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: firstname.lastname@example.org.
?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
samples after cell fractionation and were typically harvested after 24 h unless
otherwise noted. Luciferase activity was measured in accordance with the man-
ufacturer’s instructions (Promega). Luciferase activity was measured with a Wal-
lac 1420 Victor2 plate reader. Error bars in the figures represent standard
deviations from samples scored in triplicate. Graphs are for representative data
sets from at least three independent assays.
Western blotting. Samples from purified virus or cell fractionations were
loaded onto NuPage 10% bis-Tris gels and typically run for 3 h at 150 V in 1?
NuPage MOPS (morpholinepropanesulfonic acid) buffer. Electrophoresis was
performed in an XCell SureLock minicell (Invitrogen). Proteins were transferred
to a Hybond ECL membrane utilizing the XCell II Blot module (Invitrogen) for
wet transfer according to the manufacturer’s protocol. Membranes were blocked
for at least 30 min at room temperature in 5% nonfat dry milk (NFDM) dis-
solved in 1? PBS–Tween (0.1%). For detection of capsid proteins, primary
antibody (MAb B1) was diluted 1:20 with 2.5% NFDM in 1? PBS–Tween
(0.1%) and incubated for 1 h at room temperature or overnight at 4°C. After
washing with 1? PBS–Tween (0.1%), blots were incubated for 1 h at room
temperature with anti-mouse–horseradish peroxidase secondary antibody, di-
luted 1:5,000 in 2.5% NFDM with 1? PBS–Tween (0.1%). Following multiple
washes with 1? PBS–Tween (0.1%), SuperSignal West Femto Maximum Sensi-
tivity substrate (Pierce) was added to each membrane according to the manu-
facturer’s protocol for developing. Each membrane was then exposed to Amer-
sham Hyperfilm MP.
Quantifying genomes by PCR. Identically to methods stated earlier, cells were
fractionated into PNS, NA, and NU fractions as previously described (41). DNA
was isolated from fractions using DNeasy columns (Qiagen) according to the
manufacturer’s protocol. Quantitative PCR was performed on a LightCycler 480
using Sybr green (Roche) and primers designed against the firefly luciferase
transgene: 5? AAA AGC ACT CTG ATT GAC AAA TAC 3? (forward) and 5?
CCT TCG CTT CAA AAA ATG GAA C 3? (reverse). Conditions used for the
reaction were as follows: 1 cycle at 95°C for 10 min; 45 cycles at 95°C for 10 s,
62°C for 10 s, and 72°C for 10 s; and 1 cycle at 95°C for 30 s, 65°C for 1 min, and
99°C for acquisition.
Genome pulse-chase. Confluent 10-cm plates of HeLa cells were pulsed with
rAAV2 (2,000 vg/cell) for 2 h at 37°C, washed with medium, and harvested at 0,
12, and 24 h postinfection in 5 ml PBS. DNA from 200 ?l of the cell suspension
was purified using DNeasy columns and applied to a Hybond XL membrane
using a dot blot manifold. DNA specific to the viral genome was detected as
mentioned above by hybridization of a radiolabeled probe created against the
siRNA transfection. Knockdown of NCL, NPM1, and vallosin-containing pro-
tein (VCP) was obtained by transfecting validated siRNA sequences using Hip-
erfect (Qiagen) according to the manufacturers’ protocol for fast-forward trans-
fection of adherent cells. Briefly, 80,000 HeLa cells were plated into 24well plates
and mixed immediately with preincubated Dulbecco’s modified Eagle’s medium,
transfection reagent, and siRNA at a final concentration of 5 nM. Two days after
transfection, cells were trypsinized and replated at appropriate densities for
transduction assays or confocal immunofluorescence. Target sequences were as
follows: Hs_NPM1_7, 5? AAA GGT GGT TCT CTT CCC AAA 3?;
HS_NPM1_8, 5? AAT GTC TGT ACA GCC AAC GGT 3?; Hs_VCP_6, 5? AAG
ATG GAT CTC ATT GAC CTA 3?; Hs_VCP_7, 5? AAC AGC CAT TCT CAA
ACA GAA 3?; Hs_NCL_10, 5? AAG GAA ATG GCC AAA CAG AAA 3?; and
Hs_NCL_5, 5? AAG CTA TGG AGA CTA CAC CAG 3?.
Identifying and overcoming subcellular barriers that prevent
the majority of virions from delivering their payload to the
nucleus are critical steps toward developing successful vectors
for gene therapy applications. As previously reported, protea-
some inhibitors and genotoxic agents affect events of subcel-
lular processing to enhance transduction of rAAV2 in a cell
type- and serotype-specific manner (16, 20, 27, 29). The pro-
teasome inhibitor MG132 is known to reduce the rate of deg-
radation of ubiquitin-conjugated proteins by the 26S complex
of the proteasome. In contrast, the primary target of the geno-
toxic agent HU is thought to be ribonucleotide reductase. It is
unclear precisely how transduction of rAAV is enhanced fol-
lowing these treatments, but MG132 and HU are postulated to
increase efficiency of nuclear entry (20, 22, 69). Here we dem-
onstrate that the aforementioned pharmacological agents can
regulate accumulation and mobilization of intact AAV virions
in the nucleus following infection using a variety of techniques.
