Journal of General Virology (2005), 86, 2057–2068
DOI 10.1099/vir.0.80652-0
Binding and entry characteristics of porcine
circovirus 2 in cells of the porcine monocytic line
3D4/31
G. Misinzo,1 P. Meerts,1 M. Bublot,2 J. Mast,3 H. M. Weingartl4
and H. J. Nauwynck1
1
Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
9820 Merelbeke, Belgium
Correspondence
H. J. Nauwynck
2
[email protected]
Merial, Biological Research, Lyon, France
3
Veterinary and Agrochemical Research Centre, Brussels, Belgium
4
Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada
Received 29 September 2004
Accepted 24 March 2005
Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome
and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target
cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is
unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were
studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal
microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated
endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection.
It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min
post-incubation. Bound PCV2 VLPs were internalized in 47±5?0 % of cells. Internalization was
continuous, with 70?5±9?7 % of bound PCV2 VLPs internalized at 360 min post-incubation.
Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly
by chemical inhibitors that specifically blocked (i) actin-dependent processes, including
cytochalasin D (75?5±7?0 % reduction) and latrunculin B (71?0±3?0 % reduction), and (ii)
clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock
(50?2±6?3 % reduction), hypertonic medium (56?4±5?7 % reduction), cytosol acidification
(59?1±7?1 % reduction) and amantadine (52?6±6?7 % reduction). Inhibiting macropinocytosis
with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2
infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium
chloride (47?0±7?9 % reduction) and chloroquine diphosphate (49?0±5?6 % reduction).
Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via
clathrin-mediated endocytosis and requires an acidic environment for infection.
INTRODUCTION
Porcine circoviruses (PCVs) are small, non-enveloped
viruses with a single-stranded, closed-circular DNA (Todd
et al., 2000), classified in the genus Circovirus (Pringle, 1999)
of the family Circoviridae. The first porcine circovirus
(PCV1) was detected as a contaminant of the continuous
porcine kidney (PK-15) cell line (Tischer et al., 1974) and
was found to be non-pathogenic for pigs (Tischer et al.,
1986). In 1997, an agent morphologically similar to but
genetically and antigenically distinct from PCV1 was isolated from pigs with post-weaning multisystemic wasting
syndrome (PMWS) (Ellis et al., 1998; Morozov et al., 1998).
This virus was designated PCV2. The PCV2 genome contains two major open reading frames (ORFs). ORF1 encodes
0008-0652 G 2005 SGM
the replicase protein (Rep) involved in rolling-circle
PCV2 DNA replication, whilst ORF2 encodes the PCV2
capsid protein (Hamel et al., 1998; Mankertz et al., 1998;
Nawagitgul et al., 2000). The PCV2 virion has an icosahedral
T=1 structure containing 60 capsid protein molecules
arranged in 12 pentamer-clustered units (Crowther et al.,
2003). PCV2 has been associated with different diseases
and syndromes, two of which have been reproduced
experimentally: PMWS (Allan et al., 1999) and reproductive
problems (Sanchez et al., 2001).
Recently, PCV2 target cells have been shown to be cardiomyocytes, hepatocytes and macrophages (mw) during fetal
life and mainly monocytes/mw in early post-natal life
(Sanchez et al., 2003). In experimentally inoculated pigs, the
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2057
G. Misinzo and others
majority of PCV2-infected pigs showed low or moderate
levels of PCV2 replication, whilst a few showed high-level
PCV2 replication (Sanchez et al., 2003). In those pigs with
low or moderate levels of virus replication, the majority
of PCV2-infected cells appeared to be differentiated mw. In
addition to differentiated mw, infiltrating monocytes and
lymphocytes were also infected in pigs with high levels of
virus replication (Sanchez et al., 2004).
Although PCV2 target cells have been well characterized, the
mechanistic details of the early stages of PCV2 infection,
involving the attachment of virions to the cell surface by
binding to their cellular receptors followed by entry into
these target cells, are still poorly understood. The binding and entry of viruses determines the first phase of viral
infection and it is important to characterize these early
events for PCV2.
Initiation of viral infection requires entry of the virus
into the host cell by direct penetration of the plasma
membrane or, more often, through one or more of the
endocytic pathways following interaction with cell-surface
receptors. The endocytic pathways include clathrin- and
caveolae-mediated endocytosis, macropinocytosis and
clathrin- and caveolae-independent endocytosis (Conner
& Schmid, 2003; Nichols & Lippincott-Schwartz, 2001).
Non-enveloped viruses are internalized mainly via either
clathrin- or caveolae-mediated endocytosis. Among the
non-enveloped viruses, adenovirus (Meier & Greber, 2003),
adeno-associated virus (Bartlett et al., 2000), human
polyomavirus JC (Pho et al., 2000) and canine parvovirus
(Parker & Parrish, 2000) enter via clathrin-mediated endocytosis, whereas SV40 and mouse polyomavirus (Pelkmans
& Helenius, 2002) enter via caveolae-mediated endocytosis.
Actin is involved in all forms of endocytosis (EngqvistGoldstein & Drubin, 2003). Actin filaments facilitate uptake
and delivery to the degradative compartments of ligands
internalized via clathrin-mediated endocytosis (Durrbach
et al., 1996). Actin polymerization is also required for
the formation of the membrane protrusions at the site
of internalization during macropinocytosis (Grimmer
et al., 2002; Lee & Knecht, 2002), and cortical actin filaments confine and organize caveolae near the cell surface
(Mundy et al., 2002). Polymerization of filamentous actin
also occurs at endocytic sites in caveolae-mediated endocytosis (Pelkmans et al., 2002).
