Genetic Retargeting of Adenovirus: Novel Strategy Employing "Deknobbing" of the Fiber

Abstract

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.

Full text

JOURNAL OF VIROLOGY,
0022-538X/01/$04.00⫹0 DOI: 10.1128/JVI.75.16.7280–7289.2001
Aug. 2001, p. 7280–7289 Vol. 75, No. 16
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Genetic Retargeting of Adenovirus: Novel Strategy Employing
“Deknobbing” of the Fiber
MARIA K. MAGNUSSON,
1,2
SAW SEE HONG,
3
PIERRE BOULANGER,
3
AND LEIF LINDHOLM
1,2
*
Department of Medical Microbiology and Immunology, University of Go¨teborg,
1
and Got-A-Gene AB,
2
Go¨teborg,
Sweden, and Laboratoire de Virologie et Pathoge´ne`se Virale, CNRS UMR-5537,
Faculte´deMe´decine RTH Laennec, 69372 Lyon Cedex, France
3
Received 27 December 2000/Accepted 23 May 2001
For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral
tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described.
The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimer-
ization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding
recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear
import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with
antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the
exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The
recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two
to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower.
Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the
coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was un-
changed in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed
an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described
method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.
One of the general limitations for successful gene therapy
today is the difficulty of achieving in vivo gene delivery to
specific cells. Among several potent vectors used for gene
therapy is adenovirus (Ad), which benefits from being safe,
well studied, and easy to propagate (46). However, Ad has a
broad tropism and infects a wide variety of cells by binding to
the coxsackie B virus and Ad receptor (CAR) (3) and the
major histocompatibility complex class I alpha-2 domain (16).
On the other hand, cells that do not express these receptors are
often refractory to Ad transduction.
Cellular binding of Ad is mediated by the fiber protein,
which is anchored to the penton bases at vertices of the viral
icosahedron. The fiber is a homotrimer composed of three
identical fiber polypeptides arranged in a parallel orientation
(39). Trimerization is absolutely crucial for the fiber to func-
tion in attachment both to the capsid and to the cellular re-
ceptor (7) and is achieved by a trimerization signal that is
situated within the knob region (13, 27), which also contains
the ligand that binds to the cellular receptors (24).
Viral retargeting can be divided roughly into two conceptu-
ally different strategies: (i) nongenetic retargeting and (ii) ge-
netic retargeting involving engineering of viral proteins. Within
each group, expanded as well as narrowed tropism may be
achieved. The first strategy has mainly utilized bispecific anti-
bodies or peptides that block the native binding of the fiber
and redirect the virus to a new cellular receptor (9, 15).
Efforts using the second strategy include construction of a
chimeric Ad5 fiber with an Ad3 knob (22), modifications of the
penton base (43) or hexon proteins (41), and insertions of new
amino acid (aa) motifs in the fiber knob (8, 25). However, the
last approach is limited by the fact that the trimeric nature of
the fiber is sensitive to genetic alterations, so that only small
insertions are tolerated. As an example, the C-terminal inser-
tion of 24 aa (25) was tolerated, while 26 aa at the same
position totally disrupted the trimeric structure (45). Most of
the approaches mentioned retain the native binding structure
and thus broaden the viral tropism. For these vectors to work
satisfactorily in vivo, tissue-specific promoters or other regula-
tory elements are a necessity unless ablation of the native
cellular binding is achieved. Recently, Kirby et al. (21) abol-
ished high-affinity binding to CAR by point mutations in the
DG loop of the knob. However, the native conformation of the
knob will still be needed, and large insertions in flexible loops
such as DG or HI might be as badly tolerated as in the rest of
the knob. It is therefore unlikely that the use of nonbinding
fiber-knobs as molecular scaffolds or frameworks for new cell-
binding ligands will be widely useful for the construction of
genetically retargeted Ad.
The aim of this study was to genetically retarget Ad and
simultaneously remove the cell-binding ligand. In contrast to
the earlier concept of preserving and modifying the knob, we
have “deknobbed” the fiber by removing the fiber sequence
C-terminal of the seventh shaft repeat. This completely re-
moves the cell-binding ligand but also the trimerization signal.
To compensate for the loss of trimer formation, we inserted
the neck region peptide (NRP) of human lung surfactant pro-
tein D as an external trimerization signal. This 36-residue mo-
tif self-assembles into an extremely strong, tightly associated,
parallel three-stranded ␣-helical bundle (17). In its original
* Corresponding author. Mailing address: Department of Medical
Microbiology & Immunology, University of Go¨teborg, P.O. Box 435,
SE-405 30 Go¨teborg, Sweden. Phone: 46-31-3424693. Fax: 46-31-
415608. E-mail: leif.lindholm@microbio.gu.se.
7280

lung surfactant protein D context, NRP is flanked N-terminally
by collagen regions and C-terminally by C-type lectin domains,
suggesting that complex structures can be placed C-terminally
of NRP without disrupting the ability to trimerize. As a proof
of concept, we placed the short peptide motif arginine-glycine-
aspartic acid (RGD) at the C-terminal end of the fiber for
binding to cell surface integrins ␣
v
␤
3
and ␣
v
␤
5
(44) and showed
that these recombinant fibers could be rescued into functional,
infectious, retargeted virions.
(Construction of knobless fibers with a new trimerization
signal and a new cell-binding ligand was described by L.L. at
the Cold Spring Harbor meeting on Vector Targeting Strate-
gies, 1997.)
MATERIALS AND METHODS
Cells. HEK-293 cells (11) were purchased from Microbix (Toronto, Ontario,
Canada) and Cos7 cells, RD cells, and HeLa cells were obtained from the
American Type Culture Collection (ATCC, Rockville, M.). All cultures were
maintained in Iscove’s medium (Gibco-BRL) supplemented with 10% fetal bo-
vine serum (Sigma-Aldrich) and gentamicin (50 ␮g/ml) (Gibco-BRL) at 37°C
and 5% CO
2
.Spodoptera frugiperda Sf9 cells (ATCC) were propagated in TC100
medium (Gibco-BRL) with the abovementioned supplements at 28°C.
Antibodies. Three monoclonal fiber antibodies were obligingly supplied by Jeff
Engler (University of Alabama at Birmingham). Antibody 4D2.5 recognizes the
conserved linear motif FNPVYP found in most Ad fiber tails (13, 14); 2A6.36 is
specific for a conformational epitope present in fiber trimers within residues 17
to 61, as suggested by deletion mapping (13) and the lack of reactivity with
trimers of our deletion mutant AT61 (27); 1D6.14 is an antiknob, CAR-binding
blocking antibody (32) whose epitope has been mapped within residues 471 to
491 (unpublished data). Fluorescein isothiocyanate (FITC)-labeled rabbit anti-
mouse immunoglobulin G (IgG) and streptavidin-horseradish peroxidase (HRP)
were purchased from Dako. Anti-␣
v
␤
3
integrin (LM609) and anti-␣
v
␤
5
integrin
(P1F6) monoclonal antibodies were from Chemicon International, Inc. The
monoclonal antibody RL2, which is specific for O-linked GlcNAc residues (26),
was obtained from Larry Gerace via Jeff Engler. Antihexon capsomers were
produced by the 2Hx2 hybridoma, purchased from ATCC.
