Characterization of the Early Steps of Human Parvovirus B19
Silva Quattrocchi,aNico Ruprecht,aClaudia Bönsch,a* Sven Bieli,aChristoph Zürcher,a* Klaus Boller,bChristoph Kempf,a,c
and Carlos Rosa,c
Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerlanda; Department of Immunology, Paul Ehrlich Institute, Langen, Germanyb; and CSL Behring
AG, Bern, Switzerlandc
nus of the Parvoviridae family. Transmitted mainly via the respi-
childhood disease named erythema infectiosum, or fifth disease.
dromes, such as acute and chronic arthropathies, severe cytope-
nias, hydrops fetalis, and fetal death (49). The single-stranded
DNA genome of B19V is packaged into a small, nonenveloped,
icosahedral capsid consisting of 60 structural subunits, of which
approximately 95% are VP2 (58 kDa) and 5% are VP1 (83 kDa)
(17). Two other open reading frames coding for small proteins of
unclear functions have also been identified (51, 64).
action with a single receptor, others require complex interactions
with receptors and coreceptors before they can be internalized. In
ing of virus trafficking through the endosomal pathway, the cel-
lular elements involved, and the sites of escape into the cytosol
differ considerably among virus species and cells. Moreover, the
mechanisms by which parvoviruses enter the nucleus remain un-
clear. Since they are small (22 to 25 nm), they can theoretically
pass through the nuclear pore complex (NPC) without disassem-
of the outer nuclear envelope has been proposed (14, 15). For
most parvoviruses, the majority of capsids accumulate in a peri-
uman parvovirus B19 (B19 virus [B19V]) was discovered in
1975 (16) and has been classified within the Erythrovirus ge-
nuclear location from which the viral DNA is imported into the
disassembly (33). Three cell receptors/coreceptors have been
identified for B19V: the glycosphingolipid globoside (globotetra-
osylceramide [Gb4Cer]) (12), the ?5?1integrin (59), and the
erythroid progenitor cells in the bone marrow, which are also the
main target cells for the virus, and it seems to be the primary
attachment receptor. Ku80 might also function in virus attach-
as a coreceptor required for internalization (58, 59).
The mechanisms of B19V uptake and intracellular trafficking
have remained elusive (48). These studies are limited because a
well-established cell line system for B19V infection is lacking;
thus, it is not possible to propagate the virus to high titers. There-
fore, highly viremic plasma from naturally infected individuals
without virus-specific antibodies remains the main source of in-
fectious native virus. UT7/Epo cells are commonly used to study
ing of B19V are not restricted in UT7/Epo cells, the infection is
limited to a small number of cells due to intracellular factors in-
Received 25 April 2012 Accepted 8 June 2012
Published ahead of print 20 June 2012
Address correspondence to Carlos Ros, firstname.lastname@example.org.
*Present address: Claudia Bönsch, Department of Structural Biology and
Bioinformatics, Faculty of Medicine, University of Geneva, Geneva, Switzerland;
Christoph Zürcher, Institute of Veterinary Virology, Faculty of Veterinary Medicine,
University of Bern, Bern, Switzerland.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org Journal of Virologyp. 9274–9284 September 2012 Volume 86 Number 17
tion has been described in ex vivo-expanded primary human ery-
throid progenitor cells (EPCs) (21, 60), where B19V infects a
larger number of cells. The improvement operates at the level of
ing (13); however, the production and egress of infectious prog-
B19V infection is limited to a narrow time frame due to large
variations in virus receptor expression (60), UT7/Epo cells pro-
vide more stable conditions for the study of entry/trafficking
Using complementary approaches, we have studied the early
receptor globoside associate with lipid rafts, the internalization
mechanism occurs through clathrin-mediated endocytosis. Viral
particles are routed to the lysosome for degradation. Escape into
the cytosol depends on low endosomal pH and occurs without
apparent membrane damage. Nuclear entry is highly inefficient
radation of incoming viruses by blocking their transfer to lyso-
somes. The viral DNA is then efficiently imported into the nu-
cleus, while the capsids remain extranuclear.