The endocytic pathway(s) offers a low pH-dependent conformational change that triggers fusion, penetration and/or
uncoating of certain viruses. As such, some non-enveloped
viruses, e.g. adenovirus type 2 (Varga et al., 1991), rhinovirus (Prchla et al., 1994), reovirus (Martı́nez et al., 1996)
and canine parvovirus (Basak & Turner, 1992), are affected
by lysosomotropic weak-base treatments that prevent
endosomal acidification.
Several techniques have been used to determine the
entry pathways of viruses, including the demonstration of
2058
co-localization of entering viruses with components of
the cellular endocytosis machinery and the use of chemical
inhibitors that affect different pathways of endocytosis
(Sieczkarski & Whittaker, 2002). The aim of the present
study was to determine the entry route of PCV2 into the
porcine monocytic cell line 3D4/31 (Weingartl et al., 2002)
by assessing the effect of different chemical entry inhibitors on PCV2 infection and by co-localization studies of
recombinant PCV2 virus-like particles (VLPs) with components of the endocytic pathway(s) mediating PCV2
internalization by fluorescent confocal microscopy.
METHODS
Virus. PCV2 strain Stoon-1010 isolated from a PMWS-affected
piglet in Canada (Ellis et al., 1998) was used in this study. A virus
stock propagated in PK-15 cells with a titre of 105?5 TCID50 ml21
was used in all PCV2 infection experiments. Cells were inoculated
with PCV2 at an m.o.i. of 0?5.
Recombinant PCV2 VLPs. A clarified lysate of Spodoptera frugi-
perda 9 (Sf9) insect cells infected with a baculovirus recombinant
P054, expressing ORF2 of PCV2, was used as a source of PCV2
VLPs. PCV2 VLPs were purified in a caesium chloride gradient as
described previously (Nawagitgul et al., 2000). PCV2 VLPs were
used in binding and internalization studies instead of PCV2 virions
because of the difficulty in obtaining sufficient preparative amounts
of the latter, due to low PCV2 titres. The correct conformation
of PCV2 capsid proteins and PCV2 VLPs was demonstrated by
the reactivity of PCV2-specific monoclonal antibodies F217 and
F190 (McNeilly et al., 2001) and monospecific porcine polyclonal
antibodies against PCV2 (Sanchez et al., 2003), and by electron
microscopy. Negative-staining electron microscopy was performed
according to the method of Nawagitgul et al. (2000) with 1 % phosphotungstic acid. Electron microscopy revealed the presence of
single PCV2 VLPs (65 %), the rest being aggregates of two or more
PCV2 VLPs (Fig. 1f and g). In parallel, different dilutions of PCV2
VLPs were smeared onto microscope slides (Menzel-Gläser), airdried and fixed with 3 % (w/v) paraformaldehyde in PBS with calcium and magnesium (PBS+) at room temperature for 10 min.
PCV2 VLPs were then stained by using biotin-conjugated anti-PCV2
swine polyclonal antibodies (Sanchez et al., 2003) followed by fluorescein isothiocyanate (FITC)-conjugated streptavidin (Molecular
Probes) for 1 h each at room temperature. For comparison purposes, 4?561010 particles ml21 of 20 nm yellow–green-fluorescent
(wavelength 505/515 nm) and 3?66108 particles ml21 of 100 nm
red-fluorescent (wavelength 580/605 nm) carboxylate-modified
microspheres (FluoSpheres; Molecular Probes) were used. Stained
PCV2 VLPs and fluorospheres were mounted with a glycerol solution containing 1,4-diazabicyclo(2.2.2)octane (DABCO) anti-fading
agent. Digital images of stained PCV2 VLPs and fluorospheres were
acquired at the same magnification, using a Leica TCS SP2 laserscanning spectral confocal system linked to a Leica DM/IRB inverted
microscope (Fig. 1a–c). The fluorescence area of individual fluorescence spots in images of stained PCV2 VLPs and of 20 and 100 nm
fluorospheres was calculated by using image-analysis software
(SigmaScan Pro 5.0; Jandel Scientific) and their distribution is
shown in Fig. 1(d). The number of PCV2 VLP fluorescence spots
varied as a function of dilution (data not shown). The majority
(55 %) of PCV2 VLP fluorescence spots had an area corresponding
to that of 20 nm fluorospheres (Fig. 1d). However, some PCV2 VLP
fluorescence spots had sizes larger than that of 20 nm fluorospheres,
indicating the presence of small and large PCV2 VLP aggregates.
Both fluorescent confocal and electron microscopy gave a comparable PCV2 VLP distribution (Fig. 1d and e).
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Journal of General Virology 86
PCV2 binding and entry
Fig. 1. Visualization and analysis of PCV2 VLPs by fluorescent confocal and electron microscopy. (a–c) Representative
fluorescent confocal images of immunostained PCV2 VLPs (a) and 20 nm yellow–green-fluorescent (b) and 100 nm redfluorescent (c) carboxylate-modified microspheres acquired at the same magnification. Bar, 8 mm. (d) Fluorescence area
distribution of PCV2 VLPs (%) and 20 nm (thin line) and 100 nm (thick line) fluorescent carboxylate-modified microspheres
calculated from fluorescent confocal microscopy images by using SigmaScan Pro 5.0. (e) Distribution of PCV2 VLPs based
on images from electron microscopy. (f, g) Electron microscopy images of PCV2 VLPs. Bar, 500 nm.
