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Adenovirus serotype 5 hexon is critical for virus
infection of hepatocytes
in vivo
O. Kalyuzhniy*, N. C. Di Paolo*, M. Silvestry
†
, S. E. Hofherr
‡
, M. A. Barry
§
, P. L. Stewart
†
, and D. M. Shayakhmetov*
¶
*Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195-7720; †Department of Molecular Physiology and
Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232; ‡Department of Molecular and Human Genetics, Baylor College of Medicine, Houston,
TX 77030; and §Departments of Internal Medicine and Immunology, Mayo Clinic, Rochester, MN 55902
Edited by Thomas E. Shenk, Princeton University, Princeton, NJ, and approved February 19, 2008 (received for review December 14, 2007)
Human species C adenovirus serotype 5 (Ad5) is the most common
viral vector used in clinical studies worldwide. Ad5 vectors infect
liver cells in vivo with high efficiency via a poorly defined mech-
anism, which involves virus binding to vitamin K-dependent blood
coagulation factors. Here, we report that the major Ad5 capsid
protein, hexon, binds human coagulation factor X (FX) with an
affinity of 229 pM. This affinity is 40-fold stronger than the
reported affinity of Ad5 fiber for the cellular receptor coxsackievi-
rus and adenovirus receptor, CAR. Cryoelectron microscopy and
single-particle image reconstruction revealed that the FX attach-
ment site is localized to the central depression at the top of the
hexon trimer. Hexon-mutated virus bearing a large insertion in
hexon showed markedly reduced FX binding in vitro and failed to
deliver a transgene to hepatocytes in vivo. This study describes the
mechanism of FX binding to Ad5 and demonstrates the critical role
of hexon for virus infection of hepatocytes in vivo.
gene transfer 兩virus targeting
Gene delivery systems based on human species C adenovirus
serotype 5 (Ad5) are among the most frequently used in
clinical studies, which aim to correct human genetic and acquired
diseases, including cancer (1). The extreme propensity of the
virus for hepatocyte infection after its intravascular delivery has
made Ad5 the vector of choice for applications requiring high-
level transgene expression in hepatocytes in vivo (2). However,
the efficient interaction between Ad5 and liver cells, which
sequester a large proportion of the delivered vector dose (3),
represents a significant hindrance if gene delivery to extrahe-
patic cells and tissues is required. From in vitro analyses, it was
found that Ad5 infection is initiated when the minor capsid
protein, fiber, binds to the coxsackievirus and adenovirus re-
ceptor (CAR) on the cell surface (4, 5). Subsequent binding of
the penton base protein to cellular integrins facilitates internal-
ization of the attached particle into the cell (6). Although binding
of both CAR and integrin is critical for cell infection in vitro,
neither of these interactions is essential for Ad5 entry into
hepatocytes in vivo (7–9).
We and others recently identified a pathway of hepatocyte
infection by Ad5, which is mediated by the vitamin K-dependent
blood coagulation factors, including factors VII, IX, X, and
protein C (10, 11). When Ad5 particles are delivered intrave-
nously, they interact with blood coagulation factors, which
subsequently bind low-density lipoprotein receptor-related pro-
tein (LRP) and heparan sulfate proteoglycans (HSPGs) on
hepatocytes to mediate virus entry. Although rather complex,
this mechanism is very efficient and can be specifically blocked
by lactoferrin, which competes for blood factor binding to LRP
and HSPGs on hepatic cells (10). It is noteworthy that, although
all of these blood factors support CAR-independent cell infec-
tion by Ad5 vectors to varying degrees, coagulation factor X
(FX) appears the most efficient at mediating Ad5 entry into
hepatic cells in vivo (11).
Based on numerous experiments that demonstrate reduced
hepatocyte infection after i.v. delivery of fiber-modified vectors
in mice, it was suggested that coagulation factors might bind to
the Ad5 fiber to mediate hepatocyte infection. However, there
is still no definitive evidence that a fiber– coagulation factor
interaction is ultimately required to mediate CAR-independent
Ad5 entry into hepatocytes in vivo.
