Journal
of General Virology (1999), 80, 297–305. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Entry of porcine reproductive and respiratory syndrome virus
into porcine alveolar macrophages via receptor-mediated
endocytosis
H. J. Nauwynck,1 X. Duan,1 H. W. Favoreel,1 P. Van Oostveldt2 and M. B. Pensaert1
1
Laboratory of Veterinary Virology, Faculty of Veterinary Medicine, University of Gent, Salisburylaan 133, B-9820 Merelbeke, Belgium
Laboratory of Biochemistry and Molecular Cytology, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Coupure
653, B-9000 Gent, Belgium
2
Porcine alveolar macrophages (AMΦ) are the dominant cell type that supports the replication of
porcine reproductive and respiratory syndrome virus (PRRSV) in vivo and in vitro. In order to
determine the characteristics of the virus–receptor interaction, the attachment of PRRSV to cells
was examined by using biotinylated virus in a series of flow cytometric assays. PRRSV bound
specifically to AMΦ in a dose-dependent manner. Binding of PRRSV to AMΦ increased gradually
and reached a maximum within 60 min at 4 mC. By confocal microscopy, it was shown that different
degrees of PRRSV binding exist and that entry is by endocytosis. Virus uptake in vesicles is a
clathrin-dependent process, as it was blocked by the addition of cytochalasin D and co-localization
of PRRSV and clathrin was found. Furthermore, by the use of two weak bases, NH4Cl and
chloroquine, it was demonstrated that PRRSV uses a low pH-dependent entry pathway. In the
presence of these reagents, input virions accumulated in large vacuoles, indicating that uncoating
was prevented. These results indicate that PRRSV entry into AMΦ involves attachment to a specific
virus receptor(s) followed by a process of endocytosis, by which virions are taken into the cell within
vesicles by a clathrin-dependent pathway. A subsequent drop in pH is required for proper virus
replication.
Introduction
Virus infection of cells begins with the recognition and
attachment of viral particles to specific receptors on the cell
surface membrane and is followed by the delivery of viral
genomic material into the appropriate subcellular compartment. In the case of enveloped animal viruses, viruses enter
cells either by direct fusion of the viral envelope with the
plasma membrane or by a process of endocytosis, where
virions are taken into the cell within vesicles and subsequently
undergo a pH-dependent fusion event (as reviewed by Marsh
& Helenius, 1989 ; Marsh & Pelchen-Matthews, 1994). Viruses
may be taken up by clathrin-coated vesicles, as for Semliki
Forest virus (SFV) (Marsh & Pelchen-Matthews, 1994), by
caveolae, as for simian virus 40 (SV40) (Anderson et al., 1996),
or by non-coated vesicles, as for lymphocytic choriomeningitis
virus (Borrow & Oldstone, 1994).
Author for correspondence : Hans Nauwynck.
Fax j32 9 264 7495. e-mail Hans.Nauwynck!rug.ac.be
0001-5685 # 1999 SGM
Porcine reproductive and respiratory syndrome virus
(PRRSV), a member of the family Arteriviridae, is an enveloped
RNA virus (Meulenberg et al., 1993 ; Conzelmann et al., 1993).
It demonstrates a high tropism for cells of the monocyte\
macrophage lineage both in vitro and in vivo. Of the porcine
cells tested, only alveolar macrophages (AMΦ) and some
cultivated peripheral blood monocytes (BMo) support productive replication of PRRSV (Benfield et al., 1992 ; Kim et al.,
1993 ; Voicu et al., 1994 ; Duan et al., 1997 a). The state of
differentiation and activation of macrophages dramatically
affects their susceptibility to PRRSV. When BMo and AMΦ are
aged in vitro, their susceptibility to PRRSV increases significantly, whereas infection can be completely blocked by
treatment of AMΦ with phorbol 12-myristate 13-acetate
(PMA) without changing the binding of virus to the cells
(Duan et al., 1997 b). Little is known about the regulation of
macrophage tropism and susceptibility to PRRSV.
PRRSV, like other enveloped viruses, infects cells after
attachment to a cellular receptor. A putative virus receptor has
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H. J. Nauwynck and others
been identified on AMΦ that determines part of the specific
virus macrophage tropism (Duan et al., 1998 a, b). The next
steps in the entry process of PRRSV have already been studied
in a non-porcine susceptible cell line, MARC-145. It was found
that endocytosis and low pH are necessary for proper virus
replication (Kreutz & Ackermann, 1996). However, these
findings are difficult to extrapolate to AMΦ. Indeed, in contrast
to cells of the continuous cell line MARC-145, AMΦ forms a
heterogeneous population of cells with many special biological
characteristics. Furthermore, monoclonal antibodies (MAbs)
against the putative receptor of PRRSV on AMΦ did not react
with MARC-145 cells, suggesting that PRRSV enters MARC145 and AMΦ cells after binding to different receptors.
