Impaired Intracellular Trafficking of Adeno-Associated Virus Type 2 Vectors Limits Efficient Transduction of Murine Fibroblasts

Abstract

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly
used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by
AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the
coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third
obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine
fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis.
We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus
is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced
by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications
for the optimal use of these vectors in human gene therapy.

Full text

JOURNAL OF VIROLOGY,
0022-538X/00/$04.00⫹0
Jan. 2000, p. 992–996 Vol. 74, No. 2
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Impaired Intracellular Trafficking of Adeno-Associated Virus
Type 2 Vectors Limits Efficient Transduction of
Murine Fibroblasts
JONATHAN HANSEN,
1,2,3
KEYUN QING,
1,2,3
HYUNG-JOO KWON,
1,2,3
CATHRYN MAH,
4
AND ARUN SRIVASTAVA
1,2,3,5
*
Department of Microbiology and Immunology,
1
Walther Oncology Center,
2
and Division of Hematology/Oncology,
Department of Medicine,
5
Indiana University School of Medicine, and Walther Cancer Institute,
3
Indianapolis,
Indiana 46202, and Gene Therapy Center, University of Florida College of Medicine,
Gainesville, Florida 32610
4
Received 9 September 1999/Accepted 18 October 1999
Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the
more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer
and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target
cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral
second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to
the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV
receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document
that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus
is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently
transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-
mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.
Adeno-associated virus type 2 (AAV) is a nonpathogenic
human parvovirus which requires coinfection with a helper
virus, such as adenovirus, for its optimal replication (3). In the
absence of a helper virus, the wild-type AAV genome inte-
grates in a site-specific way into human chromosome 19 and
establishes a latent infection (14, 15, 27). AAV possesses a
wide host range that transcends the species barrier (18). These
properties of AAV have been instrumental in the development
of recombinant AAV vectors for their use in human gene
therapy (4–6, 18, 33–35, 40). We and others have reported
AAV-mediated transduction and transgene expression both in
vitro and in vivo (1, 2, 9, 10, 12, 13, 17, 19–26, 28, 30, 41).
However, the transduction efficiency of AAV vectors varies
greatly in different cell types. This problem has been attributed
to the following two obstacles that AAV must overcome. First,
AAV must bind to a cellular receptor as well as to a coreceptor
for successful entry into the target cell (19, 21, 24, 36, 37), and
second, because AAV is a single-stranded DNA-containing
virus, the target cell must allow for the conversion of the
single-stranded viral genome to a transcriptionally active dou-
ble-stranded intermediate (7, 8). We have documented the
existence of a host cell protein, which we have designated the
single-stranded D sequence-binding protein (ssD-BP), which
plays a crucial role in viral second-strand DNA synthesis (23,
25). The ssD-BP is phosphorylated at tyrosine residues by
epidermal growth factor receptor protein tyrosine kinase
(EGFR-PTK) activity, and the phosphorylated form of the
ssD-BP prevents viral second-strand DNA synthesis and con-
sequently AAV-mediated transgene expression (16). In this
report, we provide evidence for the existence of a third obsta-
cle, impaired intracellular trafficking into the nucleus, which
AAV must also overcome prior to high-efficiency transduction.
We recently documented that AAV binds to murine NIH
3T3 cells efficiently but that little transgene expression occurs
(24). This observation was interpreted as the inability of AAV
to enter these cells. However, Southern blot analyses (31) of
low-M
r
DNA isolated from NIH 3T3 cells soon after infection
with a recombinant AAV vector containing the human cyto-
megalovirus (CMV) immediate-early gene promoter-driven
bacterial ␤-galactosidase (lacZ) reporter gene revealed the
presence of AAV single-stranded DNA. These results are
shown in Fig. 1A. In these experiments, equivalent numbers of
Raji (nonpermissive for AAV binding and entry) (24), 293
(permissive for viral binding and entry) (23), and NIH 3T3
cells were either mock infected or infected with 10
4
particles/
cell of cesium chloride density gradient-purified recombinant
vCMVp-lacZ vector for2hat37°C, following which cells were
treated with 0.05% trypsin and washed extensively with phos-
phate-buffered saline (PBS) to remove any virus particles ad-
sorbed to cellular receptors in the plasma membrane, and
low-M
r
DNA samples were isolated. Equivalent amounts were
analyzed on Southern blots with a
32
P-labeled DNA probe
specific for lacZ gene sequences as previously described (19,
24). These data suggest that AAV does indeed gain entry into
NIH 3T3 cells. The apparent lack of transgene expression in
NIH 3T3 cells noted previously (24) was not due to the inac-
tivity of the CMV promoter, since abundant expression of the
lacZ gene could be detected in plasmid DNA transfection
experiments (data not shown).
Although treatment of NIH 3T3 cells with tyrphostin 1, a
specific inhibitor of EGFR-PTK (16), led to dephosphorylation
of the ssD-BP as determined by electrophoretic mobility shift
assays (EMSAs), transgene expression in tyrphostin 1-treated
NIH 3T3 cells could not be detected by a cytochemical staining
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, Indiana University School of Medicine, 635
Barnhill Dr., Medical Science Building, Room 257, Indianapolis, IN
46202-5120. Phone: (317) 274-2194. Fax: (317) 274-4090. E-mail:
asrivast@iupui.edu.
992