In this study, we analyzed the interactions of human FX with
human adenoviruses from species B–F. Using surface plasmon
resonance (SPR), we found that FX binds with picomolar
affinity to hexon, the major Ad capsid protein. Cryoelectron
microscopy (cryoEM) and single-particle image reconstruction
have localized the FX-binding area to the central depression at
the top of each Ad5 hexon trimer. This cryoEM result, combined
with sequence analysis of hexons that bind FX and those that do
not, indicates there are likely two alternative sites for FX binding.
One site is within hypervariable region 3 (HVR3), and the other
is within hypervariable region 7 (HVR7). Both sites are pre-
dicted to form similar binding pockets within the central de-
pression of the hexon trimer. An adenovirus mutant that binds
FX in vitro with 10,000-fold reduced affinity compared with
unmodified vector failed to deliver the red fluorescent protein
(RFP) transgene in vivo. Thus, our study describes the mecha-
nism of FX binding to Ad5 and demonstrates the critical role of
hexon for virus infection of hepatocytes in vivo.
Results
Ad5 Hexon Binds FX with Picomolar Affinity. To quantitatively
characterize binding between blood coagulation factors and the
adenovirus capsid, we used SPR analysis (BIAcore). To avoid
complications with interpretation of the results caused by the
multivalent nature of the virus particle, Ad5 particles were
immobilized on the sensor chip by cross-linking, and varying
concentrations of blood factors were injected over sensor sur-
faces and binding responses were recorded and analyzed. Fig. 1
(Ad5 column) shows the representative datasets obtained from
the analysis of blood–factor interactions together with global fits
to a 1:1 interaction model (orange lines). Among all blood
factors tested [Fig. 1 and supporting information (SI) Fig. S1],
human FX (hFX) and mouse FX (mFX) demonstrated the
highest-affinity binding to Ad5. The association constant of the
Ad5-hFX-binding reaction was determined to be 3.37 ⫻10
5
M
⫺
1䡠s
⫺1
, the dissociation constant 7.71 ⫻10
⫺5
䡠s
⫺1
and K
D
equal
to 228.7 pM. We detected no binding of FXI, FXII, and
FX-desGla to Ad5 under the conditions used (Fig. S1). Ad5-hFX
Author contributions: O.K., N.C.D.P., and M.S. contributed equally to this work; O.K.,
N.C.D.P., P.L.S., and D.M.S. designed research; O.K., N.C.D.P., M.S., S.E.H., P.L.S., and D.M.S.
performed research; O.K., S.E.H., M.A.B., and P.L.S. contributed new reagents/analytic
tools; O.K., N.C.D.P., M.S., S.E.H., M.A.B., P.L.S., and D.M.S. analyzed data; and O.K., P.L.S.,
and D.M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
¶To whom correspondence should be addressed. E-mail: dshax@u.washington.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0711757105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
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binding strongly depends on the presence of both Ca
⫹2
and Mg
⫹2
ions and could be completely eliminated in a buffer containing
3 mM EDTA (data not shown).
Because of the very high affinity of FX for Ad5 and its strong
dependence on the presence of calcium and magnesium ions, we
hypothesized that the FX-binding protein within the Ad5 capsid
could be purified from virus-infected cells using affinity chro-
matography. Indeed, using the hFX affinity column, a 98% pure
protein with an apparent molecular mass of 100 kDa (after
separation by SDS/PAGE under denaturating conditions) was
obtained from the lysate of cells infected with Ad5 (Fig. S2 and
SI Text,Materials and Methods). The affinity-purified protein
was digested with trypsin and analyzed by using data-dependent
tandem mass spectrometry. Numerous peptides corresponding
to the Ad5 hexon were readily identified by Mascot software.
The trimeric state of the purified hexon was further confirmed
by size-exclusion chromatography. The experimentally deter-
mined molecular mass of the protein was estimated to be 337
kDa, which corresponds well with the expected molecular mass
of 324 kDa for the Ad5 hexon trimer (Fig. S2 A–C).