MATERIALS AND METHODS
Cells and viruses. UT7/Epo cells were cultured in RPMI medium with
10% fetal calf serum (FCS) and were complemented with 2 U/ml of re-
combinant human erythropoietin (Epo; Janssen-Cilag, Midrand, South
Africa) at 37°C under 7.5% CO2. A B19V-infected plasma sample (geno-
type 1; CSL Behring AG, Charlotte, NC), without detectable levels of
tious virus. The virus was pelleted by ultracentrifugation through 20%
(wt/vol) sucrose, and the concentration of virions was determined using
cipitation and immunofluorescence, a monoclonal antibody (MAb)
against B19V (MAb 860-55D) was kindly provided by S. Modrow (Re-
gensburg, Germany). MAb 860-55D targets a conformational epitope in
VP2 and recognizes intact capsids exclusively (22). A rabbit polyclonal
antibody (?-VP1u) against VP1u (amino acids [aa] 142 to 163) was ob-
tained as described previously (7). Early endosomes were detected using
immunofluorescence with a rabbit polyclonal antibody against EEA1
(Abcam, Cambridge, MA). A mouse MAb against the mannose-6-phos-
phate receptor and a mouse MAb against Lamp1 (Abcam) were used to
detect late endosomes and lysosomes, respectively. Flotillin-1 was ob-
nate (FITC)-labeled cholera toxin was purchased from Sigma, and FITC-
Sigma (St. Louis, MO). Ammonium chloride (NH4Cl) was dissolved in
water; nystatin and bafilomycin were dissolved in dimethyl sulfoxide
(DMSO); and filipin was dissolved in ethanol.
Control of interfering drugs. To test drugs for cytotoxicity and spec-
of drugs added a few hours after internalization. Only doses that showed
of the doses used have an effect on the cellular S phase, which is required
for parvovirus replication, the method allows one to rule out possible
ing/internalization and trafficking.
Isolation of lipid rafts. The insolubility of lipid rafts in cold nonionic
detergents provides the most common method for lipid raft isolation.
However, the presence of detergents can produce artifacts or interfere
with sphingolipid distribution. Therefore, we used a novel detergent-free
method in which rafts are purified by shearing cells in an isotonic buffer
with cations, followed by separation along an OptiPrep gradient (34). A
detergent-free lysis buffer (1? Tris-buffered saline [TBS], pH 8), in the
presence of 1 mM CaCl2and 1 mM MgCl2to stabilize the rafts and were
supplemented with protease inhibitor cocktail (Complete Mini; Roche).
The homogenate was sheared through a 22G by 3-in needle and was cen-
trifuged at 1,000 ? g for 10 min at 4°C. The resulting postnuclear super-
natant was collected and maintained on ice. The procedure was repeated
on the pellet, and the supernatant obtained was combined with the first.
OptiPrep (Sigma) in SW60 ultracentrifuge tubes. The mixture was care-
tubes were centrifuged at 38,500 rpm (200,000 ? g) for 18 h at 4°C.
Acceleration and deceleration rates were set to zero. Fractions of 520 ?l
fraction was analyzed by Western blotting with an antibody against the
lipid raft component flotillin-1.
Immunofluorescence. UT7/Epo cells were infected with B19V at 2 ?
104viral particles per cell in RPMI medium for 1 to 2 h at 4°C to allow
binding, followed by several washing steps to remove unbound virus.
After different incubation times at 37°C, the cells were fixed with ace-
tone-methanol (1/1 [vol/vol]) for 5 min at ?20°C. Following staining
with specific antibodies, the cells were washed and mounted with
Mowiol (Calbiochem, La Jolla, CA) containing 30 mg/ml of 1,4-
diazabicyclo(2,2,2)octane (DABCO; Sigma) as an antifade agent. Confo-
scanning microscope with an inverted Zeiss microscope (Axiovert 200M;
Carl Zeiss AG, Feldbach, Switzerland).
Electron microscopy. UT7/Epo cells were infected with B19V at 3 ?
Following a washing step to remove unbound virus, the cells were incu-
bated at 37°C. After 0, 1, 5, 10, or 30 min, the cells were fixed in 2.5%
glutaraldehyde. Cells were washed in phosphate-buffered saline (PBS)
and were embedded in 3% agarose. The cubes were fixed for 1 h in
osmium tetroxide (1% in PBS) on ice, washed, and further incubated for
1 h in a tannin solution (0.05 M HEPES plus 0.01 g tannin) at room
temperature (RT). The samples were washed with a sodium sulfate solu-
added to the samples for 1 h at RT; then the samples were washed with
were incubated in Epon, embedded in gelatin capsules, and polymerized
for 48 h at 60°C. Capsules were dissolved at 70°C, and 70-nm ultrathin
finally analyzed in a Zeiss EM 902 electron microscope equipped with a
TRS digital camera.