Cells. The continuous porcine monocytic cell line 3D4/31, developed from porcine alveolar mw (Weingartl et al., 2002), was grown
in monocyte/mw medium comprising a 1 : 1 mixture of RPMI 1640
(Gibco-BRL) and minimal essential medium containing Earle’s
salts (Gibco-BRL) supplemented with 10 % fetal bovine serum
(FBS), 0?3 mg glutamine ml21, 100 U penicillin ml21, 0?1 mg streptomycin ml21, 0?1 mg kanamycin ml21 and 1 % non-essential
amino acids (1006; Gibco-BRL) in a humidified incubator at 37 uC
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in the presence of 5 % CO2. Cells were seeded at 26105 cells ml21
in monocyte/mw medium onto microscope slides mounted with
an eight-well cell-culture silicone chamber (Vivascience AG) for all
PCV2 infection experiments. For experiments involving the use of
PCV2 VLPs, cells were seeded at 1?56105 cells ml21 in monocyte/
mw medium onto microscope slides mounted with a 12-well cellculture silicone chamber (Vivascience AG). In both cases, cells were
cultivated for 24 h before the experiments were performed.
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G. Misinzo and others
Binding kinetics of PCV2 VLPs on 3D4/31 cells. In order to
establish the binding kinetics of PCV2 VLPs in 3D4/31 cells, cells
were chilled on ice and washed with cold RPMI 1640 before the
addition of PCV2 VLPs to the cells. PCV2 VLPs were added at a
concentration of 2?761010 particles in 100 ml RPMI 1640 onto 3D4/
31 cells and incubated for 0, 1, 5, 10, 15, 30 and 60 min at 4 uC.
Cells were then washed to remove unbound PCV2 VLPs before they
were fixed with 3 % (w/v) paraformaldehyde in PBS+ at room temperature for 10 min. In order to stain the PCV2 VLPs, cells were
incubated with biotin-conjugated anti-PCV2 swine polyclonal antibodies (Sanchez et al., 2003) for 1 h at room temperature before
they were washed and incubated with FITC-conjugated streptavidin
(diluted 1 : 100 in PBS; Molecular Probes) for 1 h at room temperature. The cells were mounted and analysed by acquisition of digital
images of stained PCV2 VLPs by fluorescent confocal microscopy. Successive images from the apex to the base of a single cell
were taken and merged. The total fluorescence area of PCV2 VLPs
attached per cell was calculated by using SigmaScan Pro 5.0 for
20 cells at each time point to establish their binding kinetics onto
3D4/31 cells.
Internalization of PVC2 VLPs into 3D4/31 cells. To study the
internalization of PCV2 VLPs, 3D4/31 cells were washed with RPMI
1640 at 37 uC and incubated with PCV2 VLPs at either 4 or 37 uC
for 15 min, followed by washing of unbound PCV2 VLPs with
RPMI 1640. Cells were then further incubated at 37 uC in monocyte/
mw medium without FBS. At 15, 60, 120, 180 and 360 min postincubation of cells with PCV2 VLPs, cells were fixed with 3 % (w/v)
paraformaldehyde in PBS+ for 10 min at room temperature. Cells
were subsequently washed with PBS+ and permeabilized with
Triton X-100 (0?1 % in PBS+) for 2 min at room temperature.
After washing, PCV2 VLPs were stained by incubating the cells for
1 h at room temperature with polyclonal biotin-conjugated antiPCV2 swine antibodies (Sanchez et al., 2003) followed by 1 h incubation at room temperature with FITC-conjugated streptavidin
(1 : 100 in PBS; Molecular Probes). In order to visualize the cell
border, actin filaments were stained by incubating the cells for 1 h
at 37 uC with phalloidin–Texas red (1 : 100 in PBS; Molecular
Probes). Stained cells were mounted and analysed by fluorescent
confocal microscopy. PCV2 VLPs were scored as internalized once
they crossed the cortical actin rim, based on merged confocal
images. The fluorescence area of internalized and non-internalized
PCV2 VLPs was calculated by using SigmaScan Pro 5.0 for 40 cells
at each time point to establish their internalization kinetics into
3D4/31 cells.
order to determine cell viability, cells were incubated with different
concentrations of inhibitors and washed with RPMI 1640, followed
by incubation with 20 mg propidium iodide ml21 for 10 min to
stain dead cells.