Construction of recombinant fibers. The wild-type (WT) fiber gene was am-
plified from pAB26 (Microbix) using the primers 5⬘-CTC GGA TCC GAT GAA
GCG CGC AAG ACC GTC TGA A-3⬘(5⬘oligonucleotide) and 5⬘-TTC CTC
GAG TTA TTC TTG GGC AAT GTA TGA-3⬘(3⬘oligonucleotide), introduc-
ing an upstream BamHI and a downstream XhoI site, respectively (Fig. 1).
Recombinant fibers containing the tail, different numbers of shaft repeats (1, 7,
or 22) followed by the external trimerization signal NRP (PDVASLRQQVA
ELQGQVQHLQAAFSQYKKVELFPNG) (17), a linker sequence from Staph-
ylococcus protein A (AKKLNDAQAPKSD), and RGD were constructed by
PCR amplification of the WT fiber gene, followed by splicing by overlap exten-
sion and ligation, which introduced the flanking restriction sites mentioned above
(detailed information can be requested from the authors). A recombinant fiber
with seven shaft repeats and the disulfide bond-containing, constrained ligand
RGD4C (ACDCRGDCFCG) (30) was also constructed for comparison to the
linear RGD motif. The fibers were named R1-RGD, R7-RGD, R7-RGD4C, and
R22-RGD (Fig. 1), respectively, indicating the different numbers of shaft repeats
preceding the cell ligand. NRP was ligated to the first repeat at the SphI site, the
seventh repeat at the NheI site, and the 22nd repeat after the conserved TLWT
motif at positions 400 to 403.
Protein expression and purification. For analysis of cellular localization in
mammalian cells, recombinant fibers were cloned into pcDNA3.1 (Invitrogen,
San Diego, Calif.), and Cos7 cells were transfected using Lipofectamine (Life
Technologies Inc., Gaitherburg, Md.) according to the protocol of the manufac-
turer. Two days posttransfection, the cells were harvested, centrifuged onto
cytospin slides, and dried overnight. Immunofluorescent staining was performed
after fixation in 3% paraformaldehyde and permeabilization in 0.1% Triton
X-100 in phosphate-buffered saline (PBS). The primary antibodies 4D2.5 and
2A6.36 were used as ascites fluids diluted to 1:1,000, while the secondary FITC-
labeled anti-mouse Ig antibody was used at a dilution of 1:10. Each antibody was
incubated with the specimens for 30 min at 37°C. Expression and nuclear local-
ization were observed using a Zeiss Axioskop fluorescent microscope and pho-
tographed with a Zeiss automatic camera at ⫻40 magnification.
For protein production and purification, the genes coding for fibers, WT pen-
ton base, and penton base mutant R340E (19, 20) were cloned into the baculo-
virus Autographa californica nuclear polyhedrosis virus, using the pBacPAK9
(Novagen) or BacPAK6 (Clonetech) vector. Recombinant Ad proteins were
expressed in Sf9 cells and purified according to a previously described method
(5), with some modifications. The anion-exchange chromatographic step was
performed using a high-performance liquid chromatography system (BioLogic
DuoFlow; Bio-Rad) and a DEAE-Sepharose Fast Flow column (DFF-100;
Sigma) equilibrated in 50 mM sodium phosphate buffer, pH 6.8 (PB-50). Sam-
ples (2 to 3 mg of protein) were applied to the column, and elution was obtained
by applying a 0.0 to 0.6 M NaCl gradient in PB-50. Fiber protein was eluted at
200 mM salt and penton base at 250 mM salt, respectively. Protein samples were
then further purified and concentrated using concentrator membranes with a
100-kDa cutoff (Vivaspin-100; Vivascience Ltd, Binbrook, Lincoln, England).
Final protein concentration was estimated using the Bradford assay (Bio-Rad).
Phenotypic analysis of fiber proteins. Recombinant fiber proteins were phe-
notypically analysed for their trimerization, glycosylation, and assembly in Sf9
cells with recombinant penton base to form complete pentons. Oligomerization
was assayed by means of nondenaturing sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (NDS-PAGE) and by conventional, denaturing SDS-PAGE.
NDS-PAGE differed from SDS-PAGE in that the samples were not denatured
by boiling in SDS sample buffer prior to electrophoresis. Glycosylation of re-
combinant fibers was assessed both by immunoreaction on blots using the mono-
clonal antibody RL2 and by chemical detection using the DIG Glycan detection
kit (Roche). Assembly of fiber with penton base to form penton in vivo was
assayed by coinfecting the same Sf9 cells with two recombinant AcNPV, one
expressing the penton base and the other expressing the fiber protein. The
presence of penton capsomer was detected in cell lysates 40 h postinfection and
analyzed by PAGE in native conditions at low voltage overnight with cooling, as
previously described (19). For immunological quantification of native penton,
penton base, and fiber proteins, blots were reacted with the corresponding
primary antibody (anti-penton base or antifiber), followed by [
35
S]SRL-labeled
anti-mouse or anti-rabbit whole IgG secondary antibody (Amersham Pharmacia
Biotech; 100 ␮Ci/ml; 5 ␮Ci per blot). Blots were exposed to radiographic films
FIG. 1. Schematic representation of the WT fiber and recombinant
fibers R22-RGD, R7-RGD (or RGD4C), and R1-RGD. The tail fiber
domain is shown by a stippled box, the shaft by a solid box, and the
knob domain by a hatched box. Additional structural and functional
motifs in recombinant fibers, i.e., extrinsic trimerization signal (NRP),
linker peptide, and cell ligand RGD or RGD4C, are represented as
indicated in the diagrams. The fiber sequence ends at aa M-61 in
R1-RGD, K-156 in R7-RGD, and T-403 in R22-RGD.
VOL. 75, 2001 GENETICALLY RETARGETED ADENOVIRUS 7281

(Hyperfilm Beta-max; Amersham Pharmacia Biotech), and autoradiograms were
scanned at 610 nm using an automatic densitometer (REP-EDC; Helena Lab-
oratories, Beaumont, Tex.). Alternatively, protein bands were excised from blots
and radioactivity was measured in a scintillation counter (Beckman LS-6500), as
previously described (18).
