only eight -repeats
(4), making it relatively short. Interestingly, most Ad
fibers are not perfectly rigid but exhibit a bent or kinked conformation as viewed by negative-stain electron microscopy (EM) (8). A cryo-EM reconstruction of Ad2 also indicates that
the fiber is bent (22). The central shaft domain of most
but not all Ad fibers contains a nonconsensus -repeat element that
may confer flexibility to the fiber (8). Notably, the
fiber shafts of Ad9, Ad37, and other subgroup D viruses do not have
this nonconsensus -repeat.
Several crystal structures of the fiber knobs from various serotypes
have been published (6, 28, 34), including a structure of
the Ad12 knob complexed with the N-terminal domain of CAR
(6). The structure of the complex reveals that all of the
residues involved in binding to CAR are located on the side of the knob and that the majority of the contact residues are in the AB loop. The
importance of the AB loop in CAR binding is highlighted by both the
conservation of amino acids comprising this loop in CAR binding
serotypes and the wide divergence of the AB loop sequences among
non-CAR binding serotypes.
The Ad37 fiber knob contains a CAR binding consensus sequence in its AB
loop (13), and direct binding of the recombinant Ad37
fiber knob to CAR has been demonstrated (33). Curiously, this serotype does not effectively use CAR as its attachment receptor, as shown by virus binding and infection studies with conjunctival, corneal, and lung epithelial cells (2, 11, 33). It has been suggested that sialic acid (2, 3) or an
as-yet-unidentified 50-kDa glycoprotein (33) may serve as
the attachment receptor for Ad37. Specific ocular cell binding is
likely mediated by the 50-kDa protein rather than by sialic acid
(33).
Huang et al. (11) have noted that a single lysine residue
at position 240 of the Ad37 fiber is critical for binding and infection
of conjunctival cells. Molecular modeling of the Ad37 fiber knob based
on the Ad5 fiber knob crystal structure (34) indicates
that residue 240 is exposed on the top surface of the knob, in the CD
loop (11). The CD loop does not contact CAR in the crystal
structure of the Ad12 fiber knob-CAR complex (6). Thus,
for Ad37, binding via the CD loop most likely involves a receptor other
than CAR. Wu et al. (33) have proposed that if Ad37 has a
rigid fiber, it might not be able to utilize CAR for cell entry because
of geometric constraints. Specifically, a rigid fiber would make it
impossible for the virus to present the side of the knob, with the CAR
binding AB loop, to CAR on the host cell surface.
Using fiber-pseudotyping technology (21, 31), we produced
an Ad5 vector containing the Ad37 fiber (Ad37f) and confirmed that the
chimeric virus exhibits ocular cell tropism (33). Cryo-EM combined with single-particle image reconstruction methods has proven
useful for analyzing the structure of Ad (22, 24, 25). Here we present a cryo-EM structural study of the pseudotyped Ad37f
virus. Modeling of the Ad37f structure with host cell receptors provides additional insight into why CAR is not utilized by Ad37.
Cryo-EM.
Cryo-EM images of pseudotyped Ad37f produced from two
plasmids, pDV80 and pDV121, were collected (33). The
particles produced with pDV80 contained two serendipitous mutations
(S356
P and I362T) in the C terminus of the Ad37 fiber protein.
These mutations did not affect trimerization, incorporation into Ad
particles, or receptor binding properties. The figures presented in
this paper were generated from particles with the fiber mutations. We
have also produced particles with the authentic wild-type Ad37 fiber (made with pDV121), and subsequent cryo-EM analysis confirmed that
there is no discernible structural difference between the mutated and
wild-type fibers.
Purified viral preparations of Ad37f particles were concentrated to
~200 µg/ml, and cryo-plunging was performed as described previously
(1, 7). The cryo-grids were examined in a Philips CM120
transmission electron microscope equipped with a
LaB6 filament, a Gatan cryo-transfer holder, and
a Gatan slow-scan CCD camera (1,024 by 1,024 pixels). Digital images
were collected under low-dose conditions (<20
e
/Å2) at a
nominal magnification of ×45,000 and at three different defocus values
(
0.5, 1.0, and 1.5 µm). The digital micrographs had a pixel
size of 4.1 Å on the molecular scale.