Using the SPR technique, we next analyzed the kinetic
parameters of the blood factor interaction with purified Ad5
hexon and purified Ad5 fiber knob domain, expressed in Esch-
erichia coli (Fig. S2 D–F and SI Text,Materials and Methods). The
data obtained (Fig. 1, Ad5-Hex and Ad5-FKn columns, and
Table S1) showed that, for all analyzed blood factors, interaction
with purified Ad5 hexon trimer, but not the Ad5 fiber knob
domain, closely follows the kinetics for the FX interaction with
the whole virus (Fig. 1). In addition, we analyzed blood factor
binding to another group C Ad serotype, Ad2, and commercially
available purified Ad2 hexon protein. This analysis demon-
strated that both human and mouse FX bind Ad2 particles and
purified Ad2 hexon with identical kinetics and af finity (Fig. 1 and
Table S1). Collectively, these data indicate that the Ad hexon
possesses a high-affinity binding site for vitamin K-dependent
blood factors. To further assess this possibility, we analyzed
blood factor interaction with an Ad5-based vector, Ad5BAP,
that possesses an insertion of a 71-aa biotin acceptor peptide
(BAP) in the exposed hexon loop of the HVR5 (12). Our analysis
showed that FX binding to Ad5BAP was reduced 10,000-fold
compared with unmodified Ad5 vector, and the 71-aa insertion
in HVR5 region of the hexon completely eliminated binding of
FIX and Protein C (Fig. 1 and Table S1).
FX Interaction with Different Human Ad Serotypes and Reovirus. We
next analyzed the affinity of FX binding to various human Ad
serotypes from species B–F and human reovirus T3D (13) by
using the same SPR protocol. This analysis revealed that human
Ad serotypes greatly vary in their ability to bind FX (Table 1).
The highest-affinity FX binding was observed for species C Ad5,
followed by species B Ad16, and then species C Ad2. Except for
the observation that two species C serotypes (Ad2 and Ad5)
exhibited high affinity for FX, no other correlation was noted
between the Ad group and FX affinity among the 11 serotypes
tested. Micromolar affinity binding was observed for species B
(Ad3 and Ad21), E (Ad4), and F (Ad41) serotypes. No FX
binding was observed for species B Ad35 or Ad50, species D Ad9
or Ad51 serotypes, or reovirus T3D. It is noteworthy that, in
agreement with these data, vectors based on Ad5 and Ad2
serotypes were shown to infect hepatocytes with high efficiency
(14, 15); however, Ad35-based vectors transduce hepatocytes
poorly after i.v. delivery (16, 17).
CryoEM Analysis of the Ad5-FX Complex. CryoEM and single-
particle image reconstruction were performed to visualize the
interaction of FX with Ad5. Comparison of cryoEM images of
the Ad5-FX complex with those of Ad5 alone revealed that FX
coats the virus with a layer of extra density. The FX coating
effect was also clearly observed in a 3D reconstruction of the
Ad5-FX complex (FX density is shown in red in Fig. 2 Aand B).
The reconstructed Ad5 virion appears to be covered with a
⬇70-Å-thick mesh of FX density over the entire capsid, except
within the vicinity of the pentons at the icosahedral fivefold
symmetry axes.
The Ad5-FX reconstruction compared with a previous recon-
struction of the Ad5 capsid (18) revealed that the point of
contact between FX and the Ad5 hexon is within the central
depression of each hexon trimer (Fig. 2C). Fitting of the Ad5
hexon crystal structure [PDB ID code 1P30 (19)] into the
cryoEM hexon density showed that the exposed hypervariable
Fig. 1. Kinetic response data for different blood coagulation factor binding
to Ad5, Ad2, and adenovirus capsid proteins. Experimentally obtained data
are shown by black lines. Global fits of these data to 1:1 single-site interaction
model are shown in orange. The responses are shown in instrument response
units (RU) vs. time in seconds (s). Columns represent six different ligands that
were immobilized on the CM5 sensor chip. Ad5, adenovirus type 5; Ad5,
Hex-Ad5 hexon; Ad5-FKn, Ad5 fiber knob domain; Ad2, adenovirus type 2;
Ad2-Hex, Ad2 hexon; Ad5BAP, hexon-mutated Ad5-based vector. Rows rep-
resent eight different blood factors used as analytes in Biacore experiments.
hFX, human factor X; mFX, mouse factor X; FIX, human factor IX; FVII, human
factor VII; protein C, human protein C.