ELISA. Integrins ␣
v
␤
3
and ␣
5
␤
1
(Chemicon) at 1 ␮g/ml in coating buffer (20
mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM CaCl
2
, 1 mM MgCl
2
, 1 mM MnCl
2
)
was used to coat 96-well plastic plates for 16 h at 4°C. After one wash in rinsing
buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2 mM CaCl
2
, 1 mM MgCl
2
,
1 mM MnCl
2
), the plates were blocked with 200 ␮l of blocking buffer (rinsing
buffer containing 4% nonfat dried milk) at room temperature for 2 h. Wells were
then washed three times with rinsing buffer. Five-step dilutions (1 to 0.008 ␮g/ml)
of recombinant fibers in blocking buffer were added to the wells in 100 ␮lfor1h
at room temperature. Detection of bound fibers was done with 1 ␮g of 4D2.5
(biotinylated and protein A purified), streptavidin-HRP diluted 1:2,000, and
TMB substrate (CanAg Diagnostics, Go¨teborg, Sweden) per ml. Color develop-
ment was stopped with 0.12 M HCl after 10 min, and plates were analyzed in a
microtiter plate reader set at 450 nm.
For WT fiber detection, 10 ␮g of 4D2.5 (protein A purified) per ml in 50 mM
sodium carbonate buffer (pH 9.6) was used to coat 96-well plates in 100-␮l
aliquots for 16 h at 4°C. Wells were washed once in PBS containing 0.1% Tween
20 (PBS-T) prior to addition of 200 ␮l of PBS containing 1% bovine serum
albumin (PBS-B) for2hofblocking at room temperature. After three washes in
PBS-T, recombinant fibers (diluted in PBS-B, 100 ␮l total volume) were added
to the wells and further incubated for1hatroom temperature. The CAR-
binding inhibiting antiknob antibody 1D6.14 (biotinylated and protein A puri-
fied) was used at 3 ␮g/ml, and streptavidin-HRP was used at 1:2,000. Color
development with TMB substrate and microtitration were performed as de-
scribed above.
Generation of recombinant Ad genomes. To generate recombinant Ad5 ge-
nomes, a fiber shuttle vector and a fiber rescue plasmid were constructed using
pTG3602, a plasmid which contains the WT Ad5 genome with PacI restriction
sites at both ends (6). This is schematically depicted in Fig. 4. To construct the
shuttle vector, the SacI-KpnI fragment of 3.4 kb from 84.0 to 93.5 map units
(mu), which contains the fiber gene, was cloned into pBluescript II SK(⫺)
(Stratagene). The fiber gene was then deleted from the NdeI site, located in the
fiber tail, to the MunI site, present a few nucleotides downstream of the fiber
gene. The deleted region was replaced by an annealed oligonucleotide linker
constituted of an XhoI site flanked by an NdeI site and a MunI site at its 5⬘and
3⬘ends, respectively. The resulting shuttle plasmid was referred to as pGAG3.
For construction of the fiber rescue plasmid, pBluescript II SK(⫺) was first
supplied with a PacI site. Thereafter, the SpeI-PacI fragment of 8.8 kb (75.4 to
100 mu) from pTG3602 was ligated to the pBluescript II SK(⫺)PacI site to
generate pGAG9.
Fiber constructions were subcloned into pGAG3 using NdeI and XhoI. As a
negative control, the empty pGAG3 was used to obtain a fiber deleted from the
NheI site in the tail region to the 3⬘end of the fiber gene. For further rescue into
pGAG9, homologous recombination in Escherichia coli BJ5183 (recBC sbcBC)
(6) was performed as described elsewhere (28). Briefly, 50 ng of pGAG9 re-
stricted with NheI was transformed into BJ5183 by electroporation together with
a 10:1 (insert-vector) molar ratio of SacI-KpnI-digested pGAG3. The recombi-
nant pGAG9 clones were then retransformed into the Nova Blue bacterial strain
(Novagen) to obtain large quantities of plasmid DNA.
To join the recombinant pGAG9 to the remaining 0 to 75.4 mu of the Ad5
genome, a cosmid cloning system (SuperCos 1 cosmid vector kit and Gigapack
III Gold packaging extract; Stratagene) was used after addition of a PacI site to
SuperCos1 in the BamHI site. This cosmid (SupercosA) was restricted with PacI
and XbaI and treated according to the manual. Then 0.5 ␮g of SupercosA, 0.5 ␮g
of PacI-SpeI-restricted recombinant pGAG9, and 0.75 ␮gofPacI-SpeI-restricted
fragment (0 to 75.4 mu) were used for ligation. Clones were screened by PCR
using primers specific for the fiber and restricted by HindIII and SpeI. Large
plasmid preparations were made using a plasmid maxi kit (Qiagen).
The 0 to 75.4 mu sequence of the Ad5 genome originated from recombined
pAdEasy1 and pAdTrackCMV (12), in which the deleted E1 region is replaced
by a green fluorescent protein (GFP) gene under the control of the cytomega-
lovirus (CMV) promoter (12).
Virus generation. Recombinant cosmid DNA was restricted with PacI, pre-
cipitated with ethanol, and resuspended in sterile H
2
O. Transfection into 293
cells was performed using FuGENE (Roche) according to the manufacturer’s
recommendations, with 2 ␮g of DNA and 3 ␮l of FuGENE in each 35-mm well.
Large-scale production and CsCl gradient purification of virus were performed
as previously described (10). The presence of fiber genes in virions was analyzed
by PCR with primers specific for the WT and recombinant fibers. The presence
of fiber proteins in virions was determined by Western blot analysis with 4D2.5
antitail antibody. Expression of GFP in infected cultures was verified by UV
microscopy using a Zeiss Axioskop fluorescent microscope. Infectious titers
(PFU per milliliter) were determined by plaque titration on 293 cells using an
endpoint dilution method (29), and the number of physical virus particles was
determined using the IDEIA Ad detection kit (Dako). The rate of virus growth
was determined as follows. 293 cells (4 ⫻10
4
) were infected with WT or recom-
binant virus at 10 PFU/cell for1hat37°C. After rinsing with PBS, complete
medium was added and cell samples were harvested at 24, 48, and 72 h after
infection. Cell pellets were washed in PBS, resuspended in 200 ␮l of PBS, and
freeze-thawed four times. The cell lysates were assayed for production of Ad5
proteins by the IDEIA kit, and the infectivity titer, determined by plaque titra-
tion on 293 cells as above, was plotted versus the time of infection.
Quantitative analysis of fiber content of virions. 293 cells in 24-well plates
were washed once with Iscove’s medium and infected with 10 PFU/cell in 100 ␮l
of Iscove’s medium. After1hat37°C, the cells were washed in complete medium
(Iscove’s medium with 10% fetal bovine serum and 50 ␮g of gentamicin per ml)
and incubated with complete medium at 37°C and 5% CO
2
. Cells were harvested
at 24, 48, and 72 h postinfection, collected in 0.2 ml of Iscove’s medium, and
freeze-thawed four quick rounds to release virions. Virus titer was determined by
plaque assay in 293 cells, and viral proteins were assayed using the IDEIA kit.