Infectivity assay. Cells (3 ? 105) in RPMI medium containing 10%
fetal calf serum and 2 U/ml of Epo were seeded into 12-well plates and
cases, cells were pulse-treated with drugs for specific times. As controls,
drugs were added 3 to 4 h postinfection. Cells were infected with B19V at
5 ? 103genome equivalents per cell and were further incubated at 37°C
for 24 h. The cells were transferred to RNase-free tubes (Eppendorf Bio-
pur, Hamburg, Germany) and were pelleted. The pellets were washed
twice with PBS and were stored at ?20°C until use. Total poly(A) mRNA
to the manufacturer’s instructions. The isolated viral NS1 mRNA was
reverse transcribed, and cDNA was quantified. Cells collected at 30 min
postinfection served as input controls to define the background signal.
Quantitative PCR. Amplification of B19V DNA or cDNA and real-
time detection of PCR products were performed using a LightCycler sys-
tem (Roche Diagnostics, Rotkreuz, Switzerland) with SYBR green
Early Steps of B19V Infection
September 2012 Volume 86 Number 17jvi.asm.org 9275
(Roche). PCR was performed using a FastStart DNA SYBR green kit
(Roche) according to the manufacturer’s instructions. Plasmids contain-
ing the genome of B19V were used at 10-fold dilutions as external stan-
dards. The number of cells used for each experiment was determined by
the quantification of the cellular ?-actin gene, as described previously
Immunoprecipitation. UT7/Epo cells were infected with B19V, as
described above. Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl,
150 mM NaCl, 1% NP-40, and 5 mM EDTA) supplemented with a pro-
tease inhibitor cocktail (Complete Mini; Roche). Viral particles were im-
munoprecipitated with a human MAb against intact capsids (MAb 860-
55D). After overnight incubation with 20 ?l protein G agarose beads at
4°C, the beads were washed four times (three times with PBS–1% bovine
serum albumin and once with PBS) and were resuspended either in pro-
tein loading buffer, for analysis of the immunoprecipitated capsids by
Western blotting, or in PBS, for quantification of the viral DNA by PCR.
Total DNA was extracted using the DNeasy tissue kit (Qiagen) and was
quantified as specified above.
Analysis of endosomal membrane permeabilization. To examine
tion or damage during the escape process, rhodamine-labeled dextrans
washed and were further incubated at 37°C in the presence of B19V to
fected cells were used as controls. The capacity of the endosomal vesicles
to retain the endocytosed dextrans was monitored by fluorescence mi-
croscopy at increasing incubation times.
Quantification of B19V DNA nuclear import. UT7/Epo cells were
infected as specified above. At increasing times postinfection, the cells
100 ?l EZ buffer before the addition of an additional 900 ?l EZ buffer
(Sigma). The samples were vortexed and were kept on ice for 5 min; then
they were pelleted at 2,400 rpm for 5 min at 4°C. This step was repeated.
Pellets were then resuspended in 500 ?l EZ buffer containing 0.25 M
sucrose and were layered on top of 500 ?l EZ buffer with 0.5 M sucrose.
The purified nuclei were collected by centrifugation at 5,000 rpm for 10
light microscopy after trypan blue staining. Nuclear pellets were resus-
pended in nuclear lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Tri-
ton X-100, and 1 mM EDTA [pH 7.4]) supplemented with protease in-
hibitor cocktail (Complete Mini; Roche) and were maintained on ice for
10 min. The homogenate was sheared through a 26G by 1-in needle and
was centrifuged at 8,000 ? g for 10 min at 4°C. The supernatant was used
to quantify the viral DNA or to immunoprecipitate capsids, as specified
riched in cholesterol and glycosphingolipids and play roles in
membrane receptor dynamics, signal transduction, intracellular
trafficking, cell polarization, and cell migration (31). The glyco-
orescence staining with FITC-labeled cholera toxin B (CTxB),
which binds to the ganglioside GM1, a common component of
some cells showed little or no detectable expression. Gb4Cer co-
localized partially with CTxB in cells expressing GM1 abundantly
(Fig. 1A). Like Gb4Cer, B19V partially colocalized with the GM1-
raft marker flotillin-1 (Flot-1) along OptiPrep density gradients
was examined. As expected, in uninfected cells, Flot-1 was found
in the light, buoyant membrane fractions, predominantly in frac-
tions 1 and 2. In contrast, when lipid rafts were isolated from
infected cells, Flot-1 shifted significantly to denser fractions, sug-
gesting a virus-mediated modification in the structure and/or
lipid composition of the rafts (Fig. 1B).