Image processing.
Computer image processing was carried out on
Compaq/DEC Alpha workstations (Compaq, Inc.). Individual particle
images (387 total) were extracted as 300- by 300-pixel fields by using
the QVIEW particle selection program (19). The IMAGIC
software package was used for all subsequent image processing and
reconstruction steps (27). The Euler orientations within
the icosahedral asymmetric triangle were computationally determined,
and the three defocus sets were then combined after two-dimensional
correction for the contrast transfer function (7). The
parameters of the modeled contrast transfer function equation
(spherical aberration constant [Cs] = 2 mm, fraction of amplitude
contrast = 0.1, kilovolts = 120, decay constant = 10 nm2, Fermi filter resolution cutoff = 8.1 Å, filter width = 3 Å, and defocus = 0.5, 1.0, or 1.5
µm) were chosen to minimize "ringing" in the particle images.
Three-dimensional reconstruction of the Ad37f particle was carried out
by the exact filter back-projection technique after two cycles of
anchor set refinement (7). The resolution of the Ad37f
reconstruction was assessed using the Fourier shell correlation 0.5 threshold criterion after applying "soft" masks to each
half-reconstruction (23). The calculated resolution of the
reconstruction is 24 Å. Isosurface representations of the
reconstruction were displayed using AVS graphical software (Advanced
Visualization Systems, Inc.). The Ad37f reconstruction was contoured to
correspond to a continuous, "nonholey" viral capsid. The density of
the fiber was noted to be ~50% of that of the viral capsid, and thus
the fiber is contoured with a lower isosurface value. The lower fiber
density may be attributed to partial occupancy of the fiber in the
pseudotyped Ad37f particles.
Several attempts were made to calculate a reconstruction of the Ad37f
structure without imposing full icosahedral (532) symmetry
in order to
visualize the fiber density without imposed fivefold
symmetry. First,
we tried to determine the correct Euler orientations
for the 387 particle images assuming either no symmetry or D3
(32) symmetry. In a
separate approach, we generated an anchor
set of reprojections of the
icosahedral Ad37f reconstruction evenly
spanning the D3 asymmetric unit
and used these reprojections to
search for D3 Euler angles for the
particle images. Unfortunately,
none of these methods yielded
reasonable three-dimensional reconstructions.
However, our
interpretation of the interaction of Ad37 with host
cell receptors
relies mainly on structural features (length and
rigidity) that are not
affected by the fivefold symmetrization
of the fiber
density.
Features of the Ad37f reconstruction.
The cryo-EM
reconstruction of Ad37f is presented in Fig.
1 and compared to two clear cryo-EM
particle images that show density corresponding to the knobs at the
ends of the short Ad37 fibers. In a cryo-EM particle image, all of the
viral density is projected into the two-dimensional image plane; thus,
fibers that are not overlapping with the capsid or obscured by the
carbon support film or background noise can be observed. Surface views
of the three-dimensional reconstruction were generated to match the
orientations of these two particle images. The comparison between Fig.
1A and B indicates that the two regions of strongest knob density
correspond to superimposed fiber knobs. The particle image in Fig. 1C
is oriented close to a fivefold-symmetry axis, and at least five fibers
are visible. The positions of these fibers correspond well with 5 of
the 10 fibers that are observed to project radially around the
reconstruction in this view (Fig. 1D). The fact that the reconstructed
fiber knobs are found in the same positions as the knobs visible in the
particle images suggests that the Ad37 fiber is properly reconstructed
and that the Ad37 fiber is rigid and straight.
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FIG. 1.
Cryo-EM particle images and reconstruction of the Ad37f
pseudotyped vector. (A and C) Two clear particle images showing density
around the capsid corresponding to the short fibers. (B and D) Surface
views of the three-dimensional reconstruction shown in the same
orientations as determined for the particle images in panels A and C. The arrows in panel A point to strong density from overlapping fiber
knobs. The arrows in panel C point to five fibers visible around the
capsid in this approximate fivefold view. The fiber is colored green,
the penton base is yellow, and the remaining capsid is blue. Bar, 100 Å.