Table 1. Affinity of FX binding to different viruses
Virus Immobilized RU K
d
,nM
Adenovirus
Ad5 384 0.229
Ad16 470 1.67
Ad2 352 20.9
Ad21 615 410
Ad41 347 630
Ad4 2,900 2,480
Ad3 2,973 3,000
Ad35 315 No binding
Ad51 667 No binding
Ad9 311 No binding
Ad50 256 No binding
Reovirus
T3D 486 No binding
Ad5-sCAR 7.9
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regions HVR3, HVR5, and HVR7 are in close proximity to FX
density (Fig. 2Dand Fig. S3). Amino acid sequence alignment of
the hexons from the 11 Ad serotypes tested (Table 1) indicated
that FX binding could be associated with the presence of amino
acids TET or TDT within HVR7 (Fig. 2 E). In addition, there are
two serotypes (Ad3 and Ad21) that have the sequence TDT
within HVR3. Both the HVR7 TET site in the Ad5 hexon and
the HVR3 TDT site mapped onto the Ad5 hexon point into the
central depression of hexon with a similar geometric arrange-
ment (Fig. 2D). Although HVR5 is spatially adjacent to HVR7
and is also localized in the central depression of the hexon trimer
(Fig. S3), its amino acid composition is highly diverse, and there
is no apparent correlation between the presence of particular
amino acids within HVR5 and the serotype affinity for FX.
FX-Binding-Ablated Virus Failed to Transduce Hepatocytes
in Vivo
.
Recent studies demonstrated that vitamin K-dependent blood
coagulation factors mediate efficient hepatocyte transduction by
Ad5-based vectors in vivo (10, 11). To analyze the role of the
FX–hexon interaction in Ad hepatocyte transduction, we took
advantage of a hexon-mutated vector, Ad5BAP (12). Analysis of
blood factor binding to Ad5BAP by using SPR demonstrated a
4 order-of-magnitude loss in affinity of FX binding to Ad5BAP
virions (Fig. 1 and Table S1). This suggested that this large
insertion in HVR5 near the putative FX attachment site on
hexon might sterically block FX binding or induce a conforma-
tional change within the FX attachment site that could inhibit
binding. We first analyzed Ad5BAP infectivity in vitro. The virus
particle-to-plaque forming unit ratio, determined on 293 cells,
was 75 ⫾23 and 79 ⫾10 for control unmodified Ad5RFP vector
and Ad5BAP, respectively. To further investigate whether a
large insertion in the HVR5 area of hexon may adversely affect
early steps of virus infection, we analyzed virus particle attach-
ment by using quantitative Southern blot analysis (20) (Fig.
S4A). Our analysis showed that both unmodified Ad5RFP vector
and Ad5BAP attached to cells with similar efficacy. Next, we
analyzed internalization rates for these vectors using a neutral-
izing antibody escape assay (21). The data obtained showed that
internalization rates were identical for both Ad5RFP and
Ad5BAP vectors (Fig. S4B). Confocal microscopy studies of
intracellular virus trafficking revealed that both vectors rapidly
migrated to the nuclear periphery. However, unlike the Ad5RFP
vector, infection of cells with Ad5BAP induces significant rear-
rangement of Cathepsin D-positive late endosomal/lysosomal
compartment and Ad5BAP virus particles colocalized with late
endosomes/lysosomes (Fig. S4C).