Pharmacological modulation of rAAV2 trafficking and
transduction using MG132 and HU. To establish the traffick-
ing profile of rAAV2, we tracked the subcellular fate of virions
by confocal immunofluorescence microscopy. Using a MAb
(MAb A20) that recognizes only intact capsids, we were able to
identify virions within the nucleus by 16 h after infection when
virus was continuously present in the medium (Fig. 1A). The
distinct pattern of accumulation that we observed for rAAV2
with no drug treatment was potentiated when MG132 was
present. Capsids could be detected in the nucleus as early as
4 h after infection, which corresponds to the earliest time that
we could detect gene expression. A markedly different pattern
was observed during infection following HU pretreatment,
with capsids showing a diffuse distribution in the nucleoplasm.
At 16 and 24 h, MG132 and HU had clearly contrasting effects
on capsid localization in the nucleus. This finding was intrigu-
ing considering that both MG132 and HU increase infection
efficiency over a wide range of particle numbers (Fig. 1B to D),
as measured by luciferase assays of transduction. HU increased
transduction from 5- to 10-fold, and MG132 increased trans-
duction by almost 2 log orders of magnitude.
It is noteworthy that despite the need for high particle num-
bers (20,000 vg/cell) in immunofluorescence studies using
AAV (38, 57, 67), a linear relationship between transduction
and dose was observed (Fig. 1B to D). This supports the notion
that similar trafficking patterns are occurring at a low multi-
plicity of infection (MOI) (200 vg/cell), despite these doses
being below the limit of visual detection. Nonetheless, the
striking nuclear accumulation pattern that we observed with
proteasome inhibition in immunofluorescence studies prompted
Recombinant AAV2 virions, but not empty capsids, accumu-
late in nucleoli. The nucleolus has been termed the gateway to
viral infection because of its seemingly ubiquitous involvement
in the replication dynamics of many viruses (31). For wild-type
AAV2, capsid assembly has been reported to occur in nucleoli
(8, 46, 64), but the roles of the nucleolus or nucleolar proteins
during infection of an exogenously applied vector have not
been studied. To more clearly determine whether rAAV cap-
sids accumulate in the nucleolus, we carried out immunofluo-
rescence experiments to identify intact capsids and the nucle-
olar marker NCL at 16 h after infection with 20,000 vg/cell in
the presence of MG132 (Fig. 2A). Cross-sectional analysis of
z-stacked confocal images shows that rAAV2 colocalizes with
NCL in nucleoli, although the majority of virions remain out-
side the nuclear membrane.
Remarkably, in contrast to rAAV2 virions, empty capsids
were not found to accumulate in nucleoli or to colocalize with
NCL, even in the presence of MG132. Only with samples
where DNA-containing particles were administered were we
able to detect nucleolar accumulation (Fig. 2A versus B) at any
time point after infection, despite similar levels of internaliza-
tion for rAAV2 and empty capsids as indicated by A20 stain-
ing. Nucleolar accumulation of rAAV2 could also be poten-
tiated by another proteasome inhibitor, ALLN, and was
2634 JOHNSON AND SAMULSKIJ. VIROL.
demonstrated in HEK-293 cells (data not shown), suggesting
that this phenomenon is not unique to one cell type.
Additionally, we were able to explore nucleolar accumula-
tion of rAAV2 in three dimensions by rendering z-stacked
images acquired by confocal microscopy using Volocity soft-
ware (Fig. 2C). This enabled us to digitally subtract the nucleus
by channel gating to reveal more precisely the localization of
capsids in its interior. Detection of NCL was observed diffusely
in the nucleoplasm (Fig. 2C, ii and vi), and when the channel
was gated to show only intense staining, it was found confined
to nucleoli (Fig. 2C, iii and vii). After completely subtracting
the NCL channel, a collection of virions localized to the nu-
cleolar interior could be clearly seen for rAAV2 (Fig. 2C, iv),
yet empty capsids were absent from these regions (Fig. 2C,
Capsid proteins are found in nucleolar fractions. To further
support the evidence from immunostaining that rAAV2 viri-
ons are capable of accumulating in the nucleolus, we in-
fected cells and attempted to isolate nucleoli containing
virus by subcellular fractionation. To this end, we infected
HeLa cells with 2,000 vg/cell rAAV2 or an equivalent
amount of empty capsids for 16 h in the presence of MG132.
Cells were fractionated into PNS, which contained mostly
cytoplasmic material; an NA fraction, which may have in-
cluded material attached to the outside of the nuclear mem-
brane; and a fraction enriched with highly purified nucleoli.
When analyzed by Western blotting, capsid proteins from
rAAV2 and empty capsids were represented at similar levels
in NA fractions (diluted 25-fold over fraction NU [Fig. 2B]).