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|
The dimensions of the Ad37 knob observed in the Ad37f reconstruction
(70 by 80 by 80 Å) agree well with those expected from
the crystal
structures of knobs from other Ad serotypes (
6,
28,
34). A
top view of the reconstructed vertex region shows
the fiber knob having
a fivefold-symmetric appearance, when in
reality it is trimeric (Fig.
2A). The Ad37 fiber protein, including
the knob, is observed to extend approximately 150 Å from the surface
of the capsid (Fig.
2B). In contrast, previous cryo-EM studies
of Ad2,
Ad5, and Ad12 revealed long fibers (>300 Å) in the particle
images,
yet only 60 to 100 Å of the shaft was reconstructed with
strong
density (
7,
24,
31). After this point, the Ad2 fiber
density becomes weaker and spreads out in a cone-like distribution
(Fig.
2C). This indicates a bend or kink in the fiber shaft at
roughly
the same location noted by negative-stain EM (
8). The
observations that the Ad2 fiber density tapers off significantly
while
the Ad37 fiber density is equally strong throughout its
length support
our conclusions that the Ad2 fiber is bent and
likely flexible, while
the Ad37 fiber is straight and rigid.
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FIG. 2.
Vertex region of the Ad37f pseudotyped vector and
comparison with Ad2. (A and B) Top and side views of the Ad37f vertex
region. (C) Side view of the Ad2 vertex region (7) shown
with a low isosurface value to include the weak diffuse density that
results from flexibility in the fiber shaft. The color scheme is the
same as in Fig. 1. Bar, 100 Å.
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|
Modeling with the fiber knob crystal structure.
The crystal
structure of the Ad2 fiber knob and a portion of the shaft
(28) was manually positioned within the reconstructed cryo-EM Ad37f fiber density. The amino acid sequence of the Ad37 knob
domain is 46% identical and 60% similar to that of the Ad2 knob
domain, and hence modeling with the Ad2 crystal structure is
reasonable. The fit along the fiber axis was clear, with very little
ambiguity in the height (Fig. 3A). For
the rotational fit, we positioned the prominent HI loop into a density
bulge on the side of the knob, considering only one-fifth of the
reconstructed knob density (Fig. 3B). As discussed previously, there is
a symmetry mismatch between the icosahedral reconstruction and the
trimeric fiber. Note that the AB loop, involved in CAR binding, is on
the side of the knob, while the CD loop, involved in binding to the 50-kDa receptor, is located more towards the top of the knob. These
findings are consistent with the idea that for Ads with short rigid
fibers, only the top, rather than the side, of the knob is available
for receptor binding (33).
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FIG. 3.
Modeling of the crystal structure of the Ad2 knob and a
portion of the shaft into the Ad37f cryo-EM fiber density. (A) Side
view. (B) Top view. The AB loop is colored red, and the CD loop is
blue. The arrows point to the HI loop that protrudes in the Ad2 crystal
structure (29) and fits well within a bulge of the
reconstructed Ad37f fiber knob. Bar, 100 Å.
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Ad37 uses v integrins as coreceptors.
One
consequence of the short, inflexible Ad37 fiber is that there is a
different spatial relationship between the fiber knob and penton base
receptor binding sites than found for serotypes such as Ad2 and Ad5,
with long, flexible fibers. Thus, we wondered what effect the Ad37
geometry would have on coreceptor binding. Previous studies had
indicated that wild-type Ad37 virus particles are capable of efficient
binding to soluble recombinant integrin v5 (12).
The amino acid sequence of the Ad37 penton base indicates that it
contains the conserved RGD integrin binding motif (3). To
confirm that the Ad37f pseudotyped vector uses
v integrins for infection, we performed
competition studies using the green fluorescent protein (GFP) reporter assay.
