JOURNAL OF VIROLOGY, Mar. 2009, p. 2632–2644
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 6
Enhancement of Adeno-Associated Virus Infection by Mobilizing
Capsids into and Out of the Nucleolus?
Jarrod S. Johnson and R. Jude Samulski*
Gene Therapy Center and Department of Pharmacology, The University of North Carolina at Chapel Hill,
7119 Thurston Bowles, CB 7352, Chapel Hill, North Carolina 27599-7352
Received 4 November 2008/Accepted 18 December 2008
Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the
parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain
unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate
of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection anal-
ysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and
accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remark-
ably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in
secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar
accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleo-
plasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering
RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin,
previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects
observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar
accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of
nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting
both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction
over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact
and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites
likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these
studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular param-
eters that mobilize virions in the nucleus and significantly influence AAV infection.
Adeno-associated virus (AAV) is classified as a dependovi-
rus because it requires the presence of a helper virus, such as
adenovirus or herpesvirus, in order to enter into a productive
lytic cycle (6). Because of its nonpathogenicity and ability to
promote sustained, long-term transgene expression in a wide
variety of tissues such as the brain, liver, muscle, retina, and
vasculature (51), several recombinant AAV (rAAV) serotypes
are emerging as attractive vectors for gene therapy. Despite
many advances in AAV vector design, barriers such as a pre-
existing immune response and off-target binding have necessi-
tated administration of high viral titers to achieve efficient
transduction (24, 51).
Beyond the barriers of the immune response (9, 42) and cell
surface targeting (52), researchers are becoming increasingly
aware that subcellular processing is a significant barrier to
infection (16, 29, 52). Subcellular processing may include con-
formational changes within the endosome or similar compart-
ments, endosomal escape, nuclear targeting, and uncoating,
but the factors that control these events are not well defined.
Understanding how cellular conditions affect subcellular pro-
cessing of virions will lead to improved gene delivery through
exploitation of these parameters and promote better vector
Given that the virion is an icosahedral particle only 25 nm in
diameter, rAAV must contain all of the molecular components
required to navigate through the subcellular environment in a
remarkably small structure. Wild-type AAV is a nonenveloped
parvovirus that packages a single-stranded DNA genome of
approximately 4.7 kb in length. The viral genome is flanked by
two inverted terminal repeats and contains two open reading
frames, one that codes for replication proteins and another
that codes for capsid proteins. Three capsid proteins (VP1,
VP2, and VP3) are encoded in the second overlapping reading
frame, each beginning with a different start codon but sharing
a common C terminus and stop codon. Capsids are comprised
of 60 copies of V1, VP2, and VP3 in a ratio of approximately
1:1:10, respectively (11, 43). During production, AAV capsids
are known to assemble at early time points in the nucleolus
(64), a subdomain of the nucleus and one of the oldest known
cellular structures. Intact capsids have been shown to interact
with nucleolar proteins such as nucleolin (NCL) and B23/
nucleophosmin (NPM1) in the context of assembly (8, 46), but
how these proteins affect infection or vector delivery is cur-
Initial cell surface binding of AAV capsids is mediated by
expression of glycoprotein receptors and specified by residues
in VP3 (45, 58, 59). After binding receptors on the host cell
plasma membrane, AAV serotype 2 (AAV2) is endocytosed
* Corresponding author. Mailing address: CB 7352, Gene Therapy
Center, 7113 Thurston Building, The University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599-7352. Phone: (919) 962-3285. Fax:
(919) 966-0907. E-mail: email@example.com.
?Published ahead of print on 24 December 2008.
from the cell surface in a clathrin- and dynamin-dependent
process (3, 5, 19). Following endocytosis, many AAV particles
accumulate in late endosomes, lysosomes, or other compart-
ments and do not deliver their genome to the nucleus (17).
This impediment to gene delivery is exacerbated when parti-
cles lack VP1 or contain specific mutations in the unique N
terminus of VP1 (23). The N terminus of VP1 is normally
folded inside the capsid, harboring a phospholipase domain
and putative nuclear localization signals necessary for infection
(13, 23, 74). These regions of VP1 are thought to translocate to
the capsid exterior during subcellular processing of the virus
(10, 35, 57). Even with proper capsid composition, the vast
majority of internalized particles remain clearly outside the
nuclear membrane, and although recent evidence suggests that
successful infection occurs when the capsid uncoats inside the
nucleus (57, 61), whether AAV can enter the nucleus as an
intact capsid is still vehemently debated.