Identical to what was found with immunostaining, a popu-
lation of capsids or capsid proteins was detected in NU
fractions after rAAV2 infection but not after empty capsid
infection. The fact that empty capsids could not be detected
in nucleoli by immunostaining or subcellular fractionation
suggests that accumulation of rAAV2 in the nucleus is not
an artifact but rather represents a property unique to ge-
FIG. 1. Trafficking and transduction after treatment with MG132 or HU. (A) Confocal immunofluorescence microscopy was used to visualize
subcellular trafficking of intact virions in HeLa cells with MAb A20 (yellow) with reference to the nuclear membrane marker lamin B1 (magenta).
rAAV2 virions (20,000 vg/cell) were administered for the indicated times under control conditions, in the presence of MG132 (2 ?M), or following
HU pretreatment (10 mM, 12 h). Nucleolar accumulation and diffuse nucleoplasmic localization are marked with white arrows and arrowheads,
respectively. The scale bar represents 20 ?m. (B) Luciferase assays of transduction were performed to quantify efficiency of firefly luciferase
transgene delivery. Two hundred, 2,000, or 20,000 vg/cell was administered for 2 h at 37°C, and cells were washed three times with cell medium,
harvested at the indicated times, and scored for transduction after 24 h. RLUs, relative light units.
VOL. 83, 2009 SUBNUCLEAR MOBILIZATION OF rAAV2635
nome-containing rAAV2 that is not demonstrated by empty
capsids (see Discussion).
Since the localization of intact capsids or capsid protein may
not reflect the localization of genomes if virions have already
uncoated, we chose to quantify relative amounts of capsid
proteins and genomes after subcellular fractionation. We have
also analyzed whether proteasome inhibition or HU treatment
affects capsid and genome localization using subcellular frac-
tionation, since these treatments appear to have different ef-
fects as observed by immunofluorescence. It has been reported
that uncoating is a slow and inefficient process for rAAV2 (56,
61, 75), so we predicted that the amount of genomes in nucle-
olar fractions should roughly correspond to the amount of
capsid protein detected in those fractions if the majority of
virions have not released their DNA. As expected, MG132
increased the amount of capsid protein associated with frac-
tions NA and NU (Fig. 3A, lanes 7 and 11). In contrast, HU
reduced the amount of capsid protein most notably in fraction
NU (lane 12), relative to untreated control infection (lane 10).
These findings further support our immunofluorescence data
that suggest proteasome inhibitors increase nucleolar accumu-
lation of capsids whereas HU mobilizes capsids into the nu-
cleoplasm, away from the nucleolus.
Vector genomes can be detected in isolated nucleoli. Quan-
tifying the number of genomes in these locations is just as
important in analyzing capsid distribution, since capsids could
have released their genomes prior to this point in trafficking.
The nucleolus may either be a site where potentially infectious
virions are sequestered given certain cellular conditions or
represent a site for depositing empty capsids after genome
release that would have no bearing on transduction efficiency.
To determine if genomes could be detected in fraction PNS,
NA, or NU, we purified DNA from these fractions after in-
fecting cells and subjected the samples to quantitative PCR.
FIG. 2. Accumulation in the nucleolus is unique to rAAV2. (A) Confocal z-stack analysis of infection was used to determine if the immuno-
fluorescent signal originated from within the nucleus. Serial cross-sections were captured in the z-plane through HeLa cells infected for 16 h in
the presence of MG132 (2 ?M) with either rAAV2 (i) (20,000 vg/cell) or empty capsids (ii) (equivalent amount relative to A20 and B1 staining).
Intact rAAV2 virions (yellow) were found to colocalize with nucleolar marker NCL (magenta) and are depicted within the nucleus (blue, DAPI).
(B) Subcellular fractionation of HeLa cells after infection (2,000 vg/cell rAAV2 or equivalent empty capsid amount). Capsid proteins present in
the PNS, NA, and NU cell fractions were detected by Western blotting (WB) (MAb B1). The PNS and NA fractions were diluted 25-fold over the
NU fraction. Nucleolar isolation was verified by the presence of NPM1 by Western blotting and observing purified nucleoli as dense retractile
bodies by phase-contrast microscopy (not shown). (C) Three-dimensional rendering of infection in panel A, as processed by Volocity software
(Improvision). The nucleus (blue channel) was gated to reveal nucleoplasmic staining of NCL (magenta) (ii and vi), focal nucleoli (iii and vii), and
then the presence or absence of rAAV2 and empty capsids within nucleoli (yellow) (iv and viii). White arrows depict nucleolar accumulation.
2636 JOHNSON AND SAMULSKI J. VIROL.
The trends observed for genome copy number (Fig. 3B) closely
follow those observed for capsid protein levels, with more
genomes detected in fraction NU following proteasome inhi-
bition (Fig. 3B, lane 11) and fewer detected after HU treat-
ment (lane 12), compared to an untreated control infection
(lane 10). It should be noted that fraction NU contains highly
purified nucleoli and during the isolation is concentrated roughly
25-fold over fractions PNS and NA. Thus, it is important to
realize that the majority of capsids and genomes in the cell do not
enter the nucleolus. Since most virions are likely associated with
the outside of the nuclear membrane, nuclear entry is an ineffi-
cient process even in the presence of proteasome inhibitors.
Virions accumulating in nucleoli remain infectious entities.