Analysis of virus infectivity on human lung epithelial (A549)
or hepatoma (HepG2) cells revealed that, although no differ-
ence in infectivity between Ad5RFP and Ad5BAP was found in
A549 cells, there was a marked reduction in infectivity of
Ad5BAP, compared with Ad5RFP, in HepG2 cells (Fig. S4 D
and E). When we analyzed virus infectivity on these cell lines in
the presence of blood coagulation factors, we found that the
infectivity of Ad5BAP was significantly lower compared with
Ad5RFP when blood factors were added to the infection media
(Fig. 3 Aand B). This difference was most dramatic in hepatoma
HepG2 cells, compared with A549 cells. Collectively, all these in
Fig. 2. CryoEM visualization of the Ad5-FX complex and FX-binding site on hexon. 3D single-particle reconstruction of the Ad5-FX complex. Ad capsid is shown
in blue. FX density is shown in red. The view in Ais along an icosahedral twofold axis. The view in Bis along an icosahedral fivefold axis. (Scale bar, 100 Å.) (C)
CryoEM structure of the Ad5-FX complex (blue) together with the strongest FX density (red). The FX density was generated by subtracting a cryoEM reconstruction
of the Ad5 capsid from that of the Ad5-FX complex. The strongest FX density appears in the central depression of each hexon trimer. (Scale bar, 100 Å.) (D) Crystal
structure of Ad5 hexon (PDB ID code 1P30) shown in a ribbon representation and as a density map filtered to 30-Å resolution (transparent gray). The hexon HVR7
residues 422– 424 (TET) are shown in a space-filling representation in yellow. The position of HVR3 TDT residues found in Ad3, Ad21, and Ad50 hexons is shown
by the corresponding Ad5 residues 212–214 in cyan. Both 45° tilt and top views are shown. (E) Amino acid sequence alignment of the hexon HVR3 and HVR7
regions for the 11 Ad serotypes tested for FX binding (Table 1). The positions of the two alternative sites proposed for FX binding are indicated by asterisks. The
sequences TDT and TET that correlate with FX binding are highlighted in cyan within HVR3 and in yellow within HVR7. Nearby positively charged Arg residues
that may ablate or reduce FX-binding affinity are highlighted in green. The red lines separate Ad serotypes that bind FX (above the line) from those that do not
(below the line).
Fig. 3. Transduction of A549 and HepG2 cells by Ad5RFP and Ad5BAP vectors
in the presence of human blood factors. Cells were infected with a multiplicity
of infection of 25 pfu per cell for each indicated virus in the presence of 8
g/ml
of FIX, FX, or protein C or 0.5
g/ml of FVII (physiological concentration of
these factors in human plasma). Two hours after addition of virus to cells, cells
were washed with new media, and RFP gene expression was analyzed 24 h
later by flow cytometry. In control settings (Saline), viruses were added to cells
in growth media only (n⫽6).
Kalyuzhniy et al. PNAS
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vitro studies showed that virus attachment to cells and internal-
ization into cells were identical for Ad5RFP and hexon-mutated
Ad5BAP vectors. However, cell-type-specific factors may affect
intracellular trafficking and infectivity of hexon-mutated vector.
Importantly, addition of blood factors to infection media signif-
icantly increased infectivity of Ad5RFP vector and had no effect
on infectivity of Ad5BAP in vitro.
When equal doses of unmodified Ad5RFP and Ad5BAP
vectors were injected i.v. into mice, the amounts of Ad5BAP
vector trapped in the liver1haftervirusadministration were
50% lower than for Ad5RFP (Fig. 4Aand Fig. S5A). Pretreat-
ment of mice with warfarin, which blocks
␥
-carboxylation of all
vitamin K-dependent zymogens and has been shown to reduce
liver cell transduction by Ad5-based vectors (11), did not signif-
icantly reduce Ad vector trapping in the liver tissue at this time.
Analysis of Ad5BAP DNA in the liver 48 h after virus injection
revealed that these levels were 75% lower, compared with DNA
levels of Ad5RFP. Treatment of mice with warfarin reduced
levels of Ad5RFP DNA in the liver by 85%. Preinjection of
warfarin-treated mice with FX before Ad5RFP injection re-
sulted in a remarkable 24-fold increase in vector DNA levels in
the liver at this time point (Fig. 4A). Administration of FX
before Ad5BAP injection in warfarin-treated mice did not
increase low levels of Ad5BAP DNA in the liver, confirming our
findings on reduced affinity of blood factor binding to Ad5BAP
(Fig. 1) and cell transduction in the presence of blood factors in
vitro (Fig. 3).