In order to elucidate the initial interaction between PRRSV
and macrophages further, the rate of PRRSV attachment and
the specific mode of entry into AMΦ were examined in the
present study.
Methods
Cells. Porcine AMΦ were obtained from 4- to 6-week-old
conventional Belgian Landrace pigs from a PRRSV-negative herd
according to the method previously described by Wensvoort et al.
(1991). The cells consisted of 98 % macrophages, as demonstrated
with the marker 74.22.15.
MARC-145 cells were used to produce PRRSV in large quantities.
MARC-145 cells were maintained in Eagle’s minimum essential medium
(MEM) supplemented with 10 % foetal calf serum (FCS) and antibiotics.
Virus. Two PRRSV isolates were used ; the Lelystad strain of PRRSV
was kindly provided by G. Wensvoort (Institute for Animal Science and
Health, Lelystad, The Netherlands). A Belgian isolate of PRRSV,
designated as 94V360, was adapted to grow in MARC-145 cells. A
thirteenth passage of Lelystad strain of PRRSV grown in AMΦ cells (10&n'
TCID \ml) and a fifth passage of 94V360 grown in MARC-145 cells
&!
(10'n) TCID \ml) were used in this study. No difference was found in the
&!
percentage of infected AMΦ between the two virus strains when
inoculation was performed at the same m.o.i.
Virus purification and biotinylation. One batch of culture fluids
of PRRSV-infected (94V360) MARC-145 cells was clarified by centrifugation at 4000 g for 20 min at 4 mC. Virus was collected by
centrifugation at 75 000 g for 3 h in a Beckman T35 rotor. The pellet was
resuspended in 1\100 the original volume of TNE buffer (50 mM NaCl,
5 mM EDTA, 10 mM Tris–HCl, pH 7n4) and centrifuged on a 0n5–1n5 M
discontinuous sucrose gradient in an SW41 rotor at 110 000 g for 16 h.
After centrifugation, the virus band was harvested and its purity was
determined by SDS–PAGE. The titre of the resulting virus preparation
was approximately 5i10( TCID \ml, as determined by a CPE assay in
&!
MARC-145 cells. The protein concentration was 2n5 mg\ml, as determined by the Bradford assay with BSA as a standard (Guttenberger,
1994). The number of virus particles, as determined by negative-staining
electron microscopy, was 1–2i10"! per mg virus protein.
Purified PRRSV in solution was labelled with biotin by using a protein
biotinylation kit (Amersham). The virus pellets were resuspended in
biotinylation buffer (40 mM Na CO , pH 8n6) at a protein concentration
# $
of 1 mg\ml. After brief sonication, 40 µl biotin reagent was added per
mg PRRSV protein. The mixture was shaken for 1 h at 4 mC and the
reaction was terminated by addition of Tris–HCl (pH 8n5) to a final
CJI
concentration of 50 mM. Biotinylated virus was collected after purification on a Sephadex G-25 column and diluted in PBS at a concentration
of 0n2 mg\ml. Biotinylated virions were stored at k70 mC. Biotinylation
did not decrease the virus infectivity significantly (from 5i10( to
2n5i10( TCID \ml).
&!
Analysis of PRRSV binding to AMΦ by flow cytometry. To
characterize the attachment of PRRSV to AMΦ, direct virus-binding
studies were carried out with biotinylated PRRSV. AMΦ were washed
three times with cold PBS containing 0n2 % BSA (PBSA) (Sigma) and
incubated in a 96-well V-bottomed plate on ice for 10 min. Different
concentrations of biotinylated PRRSV in PBSA were mixed with 2i10&
cells per well and incubated on ice for 60 min. To determine the timecourse of virus binding, 2i10& cells were incubated with 5 µg
biotinylated PRRSV for 10, 20, 30, 40, 50, 60, 90, 120 and 150 min. The
cells were washed three times with cold PBSA and incubated with 50 µl
1 : 50-diluted streptavidin–fluorescein isothiocyanate (FITC) conjugate
(Amersham) for 60 min on ice. The cells were washed once and
resuspended in 1 % formaldehyde in PBS at room temperature for 1 min.