Detecting viral capsids and viral genomes in the same subcel-
lular compartment does not necessitate that the former still
carry the latter. When these are observed to accumulate in the
nucleolus, the capsid could have remained intact after already
releasing its genome or the capsid could still contain the ge-
nome if the virus has not yet completed infection. To test
whether vector genomes detected in the nucleolus were pro-
tected in intact capsids or had been released during capsid
uncoating, we assayed whether virions isolated from nucleoli
after infection could successfully transduce cells in secondary
infections (Fig. 4A). In one condition, rAAV2 (2000 vg/cell)
was spiked into HeLa cell medium for roughly 1 min. This
short incubation period is not long enough for the majority of
virions to bind and internalize into cells. As shown in Fig. 4A,
condition 1 serves as an internal control to verify that the
procedure is not vulnerable to contamination from infectious
particles in the medium during wash steps and nucleolar frac-
tionation. Conditions 2 and 3 were 16-h infections (2,000 vg/
cell) with rAAV2 or rAAV2 plus MG132, respectively. This
time interval was chosen based on the immunofluorescence
evidence that prominent nucleolar accumulation is seen by
16 h after infection (Fig. 1A).
FIG. 3. Detection of capsid proteins and viral genomes in nucleoli
after subcellular fractionation. (A) Subcellular fractionation of HeLa
cells after no infection (lanes 1, 5, and 9), infection with rAAV2 at
2,000 vg/cell (lanes 2, 6, and 10), rAAV2 with Mg132 (lanes 3, 7, and
11), or rAAV2 after HU treatment (lanes 4, 8, and 12). Cells were
fractionated after 16 h into PNS, NA, and NU fractions. Capsid pro-
teins in fractions were detected by Western blotting (WB) (MAb B1).
Nucleolar isolation is indicated by the presence of NPM1 in the NU
fraction. (B) Quantification of viral genomes present in cell fractions
after infection by quantitative PCR (same sample order as for panel
A). Amplification was performed using Sybr green with primers de-
signed for the luciferase transgene.
FIG. 4. Secondary infection after nucleolar fractionation. (A) Ex-
perimental schematic representing three conditions of infection: 1,
control with virus present for only 1 min in the cell medium; 2, 16 h of
infection with rAAV2 (2,000 vg/cell); and 3, 16 h infection in the
presence of MG132 (2 ?M). Cells treated in this manner were pro-
cessed to separate the PNS, NA, and NU fractions. (B) To determine
if virions accumulating in these fractions remained infectious, a lucif-
erase assay of transduction was performed 24 h after 5-?l aliquots from
the designated fractions were administered to fresh HeLa cells. Parti-
cle numbers in samples NU-2 and NU-3 were determined to be similar
to those of purified rAAV2 (first bar, 10,000 vg/cell), with NU-3 being
slightly higher than NU-2. As a control, purified rAAV2 (10,000 vg/
cell) and samples NU-2 and NU-3 were subjected to heat treatment
(70°C, 10 min) to destroy capsid integrity. RLUs, relative light units.
VOL. 83, 2009SUBNUCLEAR MOBILIZATION OF rAAV2637
After the primary infection under the conditions listed
above, HeLa cells were fractionated into PNS, NA, and NU
fractions (Fig. 4A). To determine if virions accumulating in
these fractions remained infectious, small volumes of these
fractions were administered to fresh HeLa cells and transduc-
tion was scored 24 h later by luciferase assay. Compared to
purified rAAV2 (10,000 vg/cell), negligible levels of transduc-
tion were observed from the PNS fraction for all conditions,
from the NA fraction for condition 1, or from the NU fraction
from condition 1 (Fig. 4B). Low transduction was observed in
the NA fraction for conditions 2 and 3, and moderate trans-
duction was detected with the NU fraction for conditions 2 and
3. When these samples for conditions 2 and 3 from the NU
fraction were heated to 70°C for 10 min to disassemble capsids,
transduction levels dropped to baseline, as they did for heat-
treated purified rAAV2. These data strongly suggest that cap-
sids accumulating in nucleoli during infection still contain their
genomes and remain infectious entities.
Separate pathways can be exploited to enhance transduc-
tion. The central tenet that inefficient subcellular processing of
rAAV significantly reduces transduction has driven research
toward discovering ways to enhance processing and subcellular
trafficking of the virus. To date, no effect on capsid degradation
has been observed during proteasome inhibition, despite cap-
sid ubiquitination being demonstrated in vitro (20, 70). Addi-
tionally, knowing the total amount of virion accumulation in
cells treated with MG132 or HU would establish a baseline for
comparing the effects of each treatment. To determine if pro-
teasome inhibitors or genotoxic agents affect the amount of
vector genomes internalized and to follow how these drugs
affect the persistence of rAAV2 DNA, we performed a viral
genome pulse-chase to compare control, MG132, and HU
conditions. Cells were infected for 2 h at 37°C, washed with
medium, and harvested at 0, 12, and 24 h postinfection. The
remaining viral genomes were purified and detected by dot blot
hybridization (Fig. 5A). As previously documented for AAV2
(18), proteasome inhibition slows clearance of viral genomes
compared to untreated control samples, though the mecha-
nism of action remains unknown. Degradation of viral ge-
nomes is not impeded in samples pretreated with HU, as the
pattern of genome disappearance closely matches that of con-
Both MG132 and HU are pluripotent agents and have nu-
merous downstream effects, but since these drugs are thought
to act through different pharmacological mechanisms, we hy-
pothesized that together they may act synergistically to in-
crease rAAV transduction. Thus, we performed a luciferase
assay of transduction, comparing the effects of MG132, HU, or
the combination of the two to determine if they operate
through separate pathways. As expected, when combined,
MG132 and HU enhance transduction more than either one
independently (Fig. 5B). It should be noted that in these ex-
periments the concentration of MG132 was reduced twofold
compared to concentrations used in earlier assays, since some
toxicity was observed when it was combined with HU. These
results further corroborate the ability of MG132 and HU to act
through independent mechanisms that manifest as enhanced
nucleolar accumulation and nucleoplasmic mobilization of
AAV capsids, respectively.