Depending on the presence of the membrane protein caveo-
lin-1 (Cav-1), lipid rafts can be divided into caveolar and non-
proteins, respectively. Gb4Cer had variable colocalization with
colocalization in others (Fig. 1C). However, B19V rarely colocal-
ized with Cav-1 (Fig. 1C), indicating that the large majority of
B19V associates preferentially with noncaveolar lipid rafts.
Disruption of lipid rafts inhibits B19V infection. The infec-
tivity of B19V was analyzed in the presence of nystatin, an anti-
fungal reagent that disrupts cholesterol-enriched microdomains
(6, 46). When nystatin was added after virus binding/internal-
ization, none of the doses tested caused significant inhibition.
However, when added before virus infection, nystatin caused
dose-dependent inhibition of B19V infectivity (Fig. 1D). B19V
infectivity was also sensitive to filipin, a sterol-binding agent that
Like nystatin, filipin had no effect on the infection when added
after virus binding/internalization, suggesting that the plasma
B19V is internalized through clathrin-mediated endocyto-
sis. Electron micrographs from B19V-infected cells during the
first minutes of incubation at 37°C showed the uptake of B19V
characteristic clathrin electrodense coating (Fig. 2A). One single
virus particle was sufficient to accomplish the process of internal-
ization into clathrin-coated vesicles. No virus internalization in
the typical small flask-shaped caveolar invaginations or in other
structures was observed at any time. Furthermore, B19V did not
involved in caveola-dependent internalization (29) (data not
The uptake mechanism of B19V was followed using FITC-la-
beled human holotransferrin, which is typically internalized
through clathrin (26). B19V and transferrin did not colocalize
during the binding step at 4°C. However, when the temperature
was shifted to 37°C, B19V and transferrin colocalized intensively,
indicating a common internalization pathway (Fig. 2B). These
results, taken together, indicate that clathrin-mediated endocyto-
sis is the primary internalization mechanism used by B19V.
Incoming B19V capsids spread rapidly through the endo-
cytic pathway and are routed to the lysosomes. The progression
of intracellular capsids was examined using immunofluorescence
confocal microscopy at increasing time points after internaliza-
tion. Cells were washed, fixed, and stained with an antibody
early endosomes (EE) (EEA1), late endosomes (LE) (mannose-6-
phosphate receptor), lysosomes (Lys) (Lamp1), recycling vesicles
(GRP78 BIP). By 5 min postinternalization, B19V capsids were
detectable in early endosomes, and by 30 min, capsids were ob-
served throughout the endocytic pathway (Fig. 3A). The colocal-
ization signals from an average of 40 cells were quantified with
BioImage XD software (27). Maximal colocalization with LE oc-
Quattrocchi et al.
jvi.asm.orgJournal of Virology
FIG 1 Lipid rafts and B19V infection. (A) Colocalization of globoside (Gb4Cer) and B19V with the lipid raft marker GM1. UT7/Epo cells were incubated with
FITC-labeled cholera toxin and were infected with B19V at 4°C to allow virus binding. Cells were fixed and stained with an antibody against Gb4Cer or against
dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting for the detection of flotillin-1. (C) Colocalization of Gb4Cer and B19V with
Cav-1. (D) Effects of nystatin (15 to 40 ?M) and filipin (0.5 to 2 ?M) on B19V infection. UT7/Epo cells were preincubated with the drugs for 30 min at 37°C
before infection. As a control, the drugs were added 3 to 4 h postinfection. Data are means ? standard deviations for duplicate samples from three independent
Early Steps of B19V Infection
September 2012 Volume 86 Number 17jvi.asm.org 9277
with lysosomes occurred by 30 min (Fig. 3B). At later times
gressively. By 1 h, capsids were detected in lysosomes, but only a
few were detected in the LE. By 2 to 3 h postinternalization, cap-
sids were clearly less abundant and appeared scattered in the cy-
toplasm, and with the exception of a few capsids detectable in
lysosomes, colocalization with EE or LE was no longer observed.