The cells were washed once in PBS plus 2 % FCS plus 1 µg propidium
iodide (PI) per ml and once in PBS plus 2 % FCS only. Finally, the relative
fluorescence intensity of each sample was scanned on a FACSCalibur
flow cytometer (Becton Dickinson), in which dead cells labelled with PI
were excluded. Specificity of binding was demonstrated in a competition
experiment, in which 2i10& AMΦ were pre-incubated with different
amounts of unlabelled virus in a final volume of 100 µl before performing
the PRRSV-binding assay as described above.
Saturability of binding was analysed by incubation of a constant
number (2i10&) of cells with increasing amounts of biotinylated virus
(0n5–50 µg) for 60 min on ice, followed by measurement of the amount
of both bound and unbound virus. Quantification of AMΦ-bound virus
was performed by flow cytometry as described above. To measure the
amount of unbound virus and to demonstrate the specificity of binding
of biotinylated PRRSV to AMΦ, swine anti-PRRSV IgG-coated insoluble
protein A (anti-PRRSV protein A) was used. This was prepared by mixing
16 mg swine anti-PRRSV IgG with 10 mg insoluble protein A (Sigma)
and incubating the mixture at 37 mC for 60 min with constant shaking.
Afterwards, the protein A was collected by centrifuging at 10 000 g for
1 min and washed three times with PBSA. The capacity of the prepared
anti-PRRSV protein A to adsorb PRRSV was about 1 mg biotinylated
virus per mg protein A. Supernatants which contained unbound PRRSV
were mixed with 50 µl 1 mg\ml anti-PRRSV protein A and incubated at
37 mC for 60 min. The anti-PRRSV protein A was washed three times
with PBSA and 50 µl 1 : 50-diluted streptavidin–FITC conjugate was
added. After a further 60 min incubation and three washings, the antiPRRSV protein A was suspended in 500 µl PBS and the fluorescence
intensity was measured by flow cytometry. Fluorescence intensity was
compared with a virus particle standard curve that had been made by
adsorbing a series of known amounts of virus particles with anti-PRRSV
protein A, followed by incubation with streptavidin–FITC conjugate and
analysis by flow cytometry as described above. Specificity of adsorption
was demonstrated by using normal swine IgG-coated insoluble protein A
prepared by the same procedure as described above.
Investigation of the effect of acidotropic agents and
cytochalasin D on PRRSV infection. Stock solutions of the
acidotropic agents ammonium chloride (1 M) (UCB) and chloroquine
(100 mM) (Sigma) were prepared in RPMI medium and filter-sterilized
before use, while a stock of cytochalasin D (10 mM) (Sigma) was
prepared in ethanol. Afterwards, working dilutions of each product were
prepared in RPMI medium containing 10 % FCS. AMΦ (1i10')
were washed twice with PBS. Different concentrations of the
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Entry of PRRSV into porcine alveolar macrophages
Results
Kinetics of PRRSV binding
A series of virus-binding studies was performed to
characterize the receptor–ligand interactions involved in
attachment of PRRSV to AMΦ. The amount of cell-bound
virus was determined either directly, by measuring the
fluorescence intensity of the cells, or indirectly, by measuring
unbound virus. Data from both bound virus and free virus
measurements (Fig. 1 a, b) showed that the binding of PRRSV
(a)
800
700
Mean fluorescence intensity
Visualization of the PRRSV entry process by confocal
microscopy. Approximately 10 µg biotinylated PRRSV was added to
2i10& AMΦ that were untreated or treated with 10 mM ammonium
chloride, 50 µM chloroquine or 50 µM cytochalasin D. After 60 min
incubation on ice, the cells were washed once with cold PBSA and were
incubated with 1 : 50-diluted streptavidin–FITC for 60 min on ice. The
cells were washed once with PBSA and resuspended in 1 ml medium, and
then cultivated further in Teflon inserts (Ploy Labo) at 37 mC with 5 %
CO . Cells were collected after 0, 1, 3, 6, 9, 12, 16 and 24 h incubation
#
at 37 mC. To enumerate the dead cells, PI was added to a final
concentration of 1 µg\ml. To detect surface-bound biotinylated virions,
cells were fixed with cold 4 % paraformaldehyde for 10 min in suspension
at room temperature. To detect biotinylated virions inside the cell, cell
smears were made by using a cytocentrifuge at 140 g for 5 min and the
smears were fixed in acetone for 20 min at k20 mC. The binding and
entry process of FITC-tagged PRRSV in viable AMΦ was visualized on
a Bio-Rad MRC 1024 confocal laser scanning system linked to a Nikon
Diaphot 300 microscope and interfaced to a Compaq Prosignia 300
computer. Krypton\argon laser light was used to excite FITC (488 nm
line) and PI (568 nm line) fluorochromes. Images were recorded and
processed with Bio-Rad Comos 7.0a and Lasersharp 3.0 software.