Influence of nucleolar proteins on rAAV transduction and
localization. The fact that AAV capsids are known to assemble
in the nucleolus during replication has important ramifications
when considering steps leading to nucleolar accumulation dur-
ing infection (64). Two reports have identified nucleolar pro-
teins that colocalize and immunoprecipitate with intact capsids
(8, 46). These proteins, NCL and NPM1, are predominant
components of the nucleolus. While traditionally known for
their roles in ribosome assembly, they have also been found to
exist outside of the nucleolus, having roles as chaperone pro-
teins, and are known to relocate in response to stresses such as
HU. It is clear that NCL and NPM1 are implicated in the life
cycles of many viruses in addition to AAV, including human
immunodeficiency virus, herpes simplex virus, and coronavirus
(31). Yet, the impact of such proteins on AAV infection has
not been established.
To test whether NCL and NPM1, which bind the AAV
capsid during assembly, can also sequester virions in the nu-
cleolus after nuclear entry postinfection, we knocked down
NCL and NPM1 by transfection of corresponding siRNA in
HeLa cells. Significant reductions of targeted protein levels
were verified by Western blot analysis relative to ?-actin load-
ing controls (Fig. 6A). When transduction efficiency was com-
FIG. 5. MG132 and HU increase transduction through different
mechanisms. (A) Genome pulse-chase experiments were performed to
determine whether MG132 or HU influences total levels of vector
genome accumulation and to examine the effects of these drugs on
genome degradation. Cells were pulsed with rAAV2 (2,000 vg/cell) for
2 h at 37°C, washed with medium, and harvested at 0, 12, and 24 h
postinfection. Viral DNA was purified from the cell suspensions and
detected by dot blot hybridization using a radiolabeled probe created
against the luciferase transgene. (B) A luciferase assay of transduction
with rAAV2 (2,000 vg/cell) was performed to test for synergistic trans-
duction enhancement of MG132 and HU. Cells were treated with
either HU (10 mM), MG132 (1 ?M), or a combination of the two and
tested for luciferase activity at 24 h postinfection. RLUs, relative light
2638 JOHNSON AND SAMULSKIJ. VIROL.
pared for controls, mock-transfected cells, cells transfected
with scrambled nontargeted siRNA, and cells transfected with
targeted siRNAs, we observed significant increases in samples
with NCL or NPM1 knocked down (Fig. 6B). Samples that
were treated with siRNAs targeting a protein unrelated to
nucleolar function, VCP, showed a slight decrease in transduc-
tion. No discernible change in capsid localization was apparent
in mock-transfected samples, cells transfected with scrambled
siRNA, or cells transfected with VCP siRNA compared to
controls (data not shown). Remarkably, the localization of
capsids in samples treated with NCL siRNA was dramatically
altered and appeared similar to that in samples treated with
HU (Fig. 6C). In contrast, samples treated with NPM1 siRNA
had prominent nucleolar accumulation of capsids, similar to
what is observed during proteasome inhibition.
Many agents that augment AAV infection, such as adeno-
virus, herpesvirus, and genotoxic agents, are known to redis-
tribute nucleolar proteins away from the nucleolus (37, 40, 49).
By changing the localization of NCL or NPM1, these treat-
ments might essentially be analogous to siRNA-mediated pro-
tein depletion if the primary function of these proteins in the
AAV life cycle is associated with the nucleolus. Although ex-
pression levels are relatively unaffected by MG132 or HU up to
24 h after administration (Fig. 6D), HU treatment resulted in
partial redistribution of NCL from the nucleolus to the nucleo-
plasm (Fig. 6E, iii). Because capsid localization appears similar
in HU and NCL siRNA samples, we expect these treatments to
operate through the same pathway. Since both NCL siRNA
and NPM1 siRNA treatments increase transduction but they
have different effects on capsid localization, we hypothesized
that at least two nucleus-associated pathways can be exploited
to increase transduction of rAAV2.