No significant colocalization of incoming particles with recycling
vesicles, the Golgi apparatus, or the ER was observed at any time
point (data not shown). The lack of colocalization with any rele-
vant organelle marker suggests that following endocytic traffick-
ing, a proportion of incoming viruses escapes into the cytosol.
tion inside the endosomes is required by certain viruses as part of
their infectious entry route. All parvoviruses analyzed to date re-
quire endosomal acidification for the infection process (18, 25,
42). To investigate the requirement of B19V for a low endosomal
tive inhibitor of vacuolar ATPases, or the lysosomotropic weak
base ammonium chloride (NH4Cl). These compounds raise the
pHs of intracellular compartments, which can be restored upon
removal of the inhibitory substances (11, 36). UT7/Epo cells were
pulse-treated for different times with BafA1 or NH4Cl. Although
their mechanisms of action are different, BafA1 and NH4Cl treat-
ever, BafA1 and NH4Cl had no significant effect on infectivity
when added 1 h postinternalization, suggesting that most of the
infectious particles had escaped from the endocytic vesicles.
sids did not colocalize with endocytic markers (Fig. 4B). Further-
more, no colocalization was observed with markers of recycling
endosomes (Rab 11 or transferrin), caveolin-containing vesicles,
the Golgi apparatus, or the ER (data not shown), indicating that
FIG 3 Endocytic trafficking of B19V. (A) Confocal immunofluorescence im-
ages showing colocalization of B19V capsids with markers of the endocytic
pathway. EEA1, marker of early endosomes; M6PR (mannose 6-phosphate
receptor), marker of late endosomes; Lamp1, marker of lysosomes. (B) Kinet-
ysis was performed by BioImage XD software with Pearson’s correlation coef-
ficient. Values are means ? standard deviations for two measurements from
an average of 40 cells.
FIG 2 Clathrin-dependent B19V uptake. (A) Electron micrographs showing internalization of B19V in UT7/Epo cells by clathrin-mediated endocytosis. Bar,
to allow virus binding. Following washes to remove unbound virus, the cells were incubated at 37°C to initiate virus internalization.
Quattrocchi et al.
jvi.asm.org Journal of Virology
these capsids may be in the cytosol. However, in the presence of
BafA1, the capsids were retained inside late endocytic elements,
endosomal acidification is not essential for the progression of the
of the incoming viruses from late endosomes to lysosomes but is
required by B19V for escape into the cytosol.
A proportion of incoming capsids is degraded. During the
process of entry, B19V is routed to the lysosomes, reaching max-
imal colocalization by 30 min to 1 h after internalization (Fig. 3).
tion was not due to virus detachment from the receptor or recy-
cling, since no increase in the level of viral DNA was observed in
the supernatant of the infected cells from 1 to 5 h postinfection
(Fig. 4D). These observations indicate that during the process of
viral entry, a proportion of incoming capsids are routed to the
lysosomes for degradation.
Endosomal escape occurs without detectable membrane
cling endosomes (Rab 11 or transferrin), and no significant colo-
calization with caveolin-containing vesicles, the Golgi apparatus,
or the endoplasmic reticulum was observed at any time (data not
shown). This observation would suggest that the capsids had es-
caped into the cytosol. The mechanism by which these capsids
reach the cytosol is unknown. Increasing evidence indicates that
the phospholipase A2(PLA2) activity of VP1u plays a role by
disrupting the endosomal membrane (63). We have examined
whether B19V can induce detectable permeabilization or damage
in the endocytic membranes by monitoring the capacity of the
tran molecules. Dextrans were retained in the endocytic vesicles,
without detectable cytosolic or nuclear staining (Fig. 5A and B).
The lack of detectable dextran leakage would suggest that endo-
somal escape occurs either without membrane permeabilization/
disruption through a yet unidentified mechanism or from only a
few vesicles, resulting in undetectable leakage.
B19V nuclear entry is highly inefficient. Parvoviruses deliver
has been found to be inefficient in all parvoviruses analyzed (25).
We have shown previously that incoming minute virus of mice
(MVM) accumulates and persists in the lysosomal compartment
without noticeable cytosolic capsids and that incoming nuclear
viral DNA replication and RNA transcription in the nucleus (35).
To examine the presence of incoming B19V DNA in the nucleus,
taken 10 min postinternalization was used to define the back-
ground signal. As shown in Fig. 6A, no increase over the back-
ground was observed in untreated or BafA1-treated cells, even
after the onset of viral transcription in the nucleus (Fig. 6B).
The presence of viral particles in the nucleus was examined by
confocal microscopy. Viral capsids appeared scattered through-
tact with the nuclear envelope and nuclear invaginations, they
were not observed inside the nucleus (Fig. 6C). Immunoprecipi-
tation of capsids from total cells or from isolated nuclei further
quantities (Fig. 6D).
FIG 4 Low-pH-dependent entry of B19V. (A) Effect of bafilomycin A1
(BafA1) or ammonium chloride (NH4Cl) on B19V infection. UT7/Epo cells
were pulse-treated with BafA1 (20 nM) or NH4Cl (25 mM) for the indicated
periods. (B) Effect of BafA1 treatment on B19V endosomal trafficking. In-
fected cells were washed and were fixed 5 h postinfection in the absence or
capsids and antibodies against late endosomes (M6PR) and lysosomes
(Lamp1). (C) Analysis of B19V DNA degradation at increasing times postin-
was added 30 min before the infection and was removed 3 h postinfection.