In general, it has proven difficult to discern whether infec-
tious particles truly cross the nuclear membrane, due to the
limitations of fluorescence microscopy (5, 67). In an in vitro
setting it has been demonstrated that unmodified AAV capsids
are capable of entering purified nuclei (28), yet these condi-
tions do not accurately represent what occurs physiologically,
since virus directly microinjected into cytoplasm will not enter
the nucleus or efficiently transduce the cell (17, 57). In one
instance, single-particle tracking of AAV has been used to
follow capsids in a live-cell imaging paradigm and has found
that they can be quickly and directly transported to the nucleus
(54). However, another recent study has parsed confocal im-
ages of green fluorescent protein-tagged AAV2 particles dur-
ing infection and has reported that few if any particles enter
the nucleus during infection (38).
Although it is unclear whether capsids enter the nucleus
intact, it has been well established that nuclear delivery of the
genome is highly inefficient and significantly limits transduc-
tion. Several studies have identified agents that surmount sub-
cellular barriers to transduction (20, 22, 69). Two of the most
well-documented agents known to improve subcellular pro-
cessing are proteasome inhibitors and hydroxyurea (HU); how-
ever, their mechanisms of action remain unknown. Therefore,
we set out to determine what effect, if any, these agents had on
subcellular trafficking of rAAV2 in the hope of identifying
specific cellular parameters that promote efficient transduc-
Here we report that rAAV2 capsids accumulate in the nu-
cleolus during infection. Proteasome inhibitors were found to
potentiate nucleolar accumulation, while HU reduced nucleo-
lar accumulation and appeared to mobilize capsids to the nu-
cleoplasm. Acting independently, both proteasome inhibitors
and HU increased transduction, and together they were coop-
erative, which suggests that these treatments operate through
separate pathways to improve gene delivery. In addition, we
found that small interfering RNA (siRNA) knockdown of nu-
cleolar proteins NCL and NPM1 had effects similar to those of
proteasome inhibition or HU and increased transduction.
Based on our results, we have proposed a model wherein AAV
virions initially enter the nucleus intact and can be sequestered
in the nucleolus in stable form. Disruption of the nucleolus
subsequently mobilizes virions from the nucleolus to nucleo-
plasmic sites and likely permits uncoating.
MATERIALS AND METHODS
Cell culture. HeLa cells and HEK-293 cells were maintained at 37°C and 5%
CO2in Dulbecco’s modified Eagle’s medium that was supplemented with 10%
heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin.
Virus production. Virus was produced in HEK-293 cells as previously de-
scribed (68). Briefly, using polyethylenimine (linear molecular weight, ?25,000),
cells were triple transfected with pXR2, the pXX6-80 helper plasmid, and pTR-
CMV-luciferase containing the luciferase reporter transgene flanked by inverted
terminal repeats. At 60 h posttransfection, cells were harvested and virus purified
by cesium chloride gradient density centrifugation for 5 h at 65,000 rpm or
overnight at 55,000 rpm. Fractions that contained peak virus titers were dialyzed
against 1? phosphate-buffered saline (PBS) supplemented with calcium and
magnesium. Viral titers were determined in triplicate after treating dialyzed
fractions with DNase, digesting the capsid with proteinase K, and purifying viral
DNA by use of a DNeasy column (Qiagen) according to the manufacturer’s
protocol. Viral DNA was applied via a dot blot manifold to a Hybond-XL
membrane (Amersham) and detected with a [?-32P]CTP-labeled probe comple-
mentary to the luciferase transgene. Empty capsids were harvested from HEK-
293 cells after transfection of only pXR2 and XX6-80 without a transgene
plasmid and dialyzing from low-density cesium chloride fractions that displayed
peak monoclonal antibody (MAb) A20 reactivity. For this study, consistent
results were obtained for many different virus preparations and could also be
reproduced when virus was purified by use of an iodixanol gradient.