To test whether MG132 or HU will synergistically increase
transduction in NCL siRNA or NPM1 siRNA samples, we
FIG. 6. Impact of nucleolar proteins on rAAV2 transduction and trafficking. siRNAs targeting NCL and NPM1, nucleolar proteins known to
bind AAV capsids, were transfected into HeLa cells at a final concentration of 5 nM in parallel with mock-transfected cells (treated with
transfection reagent only), a scrambled siRNA transfection, or siRNA targeting a nonrelated protein, VCP. (A) Western blots of cell lysates taken
4 days after siRNA transfection, detecting loss of targeted protein relative to ?-actin controls. (B) Luciferase assay of transduction in HeLa cells
transfected with siRNAs targeting NCL, NPM1, or VCP. Cells were split to equal cell densities at 48 h after transfection and infected with rAAV2
(2,000 vg/cell). Transduction was scored 24 h after infection. RLUs, relative light units. (C) Immunofluorescence of rAAV2 in HeLa cells at 16 h
postinfection using MAb A20 to detect intact capsids (green), with nuclear membranes outlined relative to DAPI stain (not shown). Patterns of
localization were compared between samples treated with MG132 and NPM1 siRNA or samples treated with HU and NCL siRNA. Arrows and
arrowheads indicate nucleolar accumulation and diffuse nucleoplasmic staining, respectively. (D) Western blots of total cell protein from control
samples or following MG132 or HU administration for 2, 6, or 24 h. Levels of NCL or NPM1 remain relatively unaffected after drug treatments
compared to ?-actin controls. (E) Immunofluorescence localization of NCL (i, ii, and iii) or NPM1 (iv, v, and vi), shown in red, in untreated cells
compared to drug-treated cells after 24 h (MG132, ii and v; HU, iii and vi). Arrowheads indicate a shift toward nucleoplasmic localization of NCL
following HU treatment (iii).
VOL. 83, 2009 SUBNUCLEAR MOBILIZATION OF rAAV2639
performed luciferase assays of transduction following various
combination treatments (Fig. 7). In support of our hypothesis,
HU was found to have reduced efficacy of increasing transduc-
tion in NCL siRNA cells, suggesting that both treatments in-
fluence the same pathway. Similarly, the efficacy of MG132
appeared to decrease in NPM1 siRNA samples. Synergy was
observed in samples treated with MG132 and NCL siRNA or
samples treated with HU and NPM1 siRNA, which supports
that these treatments operate through separate pathways in the
nucleus to enhance transduction. It is noteworthy that a linear
relationship between transduction efficiency and dose was ob-
served across MOIs ranging from 200 to 20,000 vg/cell (Fig. 7A
With these experiments we have sought to elucidate steps
during subcellular trafficking as rAAV2 traverses from the cell
surface to the nucleus. We have demonstrated that rAAV2
virions enter the nucleus intact and have explored through
confocal immunofluorescence and biochemical fractionation
how changing of cellular parameters influences nucleolar ac-
cumulation. Our results suggest virions that accumulate in
nucleoli remain infectious and are sequestered there in stable
form. These data prompt us to speculate that mobilization
from the nucleolus to nucleoplasmic sites enables capsid tran-
sition from a stable environment to one where uncoating and
subsequent gene expression or genome degradation can occur
(Fig. 8). This model is particularly striking in light of the
helper-dependent nature of AAV, as the virus has likely
evolved to utilize nucleolar proteins for sequestration in a
stable compartment and exploit nucleolar disruption during
mitosis, genotoxic stress, or coinfection to trigger genome re-
lease under favorable conditions.
In these studies we employed multiple techniques to sub-
stantiate our conclusions. Initially with immunofluorescence
experiments, we examined the subcellular trafficking profile of
rAAV2 and detected accumulation of intact capsids in the
nucleolus. We also showed through secondary infections that
rAAV virions isolated from nucleoli retain infectivity. More-
over, disruption of nucleoli by genotoxic agents or siRNA
knockdown of nucleolar proteins mobilizes virions and in-
creases transduction. Our trafficking studies, like all immuno-
fluorescence experiments, were subject to limitations with re-
spect to detection sensitivity, as high particle numbers are
required for visualization. However, we are able to detect
capsid proteins in nucleoli postinfection at 10-fold-lower doses
by biochemical analyses. Our conclusions are also supported by
the fact that transduction assays display a linear trend between
200 and 20,000 vg/cell in control, drug treatment, and siRNA
studies (Fig. 1 and 7). Although we cannot empirically visualize
rAAV2 in nucleoli at 200 vg/cell, transduction studies strongly
argue for effects on nucleolar accumulation and nucleoplasmic
mobilization that are identical to those that the aforemen-
tioned treatments would have at lower MOIs.
Along with documenting accumulation of rAAV in the nu-
cleus, a significant finding from these studies is that empty
capsids are internalized into cells but cannot be detected in the
nucleus, even in the presence of proteasome inhibitors (Fig. 2).
This observation suggests that the phenotype of nuclear accu-
mulation is linked to infectious particles, or to virions that can
at least pass beyond the nuclear membrane. A potential reason
for the distinct trafficking patterns observed for empty and full
virions is that empty capsids fail to expose the N terminus of
VP1 during infection (data not shown). Indeed, Kronenberg et
al. have demonstrated similar results after limited heat treat-
ment and have shown that full and empty capsids display dif-
ferent cryoelectron microscopy profiles (35). Those studies, in
conjunction with our observations, suggest that empty capsids
are unable to expose the phospholipase domain in VP1 that is
thought to be required for endosomal escape or subsequent
steps during infection. In addition to the implications regard-
ing cell entry mechanisms, this finding poses an interesting
caveat concerning vector preparations for clinical use. High
numbers of empty particles in vector preparations may com-
pete with genome-containing particles for cell attachment and
uptake, effectively reducing the chances of gene delivery to the
nucleus. Other effects, such as the potential for empty particles
to trigger cellular recycling or degradation pathways or the
possibility that empty particles saturate extracellular and/or
subcellular binding sites, in turn could either inhibit or aug-
ment transduction. Thus, care must be taken to control the
FIG. 7. Specific combinations of drugs and siRNAs enhance transduction. HeLa cells transfected with siRNAs targeting NCL, NPM1, or VCP
were split to equal cell densities at 48 h after transfection and infected with rAAV2, rAAV2 plus MG132, or rAAV2 plus HU. Transduction
efficiency was scored by luciferase assay 24 h later. Asterisks indicate mitigated effectiveness of MG132 in NPM1 siRNA samples, and double
asterisks highlight a pronounced reduction in sensitivity to HU in NCL siRNA samples. RLUs, relative light units.