To study whether biotinylated PRRSV co-localized with clathrin or
caveolin during the process of entry, approximately 10 µg biotinylated
PRRSV was added to 2i10& AMΦ and incubated on ice for 60 min. The
cells were washed twice with cold PBS before incubation with 1 : 50diluted streptavidin–FITC. After being washed again with cold PBS, the
cells were incubated at 37 mC for 0, 5 and 30 min before fixation with
0n4 % formaldehyde for 10 min in suspension at room temperature. The
fixed cells were centrifuged and the pellet was resuspended in 100 µl
0n1 % Triton X-100 in FBS (PBS with 20 % FCS). Cells were washed in FBS
and resuspended in 100 µl FBS with 0n25 µg of either mouse anti-clathrin
heavy chain MAb (Transduction Laboratories) or affinity-purified rabbit
anti-caveolin antibodies (Transduction Laboratories). After 1 h incubation
at room temperature, cells were washed in FBS for 10 min and incubated
with 1 : 100-diluted goat anti-mouse IgG–Texas red conjugate (Molecular
Probes) in FBS or 1 : 100-diluted goat anti-rabbit IgG–Texas red conjugate
(Molecular Probes) in FBS as appropriate for 60 min at room temperature.
Finally, cells were washed and analysed by confocal microscopy at
488 nm to excite FITC and 568 nm to excite Texas red, as described
above.
600
500
400
300
200
100
0
–2
–1
0
1
2
3
4
Log10 viral particles added per cell
8000
(b)
7000
Viral particles bound per cell
reagents were added at 1 h prior to inoculation, at the time of inoculation
or at different times after inoculation, as described in the results. The
effect of different concentrations of the three agents on the viability of
AMΦ was examined by flow cytometry with PI staining before
inoculation. Treated and untreated cells were inoculated with 1 ml
Lelystad PRRSV stock containing 10&n$ TCID virus for 1 h at 37 mC, and
&!
then the inoculum was washed away and fresh medium was added. The
inoculated cells were incubated at 37 mC with 5 % CO and collected by
#
gently flushing at 0, 1, 3, 6, 9, 12, 16 and 24 h after inoculation. Viral
nucleocapsid protein (NP) expression was examined in an immunofluorescence assay. The AMΦ were detached by thorough flushing of
the bottom of polystyrene dishes and washed once with PBS. Cell smears
were made by using a cytocentrifuge at 140 g for 5 min. The smears were
fixed in acetone for 20 min at k20 mC and subsequently pre-incubated
with 1 : 20-diluted goat serum to block non-specific staining. They were
then incubated with a mixture of the anti-PRRSV NP MAbs WBE1 and
WBE4-6 (Drew et al., 1995) and then incubated with goat antimouse–FITC conjugate (Amersham). The smears were washed three
times with PBS between each incubation. To confirm the specificity of
staining, two mouse ascites fluids containing isotype-matched unrelated
MAbs against suid herpesvirus type 1 (Nauwynck & Pensaert, 1995)
were used as negative controls. Positive cells were counted with a Leica
DM RBE fluorescence microscope.
6000
5000
4000
3000
2000
1000
0
0
1
2
3
4
Log10 free viral particles per cell
Fig. 1. Semi-logarithmic graphs (Klotz plot) of the binding of PRRSV to
AMΦ. (a) PRRSV binding is presented as the mean fluorescence intensity
of bound virus vs the number of viral particles added per cell. (b) Specific
PRRSV binding was calculated by subtracting the number of unbound
viruses from the total viral particles added per cell. Each point is the
arithmetic mean pSD from three independent experiments.
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% Inhibition of biotinylated PRRSV binding
H. J. Nauwynck and others
Table 1. Effect of NH4Cl and chloroquine on PRRSV
replication
90
The effects of acidotropic agents were quantified as the percentage of
cells expressing NP at 12 h post-inoculation. Cells were treated with
NH Cl and chloroquine at the time of virus inoculation. The results
%
are expressed as the percentage of NP-positive cells (meanpSD from
three independent experiments).