Potassium depletion combined with hypotonic shock, cytosol acidification and hypertonic medium together with the appropriate controls were carried out as described by Hansen et al. (1993). Briefly,
potassium depletion combined with hypotonic shock was done by
rinsing and pre-incubating the cells for 15 min with potassiumdepletion buffer (0?14 M NaCl, 20 mM HEPES, 1 mM CaCl2, 1 mM
MgCl2, 1 mg D-glucose ml21, pH 7?4) before incubating the cells
with a hypotonic buffer (potassium-depletion buffer diluted 1 : 1 with
water to make it hypotonic) for 5 min (Hansen et al., 1993; Koval et al.,
1998). Control cells were incubated in the same buffer supplemented
with 10 mM KCl. Cells were treated with a hypertonic medium that
consisted of Dulbecco’s modified Eagle’s medium (DMEM) without sodium bicarbonate (Gibco-BRL) supplemented with 2 mM
L-glutamine, 20 mM HEPES and 0?45 M sucrose. Control cells were
incubated in hypertonic medium lacking sucrose. For cytosol acidification, 1 M acetic acid, pH 5?0 (NaOH), diluted 1 : 100 in DMEM
without sodium bicarbonate supplemented with 2 mM L-glutamine
and 20 mM HEPES, pH 5?0 (HCl) was added, whereas control cells
were incubated in DMEM without sodium bicarbonate supplemented with 2 mM L-glutamine, 20 mM HEPES, pH 5?0. After the
treatments, cells were inoculated with PCV2. At 1 h post-inoculation,
the viral inoculum was removed and cells were washed and further
incubated in monocyte/mw medium for 24 h before fixation with
methanol (10 min at 220 uC).
PCV2-infected cells were detected by immunofluorescence staining using a biotin-conjugated anti-PCV2 swine polyclonal antibody
(Sanchez et al., 2003) and FITC-conjugated streptavidin (diluted
1 : 100 in PBS; Molecular Probes). Incubations were done at room
temperature for 1 h. Cell nuclei were detected by incubating the cells
for 10 min at room temperature with Hoechst 33342 (Molecular
Probes) at a concentration of 10 mg ml21. After each of the incubations, cells were washed with PBS. Finally, stained cells were mounted
and analysis of the percentage of PCV2-infected cells was done by using
a Leica DM/RBE fluorescence microscope (see Fig. 4a). Approximately
10 000 cells were evaluated under each experimental condition and
the data presented in this work are the results of independent
triplicates. Differences between mean inhibitions with the different
chemical inhibitors were analysed by using Student’s two-tailed t-test
with SPSS (version 6.1) software. A value of P<0?05 was considered
significant.
Effect of different entry inhibitors on PCV2 infection. 3D4/
31 cells were susceptible to PCV2 infection and supported PCV2
replication with kinetics comparable to those observed in PK-15
cells (Meerts et al., 2005). In order to investigate the entry route of
PCV2 and a possible role of endosomal acidification in PCV2
infection of 3D4/31 cells, various chemicals that selectively disrupt
cellular internalization pathways were used as shown in Table 1.
Semi-confluent cells were washed and pre-incubated with twofold
serial dilutions in monocyte/mw medium of one of the following
chemicals: ammonium chloride, amantadine, chloroquine diphosphate, cytochalasin D, latrunculin B, nystatin and/or amiloride for
1 h at 37 uC. Control cells were incubated in monocyte/mw medium
for 1 h at 37 uC. Control cells and treated cells were then inoculated with PCV2 at an m.o.i. of 0?5 at 37 uC for 1 h, followed by
washing away of the inoculum with RPMI 1640. Thereafter, cells
were further incubated for 24 h in monocyte/mw medium before
they were fixed. Some of the inhibitors were maintained in the
monocyte/mw medium during the course of infection (see Table 1).
The concentrations of the inhibitors that were used were based on
concentrations described in previous studies (see references in
Table 1), provided that they were not toxic for the 3D4/31 cells. In
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Visualization of clathrin-mediated endocytosis of PCV2. Cells
were washed with RPMI 1640 and incubated with either 2?761010
PCV2 VLPs in 100 ml monocyte/mw medium without FBS containing biotinylated transferrin at 100 mg ml21 or monocyte/mw
medium without FBS alone for 5, 15, 30 and 60 min. Cells were
then washed with RPMI 1640, fixed with 3 % (w/v) paraformaldehyde in PBS+ for 20 min at room temperature, washed again first
with RPMI 1640 followed by Tris-buffered saline (20 mM Tris/HCl,
150 mM NaCl, pH 7?5) with 4?5 % sucrose and 2 % inactivated goat
serum (TBS-GS) and permeabilized with methanol for 30 s at
220 uC (Racoosin & Swanson, 1994). Clathrin was stained by incubating the cells for 1 h at 37 uC with anti-clathrin heavy-chain IgM
antibodies (ICN Biomedicals) diluted 1 : 50 in PBS supplemented
with 0?3 % gelatin (PBS-G). Afterwards, cells were washed in TBSGS and incubated for 1 h at 37 uC with biotin-labelled goat antimouse IgM (Santa Cruz Biotechnology) diluted 1 : 100 in PBS-G.