2640JOHNSON AND SAMULSKIJ. VIROL.
level of empty capsids in vector preparations and to under-
stand their effects in laboratory and clinical studies.
Although it is unlikely, capsids detected in the nucleolus
during infection could represent reassembled particles. This
could occur if capsids disassembled in the cytoplasm during
entry and subunits were transported into the nucleolus for
assembly. Similar examples of this concept have been found in
cellular systems, such as with the nuclear pore complex, which
disassembles in the cytoplasm and reassembles in the nuclear
membrane over the course of mitosis (34). However, for sev-
eral reasons it is unlikely that the virus is subject to this system.
During replication, AAV monomers and subunits can be
readily detected in cytoplasm and nuclei by antibody B1 (64,
65), but in our studies and others, researches have failed to
detect significant B1 staining during infection (references 38
and 57 and data not shown). Thus, the supply of free mono-
mers and subunits would likely not be great enough to support
reassembly during infection. Moreover, after isolating nucleoli
from cells infected with rAAV2, we found that particles in
these fractions retained infectivity (Fig. 4). These particles
could not have reassembled during infection, since replication
proteins are needed for repackaging of the genome, and these
proteins are absent from current vectors. For these reasons it
is unlikely that capsids detected in the nucleus represent a
reassembly artifact. More importantly, the most striking obser-
vation in this study is that infectious virions are sequestered in
nucleoli, supporting the existence of an “accumulation path-
way” for rAAV in the nucleus (Fig. 8A).
Roughly 40 years ago, AAV serotypes were first shown to
accumulate in nucleolar structures in the context of replication
(2, 30). Our results highlight the apparent paradox of how
AAV could assemble and disassemble in the same cellular
location. The process of uncoating can be defined as the sep-
aration of the genome from the capsid and might not require
complete disassembly of the particle. But even in this light, the
prospect of virions uncoating in the nucleus during infection
seems perplexing. Why wouldn’t all newly assembled capsids
be driven to uncoat during production? There are at least two
plausible explanations for this paradox. One possibility is that
during production, capsids will in fact uncoat as they are as-
sembled. Since these processes obey principles of hysteresis
(55), uncoating is likely to occur more slowly than assembly,
because an external impetus is needed to overcome the collec-
tive contact energies of the subunits. A few groups have sug-
gested that uncoating is not a rapid process for AAV2 (56, 61,
75). As progeny virions are assembled and packaged, a certain
number may always be uncoating, albeit at a lower rate. Al-
though this would explain how virions could assemble and
disassemble in the nucleus, it would not be efficient from an
evolutionary perspective, since unnecessary energy would be
FIG. 8. Illustrated model of two nuclear trafficking paradigms. Virions entering the nucleus are subject to an accumulation pathway (A) and
a mobilization pathway (B). Low or negligible transduction occurs when virions do not enter nuclei at high efficiency. If high particle numbers are
administered, virions can accumulate in the nucleolus in stable form (A), and favorable conditions may permit vector mobilization within the
nucleus to sites that promote uncoating in the nucleoplasm (B). Transduction can be increased by manipulating either pathway independently;
however, transduction is dramatically potentiated if more than one pathway is engaged, as exemplified by the cooperative effects of MG132 and
HU (Fig. 5B).
VOL. 83, 2009 SUBNUCLEAR MOBILIZATION OF rAAV 2641
expended during replication and slow uncoating during infec-
tion would place limits on viral survival.
A second possible explanation for this paradox has been
suggested by Sonntag et al. (57), relying on the premise that
incoming virions undergo modifications during infection that
promote uncoating. These modifications may include confor-
mational changes, such as the extrusion of the N terminus of
VP1/2 from the capsid interior to its exterior (35, 57), partial
proteolytic cleavage (1, 63), ubiquitination, or phosphoryla-
tion. Considering that these changes may confer some insta-
bility to the capsid, we could expect the AAV genome to
become more accessible to the cell as the virion matures during
entry. Indeed, with the parvoviruses minute virus of mice and
B19, viral genomes are found to be exposed during infection
but prior to capsid disassembly (15, 39). It remains to be seen
whether AAV genomes become more accessible after VP1
exposure or during subcellular processing; however, in such a
scenario the cell could distinguish between a virion assembled
in the nucleus and one entering from outside.