method (24). EMSAs were performed as previously described
(16, 25). Briefly, 10 ␮g of each whole-cell extract was preincu-
bated with 2 ␮g of poly(dI-dC), 2 ␮g of bovine serum albumin
(BSA), and 12% glycerol in HEPES buffer (pH 7.9) for 10 min
at 25°C. Following preincubation, 10,000 cpm of
32
P-labeled
D(⫺) sequence synthetic oligonucleotide (5⬘-AGGAACCCCT
AGTGATGGAG-3⬘) was added to the reaction mixture and
incubated for 30 min at 25°C. The bound complexes were
separated from the free probe by electrophoresis on 4% poly-
acrylamide gels. The ratio of dephosphorylated to phosphory-
lated forms of ssD-BP was determined by densitometric
scanning of autoradiograms with a Digital Imaging System
Alphaimager (Alpha Innotech Corp., San Leandro, Calif.).
These results are shown in Fig. 1B. Tyrphostin 1 treatment of
NIH 3T3 cells resulted in a change of the ratio of dephospho-
rylated to phosphorylated forms of ssD-BP from 0.8 to 1.2.
Accordingly, when NIH 3T3 cells were infected with the re-
combinant AAV vector (5 ⫻ 10
3
particles/cell) by a more
sensitive assay than cytochemical staining, a low level of AAV-
mediated transgene expression could be detected 48 h later in
these cells treated with higher concentrations of tyrphostin 1.
In these experiments, ␤-galactosidase activity was measured
with the Galacto-Light Plus chemiluminescent reporter assay
(Tropix, Inc., Bedford, Mass.) according to the manufacturer’s
instructions; results were within the linear range. These data,
expressed as relative light units (RLU) per microgram of total
protein, are shown in Fig. 1C. HeLa cells were used as a
positive control in these experiments, since tyrphostin 1 treat-
FIG. 1. (A) Southern blot analysis of viral DNA entry into cells. Equivalent numbers of Raji, 293, and NIH 3T3 cells were either mock infected or infected with
the recombinant vCMVp-lacZ vector (10
4
particles/cell), trypsinized, and washed extensively, and low-M
r
DNA samples were isolated and analyzed with the
32
P-labeled
lacZ DNA probe as described previously (19, 24). ssDNA, viral single-stranded DNA genomes. (B) EMSA for detection of the phosphorylation status of the ssD-BP
in NIH 3T3 cells. Equivalent numbers of cells were either mock treated or treated with the indicated amounts of tyrphostin 1 at 37°C for 2 h. Whole-cell extracts were
prepared and used in EMSAs with the
32
P-labeled single-stranded AAV DNA probe, as described previously (16, 25). Phosphorylated and dephosphorylated forms of
the ssD-BP are denoted by the arrow and the arrowhead, respectively. (C) AAV-mediated transgene expression. Equivalent numbers of HeLa (left) and NIH 3T3
(right) cells were infected with 5 ⫻ 10
3
particles/cell of vCMVp-lacZ and then treated with the indicated amounts of tyrphostin 1 for 2 h under identical conditions.
Forty-eight hours postinfection, ␤-galactosidase activity was determined as described in the text.
VOL. 74, 2000 NOTES 993