Circles, BafA1 treated; triangles, untreated. (D) Analysis of B19V release into
deviations for duplicate samples from three independent experiments.
Early Steps of B19V Infection
September 2012 Volume 86 Number 17jvi.asm.org 9279
that chloroquine (CQ) enhances B19V infection in UT7, HepG2,
and primary bone marrow mononuclear cells (8). To understand
the mechanism underlying the boosting effect of CQ, UT7/Epo
cells were pulse-treated for different times with CQ. As shown in
Fig. 7A, when added at the time of the infection for 1 h, CQ had a
h after the infection, CQ was beneficial for the infection, and this
the drug. This observation would suggest that immediately after
tion, and that the effect of CQ can be attributed to a subsequent
We next analyzed the efficiency of B19V nuclear entry in the
presence of CQ. In sharp contrast to no treatment or BafA1 treat-
the nuclei and reached a plateau by 3 h postinfection (Fig. 7B).
Comparison of the amount of viral DNA from complete cells and
from isolated nuclei at 3 h postinfection revealed that the viral
the total intracellular viral DNA (Fig. 7C). Although more than
capsids were not detectable inside the nucleus (Fig. 7D). Immu-
noprecipitation of viral capsids from total cells or from isolated
and F). These results would suggest either that viral DNA is im-
ported into the nucleus from capsids that are immediately un-
coated or that uncoating takes place prior to the import of the
DNA into the nucleus.
increase of virus nuclear import by CQ could be explained
through the particular effects of CQ on endosomal vesicles. Al-
though CQ, BafA1, and NH4Cl have similar effects on endosomal
pH, CQ has the ability to destabilize endocytic membranes, lead-
ing to vesicle swelling. The intense lysosomal dysfunction caused
by CQ results in a deficient transfer of cargo to the degradative
lysosomes, promoting escape from a prelysosomal vesicle and
avoiding degradation in lysosomes (28, 37). This ability has been
routinely used to improve the efficiency of transfection exper-
iments (32). Using confocal microscopy, we confirmed that in
CQ-treated cells, but not in untreated or BafA1-treated cells,
lysosomes appeared severely enlarged (Fig. 8A). The extensive
age of endocytosed dextrans (Fig. 8B). Confocal microscopy im-
the accumulation of intact capsids in late endosomes, but not in
Although the viral DNA was not degraded, the number of intact
capsids decreased significantly during the entry process (Fig. 8E).
FIG 5 Endosomal escape occurs without apparent membrane damage. (A)
Effect of incoming virus on endosomal membrane integrity. UT7/Epo cells
were incubated in the presence of rhodamine-conjugated, lysine-fixable dex-
tran (Mr, 3,000; 3 mg/ml) and B19V for 2 h. The cells were subsequently
washed to remove unbound virus and extracellular dextran and were further
incubated at 37°C. At the indicated times, the cells were fixed with 4% para-
experiment were fixed with methanol-acetone 7 h postinfection and were
stained with MAb 860-55D. A representative cell is shown.
FIG 6 Analysis of nuclear entry of B19V DNA and capsids. (A) Nuclei from
infected cells (untreated or BafA1 treated) were isolated at increasing times
ples. Samples taken 10 min postinfection served as background controls. Data
experiments. Filled circles, BafA1 treated; shaded triangles, untreated. (B) Ki-
netics of NS1 mRNA synthesis in infected cells. At increasing times postinfec-
10 min postinfection served as background controls. Data are means ? stan-
3 h postinfection. B19V capsids were stained with MAb 860-55D (green), and
nuclei were counterstained with 4=,6-diamidino-2-phenylindole (DAPI)
(blue). A 1-dimensional rendering made from the Z stack and orthogonal
sections are shown. (D) Immunoprecipitation of capsids with MAb 860-55D
from total cells (0 h and 3 h postinfection) and isolated nuclei (3 h postinfec-
tion). Double amounts of cells and nuclei were used for the 3-h-postinfection
Quattrocchi et al.
jvi.asm.org Journal of Virology
case for untreated cells; the more likely cause is the uncoating of
the incoming virus.