Western blots of the freeze-thawed material and CsCl-purified virus were re-
acted with 4D2.5 and 2Hx2 to assay the fiber versus hexon content, and quan-
tification was performed using the Scion Image program.
Gene transfer assay. Monolayers of HeLa and RD cells in 24-well plates were
infected as described above with 10 PFU of the recombinant viruses per cell, with
or without different blocking agents. The antiknob antibody 1D6.14 was used at
concentrations ranging from 0.001 to 10 ␮g/ml. Virus and antibody were mixed
and diluted to 0.1 ml in Iscove’s medium and incubated for1hat37°C before cell
infection, as above. The cells were examined by immunofluorescence (IF) mi-
croscopy at intervals during the course of infection for the appearance of GFP
fluorescence. For fluorescence-activated cell sorting (FACS) analysis, the cells
were harvested at 24 h postinfection and washed with ice-cold PBS, followed by
fixation with 0.5% glutaraldehyde for 15 min. After three washes in PBS, the cells
were analyzed for GFP expression using the FL1 emission channel in a FACScan
cytometer (Becton Dickinson, San Jose, Calif.).
RESULTS
Phenotypic characterization of recombinant fibers with dif-
ferent shaft lengths. The number of shaft repeats in fiber
determines the shaft length (33) and has important implica-
tions in receptor usage (31, 38). Fiber proteins with various
numbers of shaft repeats were thus compared (Fig. 1). Fiber
with one single repeat (R1-RGD) was constructed to test a
very short fiber; fiber with seven repeats (R7-RGD) resembled
the short-shafted Ad3 fiber; and fiber with 22 repeats (R22-
RGD) mimicked the Ad5 WT fiber shaft. The choice of the
number of shaft repeats for the fiber of retargetable Ad5 vec-
tors was guided by the results of phenotypic analysis of the
different fiber constructs. The recombinant fibers were ex-
pressed in both mammalian and insect cells and phenotyped
according to the following criteria: (i) their cellular localiza-
tion, (ii) the occurrence and stability of fiber trimers, (iii) their
capacity to form penton capsomers in insect cells coinfected
with a recombinant baculovirus-expressing penton base, and
(iv) their glycosylation status.
(i) Cellular localization and fiber trimerization. The cellular
localization and trimer status of the recombinant fibers were
studied by transient expression in Cos7 cells at 48 h after
transfection. The cells were fixed, reacted with monoclonal
antibodies specific for fiber tail (4D2.5) and fiber trimers
(2A6.36), and examined by IF microscopy. The IF patterns
observed showed that all our recombinant fiber proteins were
well expressed and localized in the nucleus. They were all
capable of forming trimers, just like the WT fiber (data not
shown).
7282 MAGNUSSON ET AL. J. VIROL.

The trimerization status was also tested by electrophoresis
and Western blotting of fiber samples denatured by boiling in
SDS (SDS-PAGE) or unboiled (nondenatured; NDS-PAGE).
The NDS-PAGE pattern for unboiled recombinant WT fiber
consisted of three distinct protein species, monomers, dimers,
and trimers (Fig. 2A, lane 3). For recombinant fibers R7-RGD
and R22-RGD, dimers and trimers were similarly observed
(Fig. 2 A, lanes 7 and 9). However, the oligomeric forms of
R22-RGD were barely visible, since the R22-RGD clone was a
low expressor in terms of soluble fiber protein (data not
shown). In the case of R1-RGD, no trimers were visible in
NDS-PAGE, and all detectable fiber occurred in monomeric
form (Fig. 2A, compare lanes 4 and 5). However, in gel elec-
trophoresis under native conditions, in the absence of SDS,
fiber trimers were detected for all the recombinants, including
R1-RGD, using the antitrimer antibody 2A6.36 (data not
shown). Moreover, R1-RGD fiber reacted positively with
2A6.36 within the cell, as shown by IF microcopy. This strongly
suggested that R1-RGD fiber did form trimers in vivo but that
these trimers were unstable in vitro in the presence of SDS,
even at low temperature.
(ii) Assembly with penton base to form penton capsomers.
In vivo in coinfected Sf9 cells, all our recombinant fibers were
able to assemble with penton base to form penton capsomers,
which were detectable in gels electrophoresed under native
conditions (Fig. 2B and C). Free fiber trimers migrated in the
native gel as polydispersed molecules visible as a smear on
immunoblots (Fig. 2B), whereas penton capsomers appeared
as a sharp, slow-migrating band (Fig. 2B and C). This discrete
larger protein species was revealed by both fiber and penton
base antibodies, confirming that they were penton complexes
(Fig. 2B and C).
The efficiency of penton assembly with our recombinant
fibers was quantitatively estimated by immunoblotting of Sf9
cell lysates electrophoresed in native gels, using fiber and pen-
ton base antibodies and the corresponding radioactively la-
beled secondary antibody. The radioactivity was measured and
compared for the penton capsomer band and the free fiber
band in blots probed with antifiber antibody (Fig. 2B) or for
the penton capsomer band and the free penton base band in
blots probed with anti-penton base antibody (Fig. 2C). The
data are shown in Table 1. With WT fiber, which is highly
FIG. 2. Recombinant fibers, ability to trimerize (A) and to form penton capsomers (B and C). (A) Fiber trimers were analyzed by SDS-PAGE
and NDS-PAGE and immunoblotting with 4D2.5 antibody as boiled (b) or unboiled (u) samples. WT fiber (lanes 2 and 3), R1-RGD (lanes 4 and
5), and R7-RGD (lanes 6 and 7) were all highly expressed in Sf9 cells as soluble recombinant proteins, whereas R22-RGD was a low expresser
(lanes 8 and 9). Note that no stable fiber trimer was detectable in unboiled R1-RGD lysate (lane 5). Fiber monomers are indicated by an asterisk,
dimers by 2x, and trimers by an open circle. (B and C) Assembly of fiber with penton base was assayed in lysates of Sf9 cells coexpressing
recombinant fiber and penton base (16). Native proteins were electrophoresed in an 8% nondenaturing gel and electrically transferred, and blots
were reacted with antitrimer fiber 2A6.36 monoclonal antibody (B) or polyclonal anti-penton base (C). Control samples of single expression of WT
fiber and penton base alone are shown in lanes 1 and 2, respectively. Lanes 3 to 6, lysates from cells coexpressing WT penton base with WT fiber
(lane 3), R1-RGD (lane 4), R7-RGD (lane 5), or R22-RGD (lane 6), respectively. Penton capsomer, comprised of penton base-anchored fiber,
migrates as a discrete band reacting with both antifiber 2A6.36 (B) and anti-penton base antibodies (C), and is indicated by open circles. In panel
B, free fibers, migrating as a diffuse band of polydisperse species, are indicated by an arrowhead. In panel C, monomers of penton base are
indicated by an asterisk and pentamers (penton base capsomers) by 5x.