Confocal immunofluorescence microscopy. Similarly to what we have previ-
ously described (25), HeLa cells (5 ? 104/well) were plated on poly-L-lysine-
coated 12-mm glass coverslips (no. 1.5) at 24 h before infection. Recombinant
virions were added to cell media (2 ? 104vector genomes [vg]/cell). No virus was
added to control wells. Where indicated, HU was added at 10 mM, left for 12 h,
and washed off extensively prior to virus administration. A proteasome inhibitor
(MG132; Calbiochem) was present for the duration of infection at 2 ?M, when
used. At the indicated time points, cells were washed three times with PBS and
then fixed with 2% paraformaldehyde for 15 min at room temperature. The cells
were then permeabilized with 0.1% Triton X-100 in PBS for 5 min at room
temperature. Following four washes with PBS, the permeabilized cells were
blocked with immunofluorescence buffer (IFB) (20 mM Tris [pH 7.5], 137 mM
NaCl, 3 mM KCl, 1.5 mM MgCl2, 5 mg/ml bovine serum albumin, 0.05% Tween)
for 30 min at room temperature. The cells were incubated with primary antibody
to detect intact capsids (MAb A20 [1:10], NCL [Abcam 22758, 1:1,000], or
laminB1 [Abcam 16048, 1:750]), diluted in IFB, for 1 h at 37°C or overnight at
4°C. The cells were then incubated in secondary antibody, diluted 1:5,000 in IFB
(anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 568 [Molecular Probes]),
for 1 h at 37°C. After six washes in PBS, coverslips were mounted cell side down
on glass slides with mounting medium (Prolong antifade Gold with DAPI [4?,6?-
diamidino-2-phenylindole]; Molecular Probes). Images were captured on a Leica
SP2 AOBS upright laser scanning confocal microscope and processed using
Three-dimensional rendering of infection. Confocal z-stack sections of HeLa
cells fixed 16 h after infection with either rAAV2 or empty capsids in the
presence of 2 ?M MG132 were processed and rendered in three dimensions
using Volocity software (Improvision).
Cell fractionation and nucleolar isolation. Nucleoli were isolated from cell
fractionations as previously described (41), with minor modifications allowing for
viral infection. Briefly, five 15-cm plates of HeLa cells at 90% confluence were
used for each preparation. Empty or full rAAV2 particles were incubated with
cells for 16 h at 37°C (2,000 vg/cell, with empty capsids normalized to A20
reactivity of full capsids). Cells were washed three times with ice-cold PBS and
harvested by centrifugation at 218 ? g for 5 min. The cell pellet was resuspended
in hypotonic buffer and homogenized on ice using a tight pestle to the point
where ?90% of cells were burst and leaving nuclei intact. The homogenate was
spun at 218 ? g for 5 min to separate the crude nuclear pellet from the post-
nuclear supernatant (PNS, ?5 ml). Nuclei were further purified from cytoplas-
mic contaminants by spinning through a 0.35 M sucrose cushion to give a
nucleus-associated (NA) pellet. Nuclei were lysed by limited sonication on ice,
and the nuclear suspension in 0.35 M sucrose was layered over a 0.88 M sucrose
cushion to separate nucleolar (NU) fractions (200 ?l) from NA fractions (?5
ml). Nucleoli were further purified by another spin through sucrose, and their
integrity and purity were verified by phase-contrast microscopy and Western
Transduction assays. At least 4 h prior to infection or drug treatment, HeLa
cells were plated in 24-well plates at a density of 105cells/well. For drug studies,
cells were handled as stated above. Cells were infected with purified rAAV2 at
the designated number of vector genomes per cell or 5 ?l of the indicated
VOL. 83, 2009 SUBNUCLEAR MOBILIZATION OF rAAV2633
ways. Transduction is most dramatically potentiated under
conditions where more than one pathway is engaged to over-
come subcellular barriers, such as in the case of MG132 and
HU cooperation or synergy (Fig. 5 and 7). It remains to be
seen whether nucleolar trafficking is an obligatory step in the
cascade of infectious events, yet it is clear that by disrupting the
integrity of the nucleolus using genotoxic agents or by affecting
the expression and localization of nucleolar proteins, transduc-
tion can be improved. In summary, with these studies we have
refined our understanding of AAV2 trafficking dynamics, sep-
arating two pathways that accumulate or mobilize virions in the
nucleus and significantly enhance AAV infection.
We thank members of the Samulski lab for productive discussions
and Aravind Asokan for critically reading the manuscript. We thank
Nina DiPrimio for help with generating empty rAAV2 capsids used in
this study, and we thank Swati Yadav for calculating titers by quanti-
tative PCR. Microscopy equipment and analysis software used in this
study were provided courtesy of the Michael Hooker Microscopy Fa-
This research was supported by NIH fellowships 1F31NS060688-
01A1 and 5F31NS060688-02.
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