Lipid rafts are small, heterogeneous, cholesterol- and glycosphin-
golipid-enriched domains that play important roles in cellular
processes such as membrane signaling and trafficking (31). They
are also involved in multiple stages of the virus life cycle, such as
FIG 7 Chloroquine boosts B19V nuclear import and infection. (A) Effect of
at increasing postinfection times. B19V DNA was extracted from the nuclear
samples and quantified. Samples taken 10 min postinfection served as back-
ground controls. (C) Quantification of total incoming B19V DNA from the
whole cell and from isolated nuclei at 3 h postinfection. The amount of cells
and nuclei was normalized by quantification of the human ?-actin gene. (D)
Confocal images of cells 3 h postinfection. B19V capsids were stained with
MAb 860-55D (green), and nuclei were counterstained with 4=,6-diamidino-
2-phenylindole (DAPI) (blue). A 1-dimensional rendering made from the Z
with MAb 860-55D from total cells (0 and 3 h postinfection) and isolated
the immunoprecipitation at 3 h postinfection. (F) Quantification of the total
amount of B19V DNA from the nuclear fraction (shaded bars) or following
immunoprecipitation (IP) with MAb 860-55D (filled bars) at 3 h postinfec-
two independent experiments.
FIG 8 Chloroquine prevents the transfer of B19V to lysosomes and the deg-
left untreated. After 3 h, cells were fixed and stained with an antibody against
lysosomes (Lamp1). Nuclei were stained with 4=,6-diamidino-2-phenylindole
were treated with BafA1 (20 nM) or CQ (25 ?M) or were left untreated. Cells
were incubated in the presence of rhodamine-conjugated, lysine-fixable dex-
tran (Mr, 3,000; 3 mg/ml) for 2 h. The cells were subsequently washed to
remove extracellular dextran and were further incubated at 37°C. Cells were
fixed after 2 h with 4% paraformaldehyde. (C) CQ prevents the transfer of
B19V to lysosomes. CQ-treated cells (25 ?M) were fixed 5 h postinfection.
Cells were stained with MAb 860-55D against capsids and with antibodies
against late endosomes (mannose 6-phosphate receptor [M6PR]) and lyso-
somes (Lamp1). (D) CQ was added 30 min before the infection and was re-
moved 3 h postinfection. B19V DNA was quantified at increasing postinfec-
tion times. Data are means ? standard deviations for duplicate samples from
two independent experiments. (E) CQ-treated cells were fixed 1 and 5 h
postinfection and were stained with antibody 860-55D against intact capsids.
Early Steps of B19V Infection
September 2012 Volume 86 Number 17jvi.asm.org 9281
attachment, internalization, uncoating, protein transport, assem-
bly, and budding (53, 55). Several pieces of evidence indicate that
B19V exploits lipid rafts during the process of entry: (i) colocal-
nonraft fractions in infected cells (Fig. 1B), and (iii) inhibition of
B19V infection following lipid raft disruption (Fig. 1D). The fact
that lipid raft disruption had no effect a few hours after infection
tious entry/trafficking of B19V and not for later steps. The exact
mechanism by which these membrane microdomains contribute
to virus entry is not known. Preliminary results indicate that lipid
raft disruption does not inhibit virus attachment (data not
shown). Gb4Cer is required but is not sufficient for B19V infec-
tion. Other receptors have been shown to be important in the
process of viral entry (12, 40, 58, 59). Thus, lipid rafts may act as
quired for B19V infection, as has been shown for other viruses
Clathrin-dependent endocytosis is the default uptake mecha-
nism for parvoviruses (3, 5, 20, 41, 54). To date, only adeno-
routes based on caveolae and macropinocytosis, respectively (2,
5). Since caveolae are integral parts of some lipid rafts and B19V
tion of B19V into UT7/Epo cells could be envisioned. Although
Gb4Cer was associated with both caveolar and noncaveolar rafts,
means of electron microscopy, B19V was recurrently observed in
clathrin-coated invaginations and vesicles (Fig. 2A); however, vi-
not observed. While at the attachment step B19V did not colocal-
ize with transferrin, which is internalized through clathrin-medi-
ated endocytosis (26), extensive colocalization was observed dur-
ing the internalization step (Fig. 2B). In contrast to the slow
internalization by caveolae, cargo internalized by clathrin-medi-
ated endocytosis is quickly delivered to early endosomes (39).