VOL. 75, 2001 GENETICALLY RETARGETED ADENOVIRUS 7283

expressed in insect cells (27), only 5% of the fiber assembled
with penton base, while the rest remained as free fiber trimers,
and the penton base often appeared to be the limiting factor
for penton assembly (Fig. 2C, lane 3). With R1-RGD, R7-
RGD, and even R22-RGD fiber, assembly with penton base
seemingly occurred with a higher efficiency and/or greater sta-
bility than with WT fiber (Table 1 and Fig. 2B, compare lanes
3, 4, 5, and 6). This would suggest that penton constituted of
knobless fiber and containing the NRP trimerization signal
would be more stable than WT penton capsomers constituted
of penton base and full-length, knob-terminated fiber, at least
in insect cells. It has already been suggested that the fiber knob
domain could be responsible for a certain degree of flexibility
or instability in Ad2 penton capsomer (4, 14).
When probed with penton base antibody (Fig. 2C and Table
1), the blots of Sf9 cell lysates confirmed that none of our
recombinant fibers was defective in assembly with penton base.
The apparent low efficiency of assembly shown by R22-RGD
fiber (1% of the total penton plus penton base signal; Fig. 2C,
lane 6) likely resulted from its low expression level (Fig. 2A,
lanes 8 and 9), the R22-RGD fiber protein being the limiting
factor for penton assembly.
(iii) Glycosylation status of recombinant fibers. None of our
recombinant RGD fibers showed any detectable O-GlcNAc
signal, as assayed by two different methods, Western blot anal-
ysis using RL2 antibody specific for peptide-linked O-GlcNAc
residues and a biochemical assay using the DIG Glycan detec-
tion kit. Only WT fiber was found to be glycosylated (data not
shown). This suggested that O-GlcNAc residues were dispens-
able for most of the known biological functions of the fiber.
Thus, considering that R22-RGD fiber was expressed at low
levels and that R1-RGD self-assembled into rather unstable
fiber trimers, R7-RGD was retained as the most favorable
knobless fiber construct to be reintroduced into the viral ge-
nome for the generation of knobless Ad5 vectors. Further
characterization therefore concerned R7-RGD fiber and R7-
RGD-containing virions.
Functionality and binding specificity of fiber proteins. The
binding function of WT and R7-RGD fibers was examined
by solid-phase enzyme-linked immunosorbent assay (ELISA).
Both fibers were tested for binding to antiknob monoclonal
antibody 1D6.14 and integrin ␣
v
␤
3
. WT fiber was found to bind
with a high affinity to the knob antibody compared to the
absence of significant binding by the R7-RGD fiber (Fig. 3A).
The reverse was observed with wells coated with ␣
v
␤
3
integrin;
WT fiber showed no detectable binding, while R7-RGD fiber
had a high affinity for ␣
v
␤
3
integrin (Fig. 3B). The specificity of
binding of the R7-RGD fiber to immobilized integrin ␣
v
␤
3
was
also studied by ELISA in the presence of RGD peptides or
penton base proteins as competitors. RGD peptides competed
poorly with R7-RGD fiber for integrin binding (data not
shown), probably due to a low stability of the integrin-RGD
complexes in ELISA. However, WT penton base, which carries
five RGD motifs, competed efficiently (Fig. 3C). In contrast,
penton base mutant R340E, mutated in the RGD motifs (20),
failed to compete with R7-RGD fiber, suggesting that the
competition of WT penton base with R7-RGD fiber was RGD
specific (Fig. 3C). Binding of WT and R7-RGD fibers was also
tested on ␣
5
␤
1
integrins (data not shown), and the assays gave
similar results as for ␣
v
␤
3
integrin. Thus, our results suggested
that the R7-RGD fiber had lost its affinity for the knob anti-
body but had gained the ability to bind to ␣
v
␤
3
and ␣
5
␤
1
integrins. This implied that the RGD motif on the fiber was in
the proper conformation and accessible for binding to cell
surface-exposed ␣
v
␤
3
and ␣
5
␤
1
integrins.
Rescue of functional recombinant fiber genes into infectious
virions. To generate virions with modified fibers, a strategy was
designed to insert the recombinant fiber gene exactly in the
position of the fiber gene in the Ad5 chromosome so as to
make use of the native regulation of fiber gene expression. This
was achieved by a three-step rescue system (Fig. 4), in which
the recombinant fiber was first cloned into the shuttle vector
pGAG3 containing the tail and the flanking sequences of the
fiber gene. As a negative control, the empty pGAG3 vector was
used. The fragments were then rescued into pGAG9 (75.4 to
100 mu of the WT Ad5 genome) by homologous recombina-
tion in E. coli BJ5183. As a final step, cosmid cloning was used
for ligation to the rest of the genome. For convenient detection
of gene transfer, our recombinant Ad genome contained the
GFP reporter gene cloned under the control of the CMV
promoter in the deleted E1 region. The resulting genome was
joined at both ends to the cosmid backbone by PacI and could
thus be recovered as a linear DNA fragment after cleavage
with PacI and transfected into cells to yield virus. On the
average, approximately 20% of the cosmid colonies contained
the expected recombinant Ad5 genome.
Efficiency of virus propagation. After transfection of 293
cells, approximately 8, 16, and 16 days elapsed before plaques
appeared for the viruses designated Ad5/FibWT, Ad5/FibR7-
RGD, and Ad5/FibR7-RGD4C, respectively. The genome with
the deleted fiber did not give any plaques. The recombinant
viruses were then amplified in 293 cells, and the viruses were
purified by ultracentrifugation in a self-generating CsCl gradi-
ent. After virus purification, the fiber genotype was controlled
and confirmed by PCR amplification and DNA sequencing,
using primers specific for WT and R7-RGD fibers. In addition,
the presence of fiber proteins in virions was assayed immuno-
logically by Western blot analysis using antifiber antibody. Al-
though the expected fiber sequence and signal were found in
TABLE 1. Efficiency of penton assembly in Sf9 cells coexpressing
WT penton base and recombinant fibers with various
shaft repeats and C-terminal RGD ligands
a
Recombinant fiber Avg % of total radioactivity ⫾SD
Assembled fiber
b
Assembled penton base
c
WT 5.5 ⫾0.8 51.4 ⫾7.0
R1-RGD 9.9 ⫾4.4 48.1 ⫾15.1
R7-RGD 10.2 ⫾1.5 27.8 ⫾10.3
R22-RGD 9.9 ⫾1.9 1.1 ⫾0.4
a
Penton base and fiber proteins were coexpressed in the same Sf9 cells using
two recombinant baculoviruses. Cell lysates were analyzed by gel electrophoresis
run in native conditions and immunoblotting with anti-penton base and anti-fiber
antibodies followed by radioactively labeled secondary antibody, as depicted in
Fig. 2B and C. Results are expressed as the percentage of total radioactivity
recovered from penton plus penton base and penton plus fiber bands. Data
shown are the average of four determinations ⫾standard deviation (SD).
b
The amounts of fiber engaged in penton and remaining as unbound fiber
were determined by counting the radioactivity associated with the bands corre-
sponding to penton and free fiber, respectively (refer to Fig. 2B).
c
The amount of penton base bound to fiber versus the amount of unassembled
penton base was determined by counting the radioactivity in the bands of penton
and penton base, respectively (refer to Fig. 2C).