Evaluation of hepatocyte transduction by fluorescent micros-
copy 48 h after i.v. injection of Ad5RFP or Ad5BAP revealed
(Fig. 4B) that RFP levels in hepatocytes mimicked vector
genomic DNA levels, determined by Southern blot analysis.
The Ad5BAP vector failed to express RFP after intravascular
delivery. Warfarin treatment of mice ablated RFP transgene
expression in hepatocytes after injection of Ad5RFP vector, and
this ablation could be completely reverted by restoration of
physiological levels of FX in blood (Fig. 4B). Importantly, the
Ad5BAP vector failed to transduce hepatocytes in warfarin-
treated mice even in the presence of FX. Western blot analysis
for RFP protein levels in hepatic tissue demonstrated high levels
of RFP expression in livers of mice injected with Ad5RFP (Fig.
S5 Band C). The levels of hepatic RFP expression were at the
limit of detection by Western blot analysis in mice injected with
Ad5BAP. The median of RFP band intensity was 68-fold
stronger for Ad5RFP, compared with Ad5BAP.
Discussion
Virus binding to cellular receptors is a critical early step in
initiation of infection. This interaction is under strong evolu-
tionary pressure and is often mediated by specialized virus
capsid proteins. For species C Ad serotype 5, CAR was shown
to be the high-affinity attachment receptor at the cell surface (4,
5). The presence of CAR on epithelial cells determines the
susceptibility of these cells to Ad5 infection both in vitro and in
vivo (22, 23). Although the interactions of Ad5 with cells in vitro
are known in great detail (24, 25), the pathways of cell-specific
infection by Ad in vivo remain poorly understood. Specifically,
after i.v. delivery, the vast majority of Ad particles are trapped
in the liver (26–28). Unsuccessful attempts to prevent hepato-
cyte transduction by Ad5-based vectors through ablation of CAR
and/or integrin binding implied that another mechanism must
exist to allow Ad5 virus particles to enter hepatic cells in a
CAR-independent manner (29, 30).
Recently, we and others identified a blood factor-dependent
pathway that allows Ad5 to infect hepatocytes independently of
virus binding to CAR (10, 11). These studies also demonstrated
that FX is the most efficient at supporting Ad5 entry into
hepatocytes. Despite this advance in our understanding of
pathways of hepatocyte infection with Ad5 in vivo, the precise
mechanism of blood coagulation factor interaction with Ad
particles remained unclear.
In this study, we analyzed, in a systematic way, Ad capsid
protein interaction with vitamin K-dependent coagulation fac-
tors and found that the Ad5 hexon possesses a high-affinity
binding site for human FX. The determined picomolar affinity
of FX binding to Ad5 implies that, when delivered via an
intravascular route, Ad5 will form a ver y stable complex with FX
and not other vitamin K-dependent zymogens, which bind Ad5
with lower affinity (Table S1) and are less abundant in plasma.
CryoEM visualization of the Ad5-FX complex revealed a mesh
of FX molecules covering almost the entire adenovirus capsid.
Importantly, analysis of the contact point between FX and hexon
showed that FX binds within the central depression at the top of
each hexon trimer.
The hexon-mutated Ad5BAP vector failed to transduce he-
patocyte when injected into wild-type mice or warfarin-treated
mice with physiological levels of FX in circulation. Our in vitro
data also showed that Ad5BAP infectivity cannot be increased
by addition of blood factors into infection media (Fig. 3);
however, unmodified Ad5RFP vector infectivity increases dra-
matically when FVII, FIX, or FX is added to the media during
virus infection.
