1 Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2 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
Correspondence
H. J. Nauwynck
hans.nauwynck{at}Ugent.be
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ABSTRACT |
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INTRODUCTION |
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Recently, PCV2 target cells have been shown to be cardiomyocytes, hepatocytes and macrophages (m) during fetal life and mainly monocytes/m
in early post-natal life (Sanchez et al., 2003
). In experimentally inoculated pigs, the 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 m
. In addition to differentiated m
, 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 (Engqvist-Goldstein & 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 (Martínez 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 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.
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METHODS |
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Recombinant PCV2 VLPs.
A clarified lysate of Spodoptera frugiperda 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), air-dried 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, 4·5x1010 particles ml1 of 20 nm yellowgreen-fluorescent (wavelength 505/515 nm) and 3·6x108 particles ml1 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 laser-scanning 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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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 2·7x1010 particles in 100 µl RPMI 1640 onto 3D4/31 cells and incubated for 0, 1, 5, 10, 15, 30 and 60 min at 4 °C. 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 °C and incubated with PCV2 VLPs at either 4 or 37 °C for 15 min, followed by washing of unbound PCV2 VLPs with RPMI 1640. Cells were then further incubated at 37 °C in monocyte/m medium without FBS. At 15, 60, 120, 180 and 360 min post-incubation 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 anti-PCV2 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 °C with phalloidinTexas 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.
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/m
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 °C. Control cells were incubated in monocyte/m
medium for 1 h at 37 °C. Control cells and treated cells were then inoculated with PCV2 at an m.o.i. of 0·5 at 37 °C for 1 h, followed by washing away of the inoculum with RPMI 1640. Thereafter, cells were further incubated for 24 h in monocyte/m
medium before they were fixed. Some of the inhibitors were maintained in the monocyte/m
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 order to determine cell viability, cells were incubated with different concentrations of inhibitors and washed with RPMI 1640, followed by incubation with 20 µg propidium iodide ml1 for 10 min to stain dead cells.
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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 µg ml1. 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.
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RESULTS |
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Inhibition of caveolae-mediated endocytosis and macropinocytosis does not decrease PCV2 infection significantly
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 and 25 µM 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 °C was 67·5±22·6 %. Internalized cholera toxin was reduced to 11·4±5·4 % when 3D4/31 cells were treated with 25 µM nystatin. The maximum inhibition of PCV2 infection from all tested concentrations of amiloride was 2·4±4·6 % at 1·5 mM (Fig. 5
).
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PCV2 VLPs co-localize with clathrin
Visualization of the association of PCV2 VLPs and the cellular (clathrin-mediated) endocytic machinery was done by using fluorescent confocal microscopy. Clathrin 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 co-localize 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, 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.
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DISCUSSION |
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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 (2436 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 process of PCV2 into 3D4/31 cells based on: (i) co-localization 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.
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ACKNOWLEDGEMENTS |
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Received 29 September 2004;
accepted 24 March 2005.