7284 MAGNUSSON ET AL. J. VIROL.

our virus constructs, Ad5/FibR7-RGD and Ad5/FibR7-RGD4C
virus spread through cellular monolayers occurred at a two to
three times slower rate compared to Ad5/FibWT.
Comparison of the infectivity index of WT and recombinant
virus showed that Ad5/FibR7-RGD was 20 times less infec-
tious than Ad5/FibWT; the ratio of infectious particles (detect-
ed by fluorescent plaques and expressed as PFU) to physical
particles (estimated by biochemical methods) was found to be
1:25 for Ad5/FibWT, versus 1:500 for Ad5/FibR7-RGD. Anal-
ysis of growth curves in 293 cells showed that Ad5/FibWT and
Ad5/FibR7-RGD virions grew at similar rates in a comple-
menting cell line and synthesized similar amounts of viral pro-
teins. However, the plateau of infectious progeny yields for
Ad5/FibR7-RGD was 100-fold lower than for Ad5/FibWT
(Fig. 5).
Fiber content of recombinant Ad5 virions. To further inves-
tigate the difference in virus propagation between Ad5/FibR7-
RGD and Ad5/FibWT, 293 cells were infected with aliquots
(10 PFU/cell) of each virus. The cells were harvested at differ-
ent times postinfection and lysed, and the virus proteins re-
leased were assayed in the supernatants. At all time points
the infected cells appeared identical, as judged visually. The
IDEIA assays showed that similar amounts of viral proteins
were produced by Ad5/FibR7-RGD- and Ad5/FibWT-infected
cells. Protein analysis of cell lysates showed similar amounts of
hexon yields, while probing for fiber revealed about 10 times
less R7-RGD fiber compared to WT fiber (data not shown).
CsCl-purified viruses were also probed for fiber and hexon
contents in virus samples normalized for physical particle num-
ber and infectious particle number, respectively (Fig. 6). The
results showed that similar amounts of fibers were found when
Ad5/FibR7-RGD and Ad5/FibWT virus samples were normal-
ized for infectious particles (as counted on 293 cells; Fig. 6, top
panel, compare lanes 1 and 2). However, when virus samples
FIG. 3. Fiber-binding properties in vitro tested by ELISA. (A)
Wells were coated with anti-fiber tail monoclonal antibody 4D2.5, WT
or R7-RGD fiber was added (amount of fiber protein in a total volume
of 100 ␮l), and detection was performed using antiknob blocking
antibody 1D6.14. (B) Wells were coated with ␣
v
␤
3
integrin, WT or
R7-RGD fiber was added, and integrin-bound fiber was detected using
4D2.5 antibody. (C) Wells were coated with ␣
v
␤
3
integrin (1 ␮gof
protein per well), followed by stepwise addition of recombinant penton
base (Pb) protein (WT or R340E mutant) and R7-RGD fiber (1 ␮gof
fiber protein per well in a total volume of 200 ␮l). The stoichiometric
ratio of R7-RGD fiber to penton base molecules was 1:1, 1:5, and 1:10,
indicated under the x axis as x1, x5, and x10, respectively. Results
shown represent the mean of three separate experiments, ⫾SD.
VOL. 75, 2001 GENETICALLY RETARGETED ADENOVIRUS 7285

were normalized for physical particles (assayed by hexon con-
tent; Fig. 6, lower panel), significantly fewer fibers were pres-
ent in the Ad5/FibR7-RGD than in the Ad5/FibWT sample
(Fig. 6, top panel, compare lanes 3 and 4). As shown by quan-
titative analysis, the difference ranged within 10- to 40-fold,
depending on virus preparations. This result implied that there
was a lower fiber copy number per virion in Ad5/FibR7-RGD
or, alternatively, that the Ad5/FibR7-RGD virus preparation
contained a mixture of fiberless particles with low infectivity
(23) and fully infectious virions with normal fiber content.
Cell specificity of gene transfer mediated by recombinant
Ad5. To evaluate the tropism of the recombinant viruses, the
efficiency of Ad5-mediated gene transfer to HeLa cells and RD
cells was assayed in the presence and absence of antiknob
antibody. CAR is known to be expressed by HeLa cells but not
by RD cells if they are passaged and grown to low cell density
(37). The integrin ␣
v
␤
5
was found to be expressed on HeLa
cells, and both ␣
v
␤
3
and ␣
v
␤
5
integrins were found on RD cells,
as detected by FACS analysis after staining with specific mono-
clonal antibodies (data not shown). The infectivity of Ad5/
FibWT and Ad5/FibR7-RGD was determined in both HeLa
and RD cells (Fig. 7). As expected, HeLa cells were fully
susceptible to both Ad5/FibWT and Ad5/FibR7-RGD viruses,
whereas RD cells were found to be only permissive for Ad5/
FibR7-RGD and poorly permissive for Ad5/FibWT. To verify
that cell binding of Ad5/FibR7-RGD virus was not dependent
on the knob receptor, CAR-blocking experiments were per-
formed using 1D6.14 antibody. As shown in Fig. 8, the Ad5/
FibWT virus was readily blocked by the antiknob antibody in a
dose-dependent manner, while the antibody had no effect on
the Ad5/FibR7-RGD virus.
DISCUSSION
This study presents a new concept for genetic retargeting of
Ad and demonstrates its utility for gene transfer into cells
lacking the CAR receptor. The main feature in the present
reconstruction of the fiber gene is to genetically delete the fiber
of its native binding specificity and to replace this with a new
ligand in order to retarget the virus to a new cellular receptor.
However, in order for Ad fibers to be properly assembled into
functional proteins and be encapsidated into infectious virions,
several requirements have to be fulfilled. For example, the
fibers need to be able to form parallel homotrimers, to bind to
penton base via its tail region to form penton capsomers, to
localize within the correct cellular compartment, i.e., the nu-
cleus, where capsid assembly occurs, and lastly, in the context
of the virion, to cellular receptors.