Hexon-mutated Ad5BAP virus could be efficiently propa-
gated in 293 cells, and our in vitro analyses showed it is attached
to and internalized into cells with high efficiency, which is equal
to the unmodified Ad5RFP vector (Fig. S4). However, unlike
Ad5RFP, Ad5BAP demonstrated colocalization with late endo-
Fig. 4. In vivo analysis of hexon-mutated adenovirus vector. (A) Ad DNA deposition and persistence in the liver after i.v. injection of Ad5RFP or hexon-mutated
Ad5BAP vectors. Amounts of vector genomic DNA in the liver were analyzed by Southern blot analysis and quantified after processing of band signal intensity
by phosphorimager. The intensity of Ad5RFP bands at 1 and 48 h after virus injection was designated as 100. The graphs show the averaged data obtained from
two independent experiments with four to six biological replicates per each group. *,P⬍0.01. The representative image of Southern blot hybridization is shown
in Fig. S6A.(B) Histological analysis of Ad-encoded RFP gene expression in mouse hepatocytes 48 h after i.v. injection of indicated vectors. Representative fields
are shown. RFP expression is observed as red fluorescence on fixed liver sections. Corresponding fields in DAPI channel (nucleus-specific staining) are shown.
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somal/lysosomal cathepsin D-positive compartments and vari-
able infectivity on human lung epithelial and hepatoma cells.
These data indicate that such postinternalization steps of infec-
tion as release from the endosomes or viral DNA transport into
the nucleus may be more significantly affected by cell-type-
specific factors for hexon-modified Ads when compared with
unmodified vectors.
Our earlier data suggested that FIX could efficiently support
CAR-independent liver cell infection both in vitro and in vivo,
and that the fiber knob domain is the likely moiety that binds
FIX in the Ad capsid (10). Moreover, numerous studies of
hepatocyte transduction by capsid-modified Ad vectors
showed markedly reduced transgene expression, when vectors
with short-shafted fibers were applied in vivo (29, 31, 32). Our
SPR studies demonstrated that FIX binding to Ad5 follows a
complex kinetic that cannot be described by a 1:1 single-site
interacting model (Fig. 1). Moreover, the affinity of FIX
binding to purified hexon is reduced, compared with its
binding to complete Ad5 particles (Table S1). The observed
kinetics of FIX binding to Ad5 particles fit well into a two-site
interacting model (data not shown; O.K. and D.M.S., unpub-
lished work). Although the precise localization of these sites
remained to be determined, the hexon-mutated Ad5BAP
vector failed to bind FIX in SPR assay and transduce cells in
a FIX-dependent manner in vitro (Fig. 3). These data suggest
that, similar to FX, for FIX, Ad5 hexon possesses the dominant
binding site on the Ad capsid.
Our finding is critical for understanding the interactions of Ad
with host cells in vivo and has important practical implications
for development of safe and efficient cell- and tissue-type-
specific adenovirus vectors for gene therapy applications.
Materials and Methods
Cells and Viruses. The first-generation Ad5-based vectors, expressing the
dsRED2 RFP, Ad5RFP, and Ad5BAP, were constructed and described in detail
(12, 33). Ad5BAP is described as Ad-Hexon-BAP (33). Wild-type human Ad
serotypes Ad2, Ad3, Ad4, Ad5, Ad9, Ad16, Ad21, Ad35, Ad41, Ad50, and Ad51
were obtained from American Type Culture Collection. Human reovirus T3D
was kindly provided by Terence Dermody (Vanderbilt University, Nashville,
TN). Viruses were banded in CsCl gradients, dialyzed, and stored in aliquots, as
described (34). Adenovirus genome titers were determined by quantitative
real-time PCR (for Ad5RFP and Ad5BAP) and OD260 measurement (for wild-
type viruses).