After washing with TBS-GS, cells were incubated for 1 h at 37 uC
with streptavidin–Texas red (Molecular Probes) diluted 1 : 50 in
PBS-G and washed in TBS-GS (Van de Walle et al., 2001). PCV2
VLPs were stained using anti-PCV2 swine polyclonal antibodies
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Journal of General Virology 86
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Table 1. Inhibitors of endocytosis and their modes of action
Chemical inhibitor
or method
Presence of inhibitor
Concentration
range
Stage of
inhibition
Postinoculation
Hypertonic medium
(sucrose)
+
2
Potassium depletion
combined with
hypotonic shock
Cytosol acidification
+
2
Clathrin-mediated
endocytosis
+
2
Amantadine
+
+
23?4–750 mM
Clathrin-mediated
endocytosis
Clathrin-mediated
endocytosis
Cytochalasin D
+
2
1?56–50 mM
Actin-dependent
processes
Latrunculin B
+
2
2?96–95 mM
Actin-dependent
processes
Amiloride
+
2
0?375–6 mM
Macropinocytosis
Nystatin
+
2
6?25–50 mM
Caveolae-mediated
endocytosis
Ammonium
chloride
+
+
3?75–30 mM
Endosomal
acidification
Chloroquine
diphosphate
+
+
3?12–50 mM
Endosomal
acidification
0?45 mM
Clathrin-mediated
endocytosis
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References
Blocks clathrin-coated pit formation and
interferes with interaction between
clathrin and adaptors
Arrests coated pit formation and interferes
with interaction between clathrin and
adaptors
Prevents invaginated clathrin-coated
domains from pinching off
Stabilizes clathrin-coated vesicles, thereby
limiting their fusion with other membranes
and the formation of new ones
Caps high-affinity ends of filamentous actin
(F-actin), thereby retarding actin
polymerization
Specific sequestration of monomeric actin
and binds to globular actin (G-actin)
in vitro, inhibiting polymerization of
these actin building blocks, and promotes
depolymerization of F-actin
A selective inhibitor of membrane protein
Na+/H+ antiporters, described to
selectively inhibit macropinocytosis and
membrane ruffling without affecting
clathrin-mediated endocytosis
A sterol-binding agent that depletes
membrane cholesterol, which is important
for both maintenance of caveolae and the
ability of caveolae to seal off from the
plasma membrane
A weak lysosomotropic base that diffuses
into acidic endosomes, where it becomes
protonated. Once protonated, it is unable
to diffuse out, thereby increasing the pH
A weak base that becomes protonated once
in the endosomal vesicles, raising the pH
in these compartments
Hansen et al. (1993); Heuser & Anderson
(1989)
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Hansen et al. (1993); Larkin et al. (1983)
Hansen et al. (1993); Sandvig et al.
(1987)
Gonzalez-Dunia et al. (1998); Phonphok
& Rosenthal (1991); Van de Walle et al.
(2001)
Brown & Spudich (1979); Cooper (1987);
Flanagan & Lin (1980); Fujimoto et al.
(2000); Van de Walle et al. (2001)
Fujimoto et al. (2000); Spector et al.
(1983); Van de Walle et al. (2001)
Muro et al. (2003); West et al. (1989);
Yan et al. (2004)
Akula et al. (2003); Rothberg et al.
(1992); Sieczkarski & Whittaker (2002)
Akula et al. (2003); Gonzalez-Dunia
et al. (1998); Maxfield (1982); Ohkuma
& Poole (1978); Rodrı́guez & Everitt
(1996)
Gonzalez-Dunia et al. (1998); Maxfield
(1982); Ohkuma & Poole (1978);
Rodrı́guez & Everitt (1996)
PCV2 binding and entry
Pre-incubation
and inoculation
Mode of action
G. Misinzo and others
(diluted 1 : 100 in PBS-G) and FITC-labelled goat anti-swine antibodies (Jackson ImmunoResearch Laboratories) diluted 1 : 100 in PBSG. Finally, stained cells were mounted and analysed by fluorescent
confocal microscopy.
RESULTS
Kinetics of PCV2 VLP binding to 3D4/31 cells
The time course of binding of PCV2 VLPs to 3D4/31 cells
was examined by incubating the cells with PCV2 VLPs at
4 uC for different durations, followed by staining for the
PCV2 VLPs and acquisition of serial images across the
cell by fluorescent confocal microscopy. The results of
the interaction between PCV2 VLPs and 3D4/31 cells
showed that single and aggregate forms of PCV2 VLPs
bound to all of the 3D4/31 cells. The kinetics of binding
of PCV2 VLPs to 3D4/31 cells were similar and timedependent in all cells. The binding of PCV2 VLPs to
3D4/31 cells increased quickly within 5 min and reached a
plateau starting from 15 min (Fig. 2a). These results showed
that PCV2 VLPs bound to a saturable number of receptors on the surface of 3D4/31 cells. PCV2 VLPs bound to
3D4/31 cells with a random distribution over the entire cell
surface (Fig. 2b).
Internalization of PCV2 VLPs into 3D4/31 cells
Many viruses are able to bind non-specifically to cells, which
does not usually result in virus uptake and infection of a
cell (Tardieu et al., 1982). To determine whether PCV2
VLP binding was followed by internalization, PCV2 VLPs
were bound to 3D4/31 cells at 37 uC for 15 min; unbound
PCV2 VLPs were then washed off and the cells were
incubated at 37 uC for up to 360 min (Fig. 3). Internalization assays were carried out, starting with the binding
of PCV2 VLPs at 37 uC instead of 4 uC, as it was shown that
the internalization machinery of 3D4/31 cells was damaged
after incubating them at 4 uC, as judged by transferrin
internalization. PCV2 VLPs that crossed the cortical actin
were scored as internalized (Fig. 3c). PCV2 VLPs were
internalized slowly into 3D4/31 cells, with 47?3±5?0 % of
the 3D4/31 cells internalizing PCV2 VLPs during the course
of the experiment (360 min) (Fig. 3a). In cells that showed
internalization, the number of internalized PCV2 VLPs
per cell increased gradually from 4?7±3?6 % at 15 min to
70?5±9?7 % at 360 min post-incubation (Fig. 3b). Single
and aggregate forms of PCV2 VLPs crossed the cortical
actin, indicating that both forms were internalized. Some
cells showed complete internalization of the PCV2 VLPs at
120, 180 and 360 min post-incubation.