Consistent with a rapid internalization mechanism, immunoflu-
orescence pictures taken 5 min p.i. confirmed the presence of
B19V capsids in early endosomes (Fig. 3A). Therefore, although
B19V interacts with plasma membrane rafts, internalization oc-
curs by clathrin-mediated endocytosis and does not involve cave-
olae. This mechanism of internalization based on lipid rafts and
clathrin has been observed in other viruses (19).
by blocking viral escape, resulting in the accumulation of viral
particles in the degradative lysosomes. However, CQ, which also
alkalinizes the endosomes, enhances the infection. We have
shown previously that CQ enhances B19V infection in UT7,
HepG2, and primary bone marrow mononuclear cells (8). The
case of HepG2 cells is particularly striking. These cells are consid-
ence of CQ, HepG2 cells support B19V infection (8). The reason
for this enhancement can be explained, at least in part, by the
particular effects of CQ on endosomal vesicles. CQ causes vesicle
swelling and hampers the fusion of endosomes and lysosomes,
lysosomal compartment (28, 37). Because of these particular
properties, CQ is frequently used in transfection experiments to
increase transduction efficiency (32). We confirmed that CQ, but
8A) and prevented the transfer of the incoming capsids to the
degradative lysosomes (Fig. 8C). We could also confirm that CQ,
(Fig. 8D). The capsids retained in vacuolated prelysosomal vesi-
cles would profit by progressively escaping into the cytosol.
Although the effect of CQ on endosomal vesicles is indepen-
dent of the cell type, only B19V, and no other parvovirus, can
benefit (4, 20, 44, 50). All parvoviruses studied to date exploit
endosomal acidification for capsid modifications required for
subsequent steps, primarily the externalization of N-VP1 and its
constitutive PLA2domain, which is thought to be required for
endosomal escape (35, 50, 56). In addition, nuclear localization
signal (NLS) sequences have been identified in the N-VP1 pro-
teins from some parvoviruses, which might assist in the transport
of capsids toward the nucleus (57). We have recently shown that
B19V is unique among parvoviruses in that N-VP1 becomes ex-
pH for this critical conformational rearrangement. However,
PLA2domain of N-VP1 and the endocytic membranes.
At increasing postinternalization times, incoming capsids ap-
with endocytic markers, the ER, caveolin-containing vesicles, the
Golgi apparatus, or recycling vesicles, suggesting that they had
play a role in endosomal escape; however, the mechanism is not
known. Adenovirus is able to release coendocytosed dextrans of
different sizes, implying a mechanism of escape based on endo-
(CPV) has been studied by cointernalization of alpha-sarcin,
of 3 kDa but not of 10 kDa (52). However, the effect was evident
only 20 h postinfection, while endosomal escape of CPV occurs
earlier. In addition, a parvovirus capsid would not be able to es-
cape through a pore that selectively allows the escape of dextrans
of 3 kDa but not of 10 kDa. In the case of B19V, there was no
detectable leakage of dextrans of 3 kDa at any time or in the pres-
ence of CQ (Fig. 5 and 8B). Two possibilities can be envisaged:
either capsids permeabilize only a minor amount of endocytic
vesicles or capsids escape through a yet unidentified mechanism,
which does not involve membrane damage.
Viral capsids (Fig. 6C and D) or viral DNA (Fig. 6A) was not
tion (Fig. 6B). Thus, B19V is inefficiently imported into the nu-
contrast, in CQ-treated cells, B19V DNA was efficiently imported
At this time, more than half of the total viral DNA was found
associated with the nuclear fraction (Fig. 7C). Examination of the
incoming nuclear DNA confirmed that the viral DNA was not
associated with capsids, which remained extranuclear (Fig. 7D, E,
significantly (Fig. 8E). Since the viral DNA remained stable (Fig.
8D) and a large proportion was found in the nucleus (Fig. 7B and
Quattrocchi et al.
jvi.asm.org Journal of Virology
C), the decrease in the number of intact capsids cannot be attrib-
uted to degradation, as was the case for untreated cells. These
results would suggest either that the viral DNA is imported into
the nucleus from intact capsids that are immediately uncoated or
nucleus. The second possibility seems more plausible, since no
capsids were found in the nucleus at any time point.
infection and reveal mechanisms involved in virus uptake, endo-
cytic trafficking, and nuclear import. This study also outlines
novel questions that warrant further investigation, such as the
precise role of lipid rafts in the process of virus entry, the mecha-
nism by which B19V escapes from endosomes without detectable
permeabilization/damage, and the pathway involved in the nu-
clear import of viral DNA.
We are grateful to S. Modrow and J. Lindner (Regensburg, Germany) for
kindly providing MAb 860-55D. The skillful technical assistance of R.
Eberle (Paul-Ehrlich-Institute, Langen, Germany) in EM sample prepa-
ration is gratefully acknowledged.
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