Exploring how drug treatments influence rAAV virions in
the nucleus became a major focus of this study. Proteasome
inhibitors have repeatedly been shown to increase transduction
efficiency in a cell type- and tissue-specific manner (17). Fit-
tingly, when proteasomal degradation is inhibited, we see an
increase in capsid accumulation in the nucleolus (Fig. 1 and 3)
and a decrease in the rate of genome degradation (Fig. 5A). In
one report, rAAV capsids were shown to accumulate in nucle-
oli after microinjection into the nucleus in the presence of
proteasome inhibitors (57). It is unlikely that capsid degrada-
tion is occurring in the nucleolus, since no proteasome activity
has been detected there (32, 47, 48). However, proteasome
activity in the nucleoplasm could indirectly influence process-
ing of AAV genomes and affect their degradation. An increase
in genome availability would serve to enhance transduction
intensity and also lead to more rapid transgene expression.
Studies in addition to ours have demonstrated prolonged ge-
nome persistence following administration of proteasome in-
hibitors (18), and several reports provide evidence that inter-
fering with proteasomal degradation increases AAV transduction
primarily by improving nuclear uptake of genomes (16, 71).
Certainly, both subcellular trafficking and the kinetics of trans-
duction are positively influenced by proteasome inhibitors
(Fig. 1B to D). In broad terms these results lend credence to
the hypothesis that a nuclear accumulation pathway can be
exploited to increase rAAV transduction (Fig. 8A), but we
cannot assume accumulation is due to inhibition of capsid
degradation. While it is tempting to speculate that proteasome
inhibitors block degradation of AAV capsids, the results from
these studies, or in fact from any other report to date, do not
support the conclusion that proteasomes directly operate on
intact capsids in the context of infection.
Since AAV2 is known to assemble in the nucleolus and
interact with nucleolar proteins (8, 46), it is intriguing that
recombinant virions can be detected in this compartment after
exogenous administration. In this study we have demonstrated
that in addition to being involved in AAV replication, NCL
and NPM1 are involved in pathways that influence rAAV2
trafficking and transduction. We found these pathways to in-
tersect with accumulation and mobilization pathways, which
are influenced by proteasome inhibitors and HU, respectively.
Studies of minute virus of mice, a parvovirus related to AAV,
have shown that NCL interacts directly with the viral genome
during infection (4). Although we see effects on capsid local-
ization following knockdown of NCL and NPM1, we cannot
discount the possibility that these pathways affect the genome
as well. Both NCL and NPM1 display single-stranded DNA
and RNA binding capability (36, 72), in the context of tran-
scription (73), DNA attachment to the nuclear matrix, or chro-
matin decondensation (21). NCL and NPM1 also have roles in
the DNA damage response (33, 60, 72). Recent work by
Cervelli et al. indicates that rAAV genome single-strand-to-
double-strand conversion can be detected at nuclear foci where
DNA damage response components, the MRN complex or
MDC1 protein, are recruited (14). It has been demonstrated
that DNA damage or genotoxic stress agents such as HU
reduce the associations between rAAV genomes and these
components, suggesting that machinery involved in the DNA
damage response negatively affects genome processing (14,
53), which would be consistent with our results.
Understanding why some cells are readily transduced by
viruses while other cells are recalcitrant to infection is a cor-
nerstone to general virology and research pertaining to viral
vectors. Evolutionarily speaking, it would be highly advanta-
geous for a virus to lie dormant in a cellular compartment until
favorable conditions arose that would promote successful gene
delivery. This resonates particularly well in the case of AAV,
where coinfection by a helper virus is necessary for productive
replication. Perhaps helper-dependent viruses have learned to
exploit nucleolar proteins to sequester themselves in the nu-
cleolus and use nucleolar disruption during mitosis, genotoxic
stress, or coinfection as a trigger to release of their genetic
contents into the nucleoplasm. In support of this view, a com-
mon theme emerges when studying agents that augment rAAV
infection and affect the nucleolus. Components of adenovirus
bind nucleolar proteins (50), and adenovirus protein V induces
the redistribution of NCL and NPM1 from the nucleolus to the
cytoplasm (40). Herpesvirus, another AAV helper, also mod-
ulates the spatial distribution of NCL (7, 12, 37). Additionally,
inactivation of NCL and NPM1 by siRNA results in nucleolar
disruption and cell cycle arrest (26, 62), and these phenotypes
are remarkably similar to those caused by genotoxic agents
such as HU (49). This information corroborates our hypothesis
that rAAV2 virions, initially sequestered in the nucleolus, are
subject to a “mobilization pathway” (Fig. 8B) whereby capsids
are released from a stable, protective environment to one
where the genome becomes accessible.
In summary, our results support a model wherein at least
two pathways are in play to influence AAV trafficking and
transduction in the nucleus, an accumulation pathway and a
mobilization pathway (Fig. 8). In cases where negligible or low
transduction is observed, we expect few if any virions reach the
nucleus, and those that do may be sequestered in the nucleolus
in a dormant state. This may help explain why some investiga-
tors observe a threshold effect with AAV vectors following in
vivo administration (44, 66). Favorable conditions may be cre-
ated during cell division or cell stress that would permit virus
or vector mobilization within the nucleus to sites that promote
uncoating in the nucleoplasm (Fig. 8B). Externally, these con-
ditions can be manipulated using physical or pharmacological
treatments to force virions to adopt productive infectious path-
2642 JOHNSON AND SAMULSKIJ. VIROL.
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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