Fig. 2. PCV2 VLP binding to 3D4/31 cells. Cells were incubated with PCV2 VLPs at 4 6C for 1, 5, 10, 15, 30 or 60 min
before they were washed and fixed with 3 % (w/v)
paraformaldehyde in PBS+ for 10 min at room temperature. A
graphical representation of PCV2 VLP binding kinetics is
shown in (a) and the respective pictographical representation is
shown in (b). Each value in (a) represents the mean±SD fluorescence area of attached PCV2 VLPs per cell, as calculated
by SigmaScan Pro 5.0 from 20 cells. Each panel in (b) represents an overlay of a series of confocal images taken from the
apex to the base of a single cell. Bar, 8 mm.
Maximum reductions of 73?0±6?0 and 75?5±7?0 %
were found at concentrations of 25 and 50 mM cytochalasin
D, respectively. Latrunculin B blocked PCV2 infection
by 67?4±2?6 and 71?0±3?0 % at concentrations of 47?5
and 95 mM, respectively. PCV2 infection was decreased by
51?0±5?3 and 51?2±4?4 % at 6?25 mM cytochalasin D and
11?87 mM latrunculin B, respectively.
Disruption of actin inhibits PCV2 infection
Inhibition of caveolae-mediated endocytosis
and macropinocytosis does not decrease PCV2
infection significantly
3D4/31 cells were treated with cytochalasin D and latrunculin B to investigate the effect of actin polymerization
on PCV2 infection. Cytochalasin D and latrunculin B
treatments of 3D4/31 cells reduced PCV2 infection in a
dose-dependent manner, as indicated in Fig. 4(b).
3D4/31 cells were treated with nystatin and amiloride to
investigate the involvement of caveolae-mediated endocytosis and macropinocytosis in PCV2 infection, respectively.
Amiloride and nystatin did not reduce PCV2 infection
significantly. Treatment of the cells at concentrations of 12?5
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PCV2 binding and entry
Fig. 3. Analysis of PCV2 VLP internalization. 3D4/31 cells were incubated with PCV2 VLPs at 37 6C for 15, 60, 120, 180 or
360 min. Cells were then fixed with 3 % (w/v) paraformaldehyde in PBS+ and permeabilized. Cells were analysed by
fluorescent confocal microscopy after performing a double immunofluorescence staining to visualize PCV2 VLPs (FITC signal)
and actin (Texas red signal). In (a), the percentage of cells that showed PCV2 VLP internalization at each time point is shown,
whilst (b) represents the percentage of PCV2 VLPs internalized per cell showing internalization. The images in (c) represent
single sections through a cell at 15, 60, 180 and 360 min. PCV2 VLPs (left panels) were scored as internalized (arrowheads)
once they were inside the cortical actin rim (middle panel) based on merged images (right panel). Bar, 8 mm.
and 25 mM nystatin resulted in 4?4±7?2 and 5?9±9?4 %
reduction of PCV2 infection, respectively (Fig. 5). The
inhibitory effect of nystatin on caveolae-mediated endocytosis in 3D4/31 cells was verified by using cholera toxin. The
percentage of cholera toxin that was internalized into 3D4/
31 cells after 30 min incubation at 37 uC was 67?5±22?6 %.
Internalized cholera toxin was reduced to 11?4±5?4 %
when 3D4/31 cells were treated with 25 mM nystatin. The
maximum inhibition of PCV2 infection from all tested
concentrations of amiloride was 2?4±4?6 % at 1?5 mM
(Fig. 5).
involvement of clathrin-mediated endocytosis during
the initial stages of PCV2 infection. Potassium depletion
combined with hypotonic shock decreased PCV2 infection by 50?2±6?3 %, whereas treatment of the cells with a
hypertonic medium resulted in 56?4±5?7 % reduction.
Acidification of the cytosol reduced PCV2 infection by
59?1±7?1 %. When the 3D4/31 cells were treated with
different concentrations of amantadine, PCV2 infection
was reduced in a dose-dependent manner. The percentage
inhibition of PCV2 infection ranged from 35?7±11?2 to
52?6±6?7 % when the cells were treated with concentrations ranging from 93?75 to 750 mM amantadine (Fig. 5).
Inhibition of clathrin-mediated endocytosis
reduces PCV2 infection
PCV2 VLPs co-localize with clathrin
Cells were treated with hypertonic medium, potassium
depletion combined with hypotonic shock, cytosol acidification and amantadine in order to investigate the
Visualization of the association of PCV2 VLPs and the
cellular (clathrin-mediated) endocytic machinery was
done by using fluorescent confocal microscopy. Clathrin
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G. Misinzo and others
Fig. 4. (a) Detection of PCV2-infected porcine monocytic 3D4/31 cells. 3D4/31 cells
were inoculated with PCV2 strain Stoon1010 at an m.o.i. of 0?5 for 1 h at 37 6C.