Adherent Chang C human conjunctival cells were incubated with 100 µg
of HB5 (anti-CD21) antibody, LM609
(anti-
v3) and P1F6
(anti-
v5) antibodies,
69-6-5 (anti-
v) antibody, or recombinant
Ad5
penton base per ml (
32) in Dulbecco's modified Eagle
medium
with 10% fetal bovine serum for 1 h at 37°C.
Ad5.GFP.
F/37F was
then added at 10,000 particles per cell and
incubated for 3 h
at 37°C. The cells were then washed twice and
cultured overnight.
Eighteen to 20 h later, cells were detached
with buffer containing
0.05% (wt/vol) trypsin and 0.5 mM EDTA
(Boehringer Mannheim) for
5 min at 37°C and washed with
phosphate-buffered saline, pH 7.4.
GFP fluorescence was measured with a
FACScan flow cytometer. A
threshold established by the fluorescence of
uninfected cells
was used to distinguish cells specifically expressing
GFP. As
shown in Fig.
4, pretreatment of
cells with soluble penton base
or with function-blocking monoclonal
antibodies to
v or
v3 and
v5 integrins
significantly reduced Ad37f-mediated gene delivery.
Pretreatment of
cells with a CD21-specific (control) antibody
had no effect. These
studies indicate that the pseudotyped virus
recognizes
v integrins and is capable of using these
coreceptors
for infection.
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FIG. 4.
Pseudotyped Ad with Ad37 fiber uses v
integrins for internalization. Chang C cells were pretreated with 100 µg of HB5 (anti-CD21) monoclonal antibody, LM609
(anti-v3) and P1F6
(anti-v5) monoclonal antibodies, 69-6-5 (anti-v) antibody, or recombinant Ad5 penton base (PB)
per ml in Dulbecco's modified Eagle medium with 10% fetal bovine
serum for 1 h at 37°C before infection with Ad5.GFP.F/37F.
Cell fluorescence from GFP transgene expression was measured by flow
cytometry. Error bars indicate standard deviations from three
experiments. The error for the penton base experiment was too small to
show on the graph.
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We then attempted to model the
v5 integrin cryo-EM
density over the penton base of the Ad37f reconstruction. In the
absence
of a crystal structure for
v5 integrin, we used
the cryo-EM
density observed in the
Ad12-
v5 complex for
structural modeling
(
7). In Fig.
5, we
show the integrin density positioned over
the vertex region of the
Ad37f reconstruction. The height of the
integrin density is known to
vary with the length of the RGD loop
of the penton base
(
7). In the pseudotyped Ad37f vector, the
penton base is
that of Ad5, and the length of the RGD loop of
Ad5 (80 residues) is
almost identical to that of Ad2 (82 residues).
Thus, we based the
height of the integrin density over the Ad5
penton base on the observed
height in the published
Ad2-
v5 complex
reconstruction (
7). We chose to use the integrin density
from
the Ad12 complex, rather than that from the Ad2 complex, since
the
longer and more flexible Ad2 RGD loops resulted in weak and
diffuse
reconstructed integrin density (
7). The integrin density
shown corresponds to the extracellular portion of
v5, and the
position
of the host cell membrane is presumed to be directly
above it in vivo.
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FIG. 5.
Interaction of Ad37 and Ad2 with their cell
receptors. (A) The geometry of Ad37 with short, rigid fibers (green)
prevents the side of the fiber knob from contacting the appropriate
surface of CAR (black). (B) If the side of the fiber knob on Ad37 were
to bind CAR, there would be steric hindrance between the viral capsid
and the host cell membrane (orange). The clash is indicated by
transparent density. (C) Ad37 is shown binding a 50-kDa receptor that
is preferentially expressed on conjunctival cells (33).
Modeling indicates that concurrent binding of the fiber with the 50-kDa
receptor (purple) and of the penton base (yellow) with v
integrin (red) should be possible. (D) The Ad types with long, bent
fibers, such as Ad2 and Ad5, contact CAR with the side of the fiber
knob.
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|
The model indicates that the height of the extracellular
v5 integrin density
is roughly equivalent to that of the Ad37 fiber.