ment has previously been shown to significantly augment
AAV-mediated transgene expression in these cells by decreas-
ing the ratio of phosphorylated to dephosphorylated forms of
ssD-BP (16). Whereas there was a dose-dependent increase in
AAV transduction efficiency in both cell types, the extent of
transgene expression was roughly 2 orders of magnitude lower
in NIH 3T3 cells. These results corroborate that AAV does
indeed enter the NIH 3T3 cells.
Entry of the virus into NIH 3T3 cells was further confirmed
with AAV that was fluorescently labeled with Cy3 (Amersham
Life Sciences, Pittsburgh, Pa.) as described previously (2). 293
and NIH 3T3 cells were plated onto polylysine-treated cover-
slips and 24 h later were infected at 37°C with 10
4
particles per
cell of Cy3-labeled AAV in medium containing 0.1% BSA. At
various time points, cells were washed three times with me-
dium containing 0.1% BSA and fixed at 4°C for 20 min with
PBS containing 2% formaldehyde and 0.2% glutaraldehyde.
Cellular nucleic acid was subsequently counterstained with 1
␮M Syto-16 (Molecular Probes, Inc., Eugene, Oreg.) for 30
min at 25°C. Cells were then washed three times with PBS,
mounted on glass slides, and visualized with a Zeiss LSM 510
confocal microscope. A series of 0.3-␮m optical sections were
made through the cells and images representative of the center
of the cells were compared to assess the localization of viral
particles. These results are shown in Fig. 2. It is evident that
within 15 min, AAV could bind efficiently to both 293 (Fig. 2,
panel 1) and NIH 3T3 (panel 3) cells. By 2 h, entry of AAV
into 293 (panel 2) and NIH 3T3 (panel 4) cells was also clearly
seen. As expected, M07e cells, known not to bind AAV due to
lack of expression of one of the coreceptors (16, 21, 24), failed
to bind the fluorescent-labeled virus (data not shown).
Since these data are more qualitative than quantitative and
FIG. 2. Confocal microscopy for localization of AAV particles in 293 and NIH 3T3 cells. At 15 min (panels 1 and 3) and 2 h (panels 2 and 4) postinfection with
10
4
particles/cell of Cy3-labeled AAV, 293 (panels 1 and 2) and NIH 3T3 (panels 3 and 4) cells were visualized as described in the text. Cy3-labeled AAV (red) and
cellular nucleic acids (green) are shown in images representative of the center of the cells. Magnification, ⫻630.
994 NOTES J. VIROL.