The viral inoculum was then washed off
and cells were incubated in monocyte/mw
medium before they were fixed at 24 h
post-inoculation. The left panel shows 3D4/
31 cells stained with anti-PCV2 swine polyclonal antibodies to detect PCV2-infected
cells. The middle panel shows 3D4/31 cell
nuclei following Hoechst 33342 staining,
acquired at the same microscopic field as in
the left panel. The merged image of both
stainings in order to obtain the percentage
of PCV2-infected cells is shown in the right
panel. (b) Effect of actin disruption by
cytochalasin D and latrunculin B on PCV2
infection. Semi-confluent monolayers of 3D4/
31 cells were incubated with monocyte/mw
medium or monocyte/mw medium containing
various concentrations of cytochalasin D (&)
and latrunculin B (%) for 1 h at 37 6C. Cells
were then infected with PCV2 in the presence or absence of the inhibitors at 37 6C
for 1 h. The viral inoculum was then washed
away and cells were further incubated with
monocyte/mw medium for 24 h at 37 6C.
Infection was evaluated by counting viral
antigen-positive cells. Data are presented
as percentage inhibition of virus infection
compared with control cells (incubated
with monocyte/mw medium in the absence
of inhibitors). Each point represents the
mean±SD of three experiments.
co-localized with PCV2 VLPs in 3D4/31 cells at 30 and
60 min post-incubation, as indicated in Fig. 6. At earlier
times before 30 min, few or no PCV2 VLPs were seen to colocalize with clathrin. Clathrin, but not PCV2 VLP, staining
was observed in 3D4/31 cells not incubated with PCV2
VLPs and stained for clathrin and for PCV2 VLPs (Fig. 6).
Similarly, cells incubated with PCV2 VLPs and stained
for clathrin and PCV2 VLPs using negative swine serum
instead of anti-PCV2 swine polyclonal antibodies also
showed clathrin, but not PCV2 VLP, staining. As a control,
Fig. 5. Effect of inhibition of clathrin- and caveolae-mediated
endocytosis and macropinocytosis by amantadine, nystatin
and amiloride, respectively, on PCV2 infection. Semi-confluent
monolayers of 3D4/31 cells were incubated with monocyte/mw
medium or monocyte/mw medium containing various concentrations of amantadine (#), nystatin (%) or amiloride (&) for 1 h
at 37 6C. Cells were then infected with PCV2 in the presence
or absence of the inhibitors at 37 6C for 1 h. Subsequently, the
viral inoculum was washed away and cells were further incubated with monocyte/mw medium for 24 h at 37 6C. Infection
was evaluated by counting viral antigen-positive cells. Data are
presented as percentage inhibition of virus infectivity observed
when the cells were incubated with the virus in monocyte/mw
medium in the absence of inhibitors. Each point represents the
mean±SD of three experiments.
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Journal of General Virology 86
PCV2 binding and entry
Fig. 6. PCV2 VLP co-localization with clathrin. 3D4/31 cells were incubated with
PCV2 VLPs in monocyte/mw medium without
FBS at 37 6C for 30 or 60 min before they
were fixed with 3 % (w/v) paraformaldehyde
in PBS+ for 20 min at room temperature,
washed and permeabilized with methanol for
30 s at ”20 6C. Control cells were incubated in monocyte/mw medium without FBS
and PCV2 VLPs for 30 min before they
were fixed and permeabilized. Double
immunofluorescence staining to detect
PCV2 VLPs (left panels) and clathrin (middle
panels) was performed in cells incubated
with and without PCV2 VLPs. Cells were
washed, mounted and analysed by fluorescent confocal microscopy. Arrowheads indicate co-localization between PCV2 VLP and
clathrin in merged images (right panels).
Bar, 8 mm.
co-localization of transferrin with clathrin was studied.
Transferrin was internalized and co-localized with clathrin
at 5, 15 and 30 and 60 min post-incubation.
Inhibition of acidification of endosomes
reduces PCV2 infection
Cells were treated with the lysosomotropic weak bases
chloroquine diphosphate and ammonium chloride in order
to establish the role of endosomal acidification on PCV2
infection. The degree of PCV2 infection inhibition by
both inhibitors was dose-dependent, as shown in Fig. 7.
Treatment of the cells with ammonium chloride reduced
PCV2 infection maximally by 47?0±7?9 % at 30 mM and
chloroquine diphosphate by 49?0±5?6 % at 50 mM.
DISCUSSION
The present study was undertaken to determine the entry
pathway of PCV2 VLPs and virions into the porcine
monocytic cell line 3D4/31. By using fluorescent confocal
microscopy, it was demonstrated in this study that PCV2
VLPs: (i) attached to the plasma membrane of all 3D4/
31 cells in a rapid, time-dependent manner, giving rise to
a random distribution pattern on the cell surface; (ii)
internalized in 47?3±5?0 % of the 3D4/31 cells; and (iii) colocalized with clathrin during entry. Furthermore, it was
observed that PCV2 infection of 3D4/31 cells was inhibited by: (i) chemical inhibition methods that perturbed
clathrin-mediated endocytosis (including hypertonic
medium, potassium depletion combined with hypotonic
http://vir.sgmjournals.org
Fig. 7. Effect of inhibition of endosomal acidification by ammonium chloride and chloroquine diphosphate on PCV2 infection.
Semi-confluent monolayers of 3D4/31 cells were incubated
with monocyte/mw medium or monocyte/mw medium containing
various concentrations of ammonium chloride (&) or chloroquine diphosphate (%) for 1 h at 37 6C. Cells were then
infected with PCV2 in the presence or absence of inhibitor at
37 6C for 1 h. The viral inoculum was then washed away and
cells were further incubated with monocyte/mw medium for
24 h at 37 6C. Infection was evaluated by counting the number
of viral antigen-positive cells. Data are presented as percentage inhibition of virus infectivity observed when the cells were
incubated with the virus in monocyte/mw medium in the
absence of inhibitors. Each point represents the mean±SD of
three experiments.