Concurrent binding of
the knob with a cell receptor and penton
base with integrin could occur
if the Ad37 fiber receptor contains
a relatively small ectodomain
and/or this domain lies nearly flat
against the cell surface. For CAR
binding Ad serotypes with long,
flexible fibers, concurrent binding of
penton base with
v integrin
at the same vertex
is feasible if a maximum of four integrin binding
sites are occupied on
the penton base, thus leaving room for the
bent fiber shaft (Fig.
5).
Implications for other Ad serotypes.
Shayakhmetov and Lieber
(20) showed that modified Ad5 particles containing the
short Ad9 fiber shaft have reduced CAR binding and infection.
Experiments were carried out using Ad5 vectors with one of two
different CAR binding fiber knobs, Ad5 or Ad9, together with either the
long shaft of Ad5 or the short shaft of Ad9 (20). Our
cryo-EM studies of Ad5 (30) indicate that the Ad5 fiber
shaft is bent. We presume from published sequence data that the short
Ad9 fiber shaft is rigid, since it does not have the nonconsensus
-repeat motif found in flexible Ad fibers (8). This
presumption is corroborated by the fact that the Ad37 fiber shaft is
also missing the nonconsensus -repeat, and the results presented
here show that the Ad37 fiber is straight. These observations add a new
twist to the study by Shayakhmetov and Lieber in that not only were the
lengths of the shafts different but also one shaft was bent while the
other most likely was straight. They found that for Ad5 vectors with
either CAR binding knob, a long fiber shaft was important for efficient
adsorption and transduction of CAR-v
integrin-expressing cells. In light of the cryo-EM studies, fiber shaft
flexibility, as well as length, plays a role in receptor usage.
It is likely that all of the fiber proteins of subgroup B Ads, which
lack CAR binding activity, have straight, rigid structures.
In support
of this, a cryo-EM reconstruction of the Ad3 penton
base-fiber
dodecahedron shows that the fiber is rigid and even
shorter than those
of Ad37 and Ad9 (
18). It is known that Ad3
does not bind
to CAR and does not contain the CAR binding consensus
sequence
(
6). From our results, we predict that Ad3, like Ad37,
uses a non-CAR fiber receptor that binds to the top surface of
the
knob.
Others have shown that Ad serotypes with fiber knobs containing a
consensus CAR binding sequence do not necessarily bind CAR
on cells
(
2). Our results indicate that the flexibility or
rigidity
of the Ad fiber plays a role in determining which fiber
receptors can
be utilized for cell entry. These structural and
modeling studies show
that the geometrical constraints imposed
by a short rigid fiber
protruding from an icosahedral viral capsid
effectively prevent use of
the side of the fiber knob for receptor
binding. Thus, fiber
flexibility, as well as length, appears to
play a heretofore
unappreciated role in receptor selectivity and
in viral tropism. The
knowledge gained from these studies improves
our understanding of the
structural basis of Ad-receptor interactions
and may serve as a guide
for retargeting of Ad vectors to specific
cell types in
vivo.
| |
ACKNOWLEDGMENTS |
We thank Shuang Huang for helpful advice on Ad37 receptor interactions.
This work was supported by grants from the National Institutes of
Health (AI42929 to Phoebe L. Stewart and HL54352 and EY11431 to Glen R. Nemerow) and by Genetic Therapy Inc./Novartis grant SFP1089. Charles
Chiu was supported by an NIH-MSTP training grant (GM08042) and the
Aesculapians fund of the UCLA School of Medicine.
| |
FOOTNOTES |
*
Corresponding author. Mailing address for Phoebe L. Stewart: Department of Molecular and Medical Pharmacology, UCLA School of Medicine, A-324 CIMI, Box 951770, Los Angeles, CA 90095-1770. Phone: (310) 206-7055. Fax: (310) 206-8975. E-mail:
pstewart{at}mednet.ucla.edu. Mailing address for Glen
R. Nemerow: Department of Immunology, The Scripps Research Institute,
La Jolla, CA 92037. Phone: (858) 784-8072. Fax: (858) 784-8472. E-mail:
gnemerow{at}scripps.edu.
| |
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Journal of Virology, June 2001, p. 5375-5380, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5375-5380.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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