do not take into account the kinetics of intracellular trafficking
and uncoating of AAV in the two cell types being compared,
we hypothesized that despite successful entry, a significant
fraction of AAV vectors fails to enter the nucleus in NIH 3T3
cells. This hypothesis was experimentally tested as follows.
Southern blot analyses of low-M
r
DNA isolated at various
times postinfection were carried out with cytoplasmic as well as
nuclear fractions isolated from 293 and NIH 3T3 cells infected
under identical conditions with the CMVp-lacZ vector (10
4
particles/cell) as described above. Nuclear and cytoplasmic
fractions were prepared as described previously (32) from
equivalent numbers of cells, followed by isolation of low-M
r
DNA. These results are shown in Fig. 3A. It is evident that
within 2 h, a substantial amount (⬃76%) of the input single-
stranded AAV DNA is present in the nucleus in 293 cells,
whereas essentially all (⬃99%) of the signal is detected in the
cytoplasm in NIH 3T3 cells, as determined by densitometric
scanning of autoradiographs. By 48 h, a small portion (⬃18%)
of the input AAV DNA does enter the nucleus in NIH 3T3
cells, but the majority (⬃82%) of the signal is still in the
cytoplasm. In contrast, ⬃78% of the input viral DNA is in the
nucleus, and ⬃22% of the signal is in the cytoplasm of 293 cells
48 h postinfection. The purity of each fraction was determined
to be ⬎95%, as measured by the absence of acid phosphatase
activity (in the nuclear fraction) and the absence of histone H3
(in the cytoplasmic fraction), by Western blot analysis with
␣H3 antibody (Upstate Biotechnology, Lake Placid, N.Y.)
(data not shown).
We have previously reported that while AAV binds to HeLa
and KB cells more efficiently than to 293 cells, AAV-mediated
transduction of HeLa and KB cells is significantly lower than
that of 293 cells (23). Therefore, we compared the efficiency of
AAV trafficking into the nucleus of these three cell types.
These results are shown in Fig. 3B. It is clear that despite
less-efficient binding, the extent of AAV entry as well as traf-
ficking into the nucleus in 293 cells is significantly higher than
that in HeLa and KB cells. Thus, in addition to the phosphor-
ylation status of the cellular ssD-BP (23), AAV transduction
efficiency among permissive human cells also correlates well
with the extent of viral trafficking into the nucleus. Taken
together, these studies establish that efficient translocation to
the nucleus is essential for successful transduction of cells by
AAV vectors.
It is interesting to note that although AAV transduction
efficiency of murine cells in general has been reported to be
low (18), the exceptions include the muscle and the brain
tissues (10, 12, 17, 41). We have previously suggested that this
might be due to overabundant expression of fibroblast growth
factor receptor 1, a coreceptor for AAV infection (24), and the
lack of expression of the EGFR, PTK activity of which cata-
lyzes the phosphorylation of the ssD-BP, resulting in failure to
synthesize the viral second-strand DNA (16). It would be of
interest to now examine the kinetics of AAV trafficking into
the nucleus in primary murine cells as well as human cells that
are transduced differentially by AAV vectors, to substantiate
the observations reported here. Indeed, our recent studies
suggest that impaired intracellular trafficking of AAV into the
nucleus in primary murine hematopoietic progenitor cells lim-
its high-efficiency transduction of these cells, both in vitro and
in vivo (39).
Virtually nothing is known about the intracellular trafficking
of AAV particles following infection. However, a wealth of
information is available on the underlying mechanisms of cy-
toplasmic transport and nuclear import of other viruses (11).
For instance, herpesvirus binds to the cellular protein, dynein,
a minus-end-directed motor protein, which transports the viral
particle along microtubules toward the nucleus where the viral
DNA is released through the nuclear pore complex into the
nucleus (29). Adenovirus, on the other hand, first enters the
endosomal pathway and, after acidification of the endosome, is
released into the cytoplasm where it appears to bind microtu-
bules prior to nuclear entry (38). Since both herpesviruses and
adenoviruses provide the helper function for a productive in-
fection by AAV (3, 18) and since both adenovirus and AAV
use ␣V␤5 integrin as a coreceptor (36), it is reasonable to
suggest that trafficking of AAV into the nucleus might be
accomplished by similar mechanisms as those employed by its
helper viruses. Thus, further studies on the mechanisms of
postreceptor entry, transport into the nucleus, and uncoating
of AAV should allow a clearer understanding of molecular
events involved in AAV-mediated high-efficiency transduction
which, in turn, should lead to improvements in the optimal use
of AAV vectors in human gene therapy.
We thank Hal E. Broxmeyer for a critical review of the manuscript
as well as for his support. We also thank Johnny He for his helpful
suggestions.
This research was supported in part by Public Health Service grants
(HL-53586, HL-58881, and DK-49218; Centers of Excellence in Mo-
lecular Hematology) from the National Institutes of Health and a
grant from the Phi Beta Psi Sorority.
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