Proteins. All coagulation blood factors were purchased from Haematologic
Technologies. All supplied blood factors were at ⱖ95% purity: human FX,
HCX-0050, lot T1206; mouse FX, MCX-5050, lot R0903; human FIX, HCIX-0040,
lot U1206; human FVII, HCVII-0030, lot U1206–0.02; human protein C, HCPC-
0070, lot W0822; human FXI, HCXI-0150, lot U0607; human FXII, HCXII-0155,
lot U1020; and human Gla-domainless FX, HCX-GD, lot U0629. Ad2 hexon
(catalog no. R14800) was purchased from Meridian Life Sciences. Ad5 hexon
and Ad5 fiber knob domain purification is described in detail in SI Text,
Materials and Methods. BSA, catalog no. A0281-5G, was purchased from
Sigma.
SPR Analyses. All analyses were carried out on a Biacore 2000 machine.
Research grade CM5 sensor chips, N-hydroxysuccinimide, N-ethyl-N⬘-(3-
diethylaminopropyl)carbodiimide, ethanolaminehydrochloride, HBSP run-
ning buffer, and HBSEP regeneration buffer were purchased from the man-
ufacturer (Biacore). All data were collected at 1 Hz by using two or three
replicate injections for each concentration of analyte. Data processing and
kinetic analysis were performed by using Scrubber software (version 2.0,
BioLogic Software). Data processing included double referencing (35). Pro-
cessed data were globally fit to a simple 1:1 interaction model.
Cryoelectron Microscopy. An Ad5-FX complex was prepared by mixing 100
l
of the Ad5S vector (34), which has a short-shafted fiber (1 ⫻1013 virus particles
per ml) with 1
l of FX (2 mg/ml). Six-microliter aliquots of the Ad5-FX complex
were applied to Quantifoil R2/4 holey carbon grids (Quantifoil Micro Tools)
and a Vitrobot cryofixation device (FEI) was used for blotting and sample
vitrification in liquid ethane. Data collection was performed on an FEI Tecnai
12 (120 kV, LaB6) transmission cryoelectron microscope equipped with a Gatan
cryoholder and Gatan UltraScan 2kx2k charge-coupled device camera (Gatan).
Twenty-six cryoelectron micrographs were collected at a nominal magnifica-
tion of ⫻67,000, from which 40 particle images were selected by using the
program VIRUS (36). Initial estimates for the defocus and astigmatism param-
eters were determined with CTFFIND3 (37). The FREALIGN package (38) was
used for refinement of the orientational, defocus, and astigmatism parame-
ters of each particle image and to calculate 3D reconstructions. The resolution
of the icosahedral capsid is estimated to be 38 Å by the Fourier Shell Corre-
lation 0.5 threshold with applied inner and outer spherical masks (radii ⫽300
and 463 Å). The resolution of the reconstruction including FX and fiber density
(outer radius ⫽613 Å) is 40 Å.
Adenovirus Infection
in Vivo
.All experimental procedures involving animals
were conducted in accordance with the institutional guidelines set forth by
the University of Washington. C57BL/6 mice (Charles River) were housed in
specific pathogen-free facilities. For analysis of Ad-mediated gene transfer
into liver cells, 1011 Ad particles in 200
l of PBS were injected by tail-vein
infusion. For in vivo transduction studies, mice were killed 1 or 48 h after virus
infusion, and livers were processed for DNA, protein, and histological analy-
ses. For analysis of Ad genome accumulation in the liver tissue 1 h after Ad
vector administration, the blood was flushed from the liver by a cardiac saline
perfusion, livers were harvested, and total DNAs were purified as described
(39). Where applicable, mice were injected with warfarin and FX, as described
in ref. 11.
Histological Analysis. Liver samples from mice injected with Ad vectors were
fixed, dehydrated in sucrose, and frozen in OCT compound. RFP gene expres-
sion in hepatic cells was analyzed on frozen sections using conventional UV
fluorescent microscopy.
ACKNOWLEDGMENTS. We thank Daniel Stone for critical reading of this
manuscript and Terence Dermody for providing reovirus T3D. This work was
supported by funding from the National Institutes of Health (Grants AI062853,
AI064882, and AI065429, to D.M.S.; Grant AI042929, to P.L.S.) and by grants to
M.A.B. from the Muscular Dystrophy Association and the Propionic Acidemia
Foundation.
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