The genetically modified knobless fibers that we have con-
structed here were designed to meet the abovementioned re-
quirements. As evaluated from the results obtained, it seemed
feasible to compensate for the lack of biological functions
carried by the fiber knob at the virion level by inserting an
FIG. 4. Generation of recombinant Ad5 genomes. (A) Construction of pGAG9 and pGAG3 from pTG3602. (B) Three-step rescue system. Step
1, ligation of recombinant fiber to pGAG3. Step 2, homologous recombination into pGAG9. Step 3, cosmid cloning to join the modified fragment
from step 2 to the PacI-SpeI fragment of 24 kb from the recombined pAdTrackCMV/pAdEasy. Detailed procedure is described in Materials and
Methods.
FIG. 5. Comparative growth curves of Ad5/FibWT virus (solid line)
and Ad5/FibR7-RGD recombinant (dotted line). HEK-293 cells were
infected with the same input multiplicity (10 PFU/cell) for1hat37°C,
then cells were harvested and lysed at different times postinfection, as
indicated. Infectious titer of each sample was determined by fluores-
cent plaque assay in 293 cell monolayers. Data shown are the average
of three separate experiments ⫾SD.
7286 MAGNUSSON ET AL. J. VIROL.

extrinsic trimerization motif and a new cell ligand which com-
pletely changed the tropism of the virus. We introduced a
36-aa peptide from the human lung surfactant protein D
(NRP) as an extrinsic trimerization motif and an RGD peptide
as a cell ligand. To allow more flexibility and accessibility, a
linker sequence from Staphylococcus protein A was inserted
between the NRP and the cell surface ligand RGD. The re-
combinant fibers could be made to contain different numbers
of shaft repeats. We have been able to generate infectious
viruses with functional fibers containing one and seven shaft
repeats (this study), as well as three and five repeats (unpub-
lished results), but not with recombinant Ad5 fibers containing
the full-length shaft (22 repeats). This was in apparent contra-
diction to a recent work which showed the rescue of knobless,
His-Myc epitope-tagged, full-length fibers into virions (40).
However, the authors had cotransfected cells with a plasmid
expressing both WT fiber and a knobless, full-length fiber con-
struct, and both fibers were thus rescued into chimaeric Ad5
virions with two distinct fiber species, WT and knobless mu-
tant. This difference in strategy could explain the yield of
infectious virus progeny using knobless full-length fibers.
Among several of our shortened fiber constructs, the knobless
fiber with seven shaft repeats (R7) and an RGD peptide motif
at its C terminus (linear, as in R7-RGD, or constrained, as in
R7-RGD4C), was found to be the one which retained the
essential functions of the fiber while carrying a new cell-bind-
ing specificity. The reason for this observation is presently
under investigation, but it constitutes the rationale for the use
of R7 fibers in the present work.
The recombinant R7-RGD fiber was found to bind to ␣
v
␤
3
,
␣
v
␤
5
, and ␣
5
␤
1
integrins. As shown previously using different
approaches, the RGD motif has the ability to target Ad virions
to otherwise refractory cell lines after being incorporated into
the fiber or the hexon protein (8, 41). The failure to block the
virus with the antiknob antibody further demonstrated that
Ad5/FibR7-RGD was not longer dependent on the CAR re-
ceptor for cell attachment and infection. The recombinant
Ad5/FibR7-RGD and Ad5/FibR7-RGD4C viruses may there-
fore be used as gene transfer vectors for cells expressing inte-
grins but not CAR (Fig. 7), thereby broadening the tropism of
Ad vectors. Since the integrin ligand motif RGD is also present
in the penton base (1, 2, 44), it was legitimate to ask to what
extent penton base RGD could contribute to the cellular inte-
grin binding of Ad5/FibR7-RGD, considering that the fibers
are shortened and the RGD in penton base could theoretically
FIG. 6. Protein analysis of virus particles purified by ultracentrifu-
gation in CsCl gradient. Virus samples from CsCl gradient fractions
were analyzed by SDS-PAGE, and viral proteins were electrically
transferred to nitrocellulose membranes. Blots were reacted with an-
tifiber (4D2.5; upper panel) or antihexon (2Hx2; lower panel) antibody.
Lane 1, Ad5/FibR7-RGD (5 ⫻10
5
PFU); lane 2, Ad5/FibWT (5 ⫻10
5
PFU); lane 3, Ad5/FibR7-RGD (5 ⫻10
5
PFU); lane 4, Ad5/FibWT (1 ⫻
10
7
PFU); lanes 5 to 7, Ad5/FibWT at 10
7
,10
6
, and 10
5
PFU, respectively.
Note that lanes 1 and 2 had equal loads of infectious particles (PFU,
as counted on 293 cells), while virus loads in lanes 3 and 4 had been
normalized to equal numbers of physical particles, based on the hexon
protein content (see the hexon signal in the lower panel).
FIG. 7. Susceptibility of HeLa (CAR positive) and RD (CAR neg-
ative) cells to Ad5/FibWT virus (dark columns) and Ad5/FibR7-RGD
recombinant (light columns), as determined by the level of expression
of reporter gene GFP. Cells were infected with an equal multiplicity of
infection (10 PFU/cell) for1hat37°C. The efficiency of Ad5-mediated
gene transfer was estimated by counting the GFP-positive cells by
FACS analysis at 24 h postinfection, and results are expressed as the
percentage of positive, fluorescent cells (mean of three separate ex-
periments ⫾SD).
FIG. 8. Influence of neutralizing antiknob monoclonal antibody
1D6.14 on Ad5/FibWT-mediated (solid line) and Ad5/FibR7-RGD-
mediated (dotted line) gene transfer to HeLa cells. Aliquots of serial
dilutions of antibody were incubated with an equal multiplicity of
infection (10 PFU/cell) of virus for1hat37°C before infection. The
level of inhibitory effect was estimated by counting the GFP-positive
cells by FACS analysis at 24 h postinfection, and results are expressed
as the percentage of positive, fluorescent cells (mean of three separate
experiments ⫾SD).
VOL. 75, 2001 GENETICALLY RETARGETED ADENOVIRUS 7287

protrude approximately to the top of the shortened fiber (36,
42). Although the participation of penton base RGD could not
be excluded, some arguments suggested that the major cell-
binding determinants for Ad5/FibR7-RGD still resided in its
recombinant fiber capsomer. (i) Fiberless virions have been
reported to be far less infectious than WT, with 10,000-fold
difference in infectivity (23). In our case, Ad5/FibR7-RGD and
Ad5/FibWT showed only a 20-fold difference in infectivity. (ii)
When the RGD motif was replaced by the sequence of epi-
dermal growth factor (EGF) in R7 fiber, no viable Ad5/FibR7-
EGF virus could be isolated, although R7-EGF recombinant
fiber self-assembled into trimers which were detected in pen-
ton complexes (Magnusson et al., unpublished data). This sug-
gested that RGD in penton base could not compensate for the
lack of initial cell binding of the virus via the fiber.