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2065
G. Misinzo and others
shock, cytosol acidification and amantadine); (ii) actin
disruption using cytochalasin D and latrunculin B; and
(iii) lysosomotropic weak bases (ammonium chloride
and chloroquine diphosphate) that prevent endosomal
acidification.
To characterize the entry pathway of PCV2 VLPs into 3D4/
31 cells, the attachment of PCV2 VLPs to the plasma
membrane, an initial step in the virus infectious cycle, was
first studied. PCV2 VLPs bound to the plasma membrane
of all cells in a time-dependent manner, saturating the
receptors within the first 15 min of incubation. The saturation of binding of PCV2 VLPs to 3D4/31 cells indicated
that the PCV2 VLPs were interacting with cell-surface
receptors. One of the criteria for viral recognition sites as
receptors is saturability, the other criterion being specificity (Tardieu et al., 1982). The binding of PCV2 VLPs to
all cells indicated that all cells expressed the attachment
receptors for PCV2 at the time that the binding study
was performed (24 h post-seeding). Furthermore, it was
observed that PCV2 VLPs were distributed randomly
throughout the surface of the plasma membrane of 3D4/
31 cells, indicating that PCV2 attachment receptors have
a similar distribution pattern. A specific, saturable binding
of virions to the cell surface does not necessarily result in
virus uptake, as some viruses require interaction with more
than one cell-surface molecule to be internalized (Li et al.,
1995; Roden et al., 1994). It was examined whether PCV2
VLPs that bound to the surface of 3D4/31 cells could subsequently be internalized by 3D4/31 cells. The results
obtained in this study showed that, after 360 min incubation, only 47?3±5?0 % of the 3D4/31 cells internalized
PCV2 VLPs. Although internalization of the PCV2 VLPs
into cells showed a high variation, a time-dependent course
was found. The binding and internalization of purified
PCV2 virions in 3D4/31 cells closely resembled that of
PCV2 VLPs (data not shown). It remains to be examined
whether other cells known to be susceptible to PCV2
infection internalize PCV2 VLPs with characteristics similar to those described in this study with 3D4/31 cells.
Together with the need for cellular polymerase, the restricted number of cells that allowed complete internalization of PCV2 VLPs may be an important cause of the very
low percentage of target cells that are infected with PCV2 in
vitro. Furthermore, the slow internalization may be the
basis for the long replication cycle (24–36 h) of PCV2 in
susceptible cells (Meerts et al., 2005). Whether the internalization of PCV2 VLPs observed in 47?3±5?0 % of the
cells is sufficient to lead to infection in all of these cells could
not be examined in this study. The proportion of PCV2
antigen-positive cells observed after in vitro inoculation of
3D4/31 cells with PCV2 at an m.o.i. of 5 was approximately 6 %, implying that other factors, in addition to the
internalization step, probably also govern the outcome of
PCV2 infection.
The experimental results presented here demonstrate that
clathrin-mediated endocytosis is involved in the entry
2066
process of PCV2 into 3D4/31 cells based on: (i) colocalization of clathrin with PCV2 VLPs, shown by fluorescent confocal microscopy; and (ii) inhibition of PCV2
infection by inhibitors of clathrin-mediated endocytosis.
The blocking of PCV2 infection following the disruption of
actin in 3D4/31 cells suggests that actin reorganization is
involved in PCV2 entry. A role for the actin cytoskeleton
in clathrin-mediated endocytosis has been shown by
numerous studies (Durrbach et al., 1996; Jeng & Welch,
2001; Merrifield et al., 2002). However, disruption of the
actin cytoskeleton gave greater inhibition of PCV2 infection than inhibitors for clathrin-mediated endocytosis. This
suggests the involvement of other actin-dependent processes, in addition to clathrin-mediated endocytosis, in
PCV2 entry. Inhibition of macropinocytosis with amiloride
and caveolae-mediated endocytosis with nystatin did not
affect PCV2 infection significantly, suggesting that these
endocytic pathways are not involved in PCV2 internalization into 3D4/31 cells.
Early endosomes arise following the ATP-driven uncoating
of clathrin-coated vesicles, and these vesicles mature into
late endosomes and lysosomes. This is accompanied by
a gradual pH drop that aids uncoating and escape into
the cytoplasm for some viruses (Bomsel & Alfsen, 2003;
Kirchhausen, 2000). The lysosomotropic weak bases ammonium chloride and chloroquine diphosphate clearly reduced
PCV2 infection, indicating that endosomal acidification is
necessary for successful PCV2 infection.
It can be concluded from these results that PCV2 enters
monocytic cells predominantly via clathrin-mediated endocytosis and that endosomal acidification is an important
requirement in PCV2 infection. Understanding the entry
pathway of PCV2 can serve as an important basis for the
screening of antiviral agents and the future development
of antiviral strategies.
ACKNOWLEDGEMENTS
The authors wish to acknowledge Francis McNeilly and Gordon Allan
(Department of Agriculture and Rural Development for Northern
Ireland, Veterinary Sciences Division, UK) for kindly providing the
PCV2-specific mAbs F217 and F190. The authors would also like to
thank Chris Bracke, Carine Boone and Chantal Vanmaercke for their
excellent technical assistance.
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