As mentioned in the Results, the amount of viral capsid
proteins synthesized after infection with the same multiplicity
of infection (equal number of PFU/cell) was equivalent for
viruses containing the WT fiber and the R7-RGD fiber, al-
though their respective infectivities were significantly different.
Several hypotheses could be proposed to explain the 20-fold
difference in infectivity between Ad5/FibWT and Ad5/FibR7-
RGD viruses. (i) A lower ability for recombinant fibers to
assemble with penton base compared to WT fiber and/or a
lower stability of penton capsomers containing the knobless R7
fibers would be unlikely on the basis of their efficient assembly
with coexpressed recombinant penton base in insect cells (Fig.
2B and C). (ii) Likewise, the recombinant fibers used in our
study were C-terminally truncated, and it has been suggested
that deletion of portions of the fibers could remove some
function(s) necessary for virus maturation (23, 42). However,
we have also constructed R7 fibers with ligands other than
RGD which yield viruses that are as infectious as WT virus
(Strand et al., unpublished data). This suggested that the re-
duction in shaft length to seven repeats does not per se seem
to significantly impair the capacity of the virus to infect cells or
that the influence of shaft length can vary with the ligand used.
(iii) At late times postinfection, Ad5/FibR7-RGD-infected
cells yielded significantly lower levels of R7-RGD fiber than
WT Ad5-infected cells (about 10-fold less), and the Ad5/
FibR7-RGD virus samples contained 10 to 40 times fewer
fibers per virion than WT Ad5 (Fig. 6). The low R7-RGD fiber
content of infected cells was unexpected, since the tail and all
the sequences upstream of the fiber gene were left intact in
order not to disturb the normal regulation of fiber expression.
However, if R7-RGD fiber protein was present in limiting
amounts, this could negatively affect both the equilibrium of
the penton assembly reaction (5) and the overall fiber content
of the virus. The resulting lower number of cell-binding sites in
the recombinant virus would in turn contribute to the lower
infectivity of Ad5/FibR7-RGD. (iv) Even though there was a
direct relationship between the low fiber content of recombi-
nant viruses and the low fiber yields of Ad5/FibR7-RGD-in-
fected cells, the molecular mechanisms behind the latter re-
main unclear. It has been suggested that O-GlcNAc might be
important for Ad2 and Ad5 fiber assembly and stabilization
(26), and a major O-GlcNAc site has been mapped within
residues 61 and 410 of the Ad2 fiber shaft (27). Further dele-
tion mapping analyses have narrowed down the site(s) to
within residues 61 to 260 (13), then to 61 to 216 (35). Our
biochemical and immunological analyses showed that recom-
binant knobless R7-RGD fibers expressed in Sf9 cells and the
ones encapsidated into virions were not glycosylated, suggest-
ing that both lacked a functional O-glycosylation site(s) from
residues M-1 to K-156. Although most of the known functions
assumed by the fiber were still apparently effectuated by our
R7-RGD fiber, it could not be totally excluded that its lack of
O-glycosylation would have altered one of its subtle but critical
biological properties.
(v) Results from a recent comparative study of infectivity of
Ad containing fiber shafts of various lengths suggested that the
observed weak cell attachment of short-shafted fiber-contain-
ing Ad vectors could be due to repulsive acidic charges carried
by the Ad capsid and present on the cell surface (38). (vi)
Another factor that may affect the ability of RGD-targeted
viruses to infect cells is the efficiency of the interaction between
fiber RGD and cellular integrins in promoting virus entry (31).
It has been reported (44) that the interaction between the fiber
and CAR has a 30-fold-higher affinity than the penton-integrin
interaction. Furthermore, the particular context in which the
RGD is presented to integrins might largely influence the
binding (34). It is therefore possible that other RGD-contain-
ing peptides incorporated into the fiber would yield more ef-
ficient or competent viruses than the present ones. A compar-
ison between RGD and RGD4C fibers, which may differ in this
regard, is under way. (vii) In the same line of reasoning, it is
very possible that at the multiplicity of infection used with the
Ad5/FibR7-RGD recombinant, surface integrins were ap-
proaching saturation with the RGD peptide in the fiber. This
would lead to reduced ability of the penton RGD to interact
with another integrin molecule, precluding viral penetration.
Thus, at 10 PFU/cell, equivalent to 5,000 physical particles of
Ad5/FibR7-RGD per cell, and based on a theoretical full copy
number of fiber and penton base subunits in intact Ad5/FibR7-
RGD capsids (i.e., with 12 complete apex structures), a total of
180,000 RGD/cell (5,000 ⫻12 ⫻3) would be presented by the
fiber projections and 300,000 RGD/cell (5,000 ⫻12 ⫻5) would
be presented by the penton base capsomers. (viii) These hy-
potheses are not mutually exclusive, and the factors described
in iii, iv, v, vi, and vii could combine and account for the lower
infectivity of Ad5/FibR7-RGD compared to WT Ad5.
It is our hope that the radical fiber-engineering approach
described in this study will enable insertions of larger ligands
than those that are tolerated by the native fiber knob, for
example, single-chain antibodies and other complex, folded
binding structures. This could be reasonably envisaged, given
that conditions for correct domain folding are maintained in
our recombinant fiber constructs harboring the trimerization
motif NRP. Indeed, in its natural environment within the hu-
man lung surfactant protein D, NRP is bordered by N-terminal
collagen regions and C-terminal C-type lectin domains (17),
two domains which have structural and functional similarities
with the shaft and knob domains, respectively. Our novel ap-
proach to genetic retargeting of Ad could then open up new
possibilities for gene therapy.
ACKNOWLEDGMENTS
The work in Gothenburg (M.K.M. and L.L.) was supported by the
Swedish Medical Research Council (grant no. K98-06X-12624-01), the
Swedish Cancer Society (grant no. 0512-B99-12XCC), and the Inga-
7288 MAGNUSSON ET AL. J. VIROL.

Britt and Arne Lundberg Foundation (grant no. 205/96) and by grants
from Got-A-Gene AB, Gothenburg, Sweden. The work in Lyon
(S.S.H. and P.B.) was financially supported by the French Ministe`re de
la Recherche et de la Technologie (PRFMMIP AO-98) and the
French Foundation for Cystic Fibrosis (Vaincre la Mucoviscidose).
The expert technical contribution of Elisabeth Pettersson and Petra
Strand is gratefully acknowledged. Bert Vogelstein is thanked for the
generous supply of the vectors pAdEasy and pAdTrack.
ADDENDUM IN PROOF
After submission of the manuscript, an article taking a sim-
ilar approach was published by Krasnykh et al. (V. Krasnykh,
N. Belousova, N. Korokhov, G. Mikheeva, and D. T. Curiel, J.
Virol. 75:4176–4183).
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