80
70
60
50
40
30
Concentration
20
10
0
0
0·4
2
10
Untagged PRRSV (lg)
50
Mean fluorescence intensity
Fig. 2. Competition between biotinylated PRRSV and untagged PRRSV for
binding to AMΦ. AMΦ (2i105) were pre-incubated with different
amounts of unlabelled virus before adding 5 µg biotinylated PRRSV. Each
bar represents the mean pSD from three determinations.
8
7
6
5
4
3
2
1
0
0
20
40
60 80 100 120 140 160
Time (min)
NH4Cl
0n1 mM
0n2 mM
0n5 mM
1 mM
2 mM
5 mM
10 mM
Chloroquine
1 µM
2 µM
5 µM
10 µM
20 µM
50 µM
100 µM
No treatment
% NP-positive cells
23n8p4n7
22n1p5n4
12n4p4n0
6n6p1n7
3n9p0n6
0
0
28n5p3n3
12n3p1n4
7n9p1n6
0
0
0
0
30n2p3n2
to AMΦ was dose dependent. However, saturation of PRRSV
binding was not reached under the conditions used.
In studying the specificity of the PRRSV interaction with
the AMΦ cell surface, it was found that unlabelled PRRSV
competed with labelled virus in a dose-dependent manner and
that binding of 75–87 % of biotinylated PRRSV was inhibited
when 50 µg unlabelled PRRSV was used (Fig. 2).
The results of our studies of the dynamics of the PRRSV
interaction with AMΦ show that PRRSV binding to AMΦ
increased gradually in the initial 10–50 min and reached a
maximum within 60 min (Fig. 3). Longer incubation times did
not increase the amount of virus that bound, indicating that
binding was complete in approximately 60 min.
30 mM NH Cl or 100 µM chloroquine, 95 % of the cells were
%
viable after 24 h continuous treatment of AMΦ. Both agents
inhibited PRRSV replication in a concentration-dependent
manner and as little as 5 mM NH Cl or 10 µM chloroquine
%
was sufficient to block virus replication completely (Table 1).
PRRSV replication was completely blocked when AMΦ
were treated with 10 mM NH Cl and 50 µM chloroquine at
%
1 h before and at 0, 1, 2, 3 and 4 h after inoculation. The
number of PRRSV-positive cells was identical in cells treated
6 h or later after inoculation compared with untreated cells
(Table 2). Finally, it was demonstrated that NH Cl and
%
chloroquine treatment of AMΦ did not affect binding of
biotinylated PRRSV to AMΦ, as determined by flow cytometry analysis, after 1 and 24 h continuous treatment of AMΦ
with 10 mM NH Cl or 50 µM chloroquine. Therefore, it was
%
concluded that the effect of NH Cl and chloroquine on PRRSV
%
replication was achieved by blocking post-adsorption steps in
the entry process of the virus.
The effect of acidotropic agents on PRRSV infection
The effect of cytochalasin D on PRRSV replication
To investigate whether a pH drop was an essential step in
the infectious entry of PRRSV into AMΦ, the effect of NH Cl
%
and chloroquine on PRRSV infection was examined by
quantifying PRRSV NP-positive cells after PRRSV inoculation
of AMΦ treated with these agents. At concentrations below
AMΦ were not infected by PRRSV in the presence of
20–100 µM cytochalasin D. In the presence of low concentrations of cytochalasin D (1–10 µM), only 1–5 NP-positive
cells were detected in an AMΦ smear containing approximately 1i10& cells at 12 h after PRRSV inoculation. Concen-
Fig. 3. Time-course of binding of biotinylated PRRSV to AMΦ. Each point
shown is the arithmetic mean pSD from three independent experiments.
DAA
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Entry of PRRSV into porcine alveolar macrophages
Table 2. Effect of NH4Cl, chloroquine and cytochalasin D added at various times pre- or
post-inoculation on PRRSV replication
Acidotropic agents were added at 10 mM (NH Cl) or 50 µM (chloroquine and cytochalasin D). Percentage
%
inhibition of virus replication was calculated as 100i(% cells expressing NP in the absence of agent k
% cells expressing NP in presence of agent)\(% cells expressing NP in absence of agent). Data are means
pSD from three independent experiments.
% Inhibition of virus replication
Time of addition
(h post-inoculation)
k1
0
j1
j2
j3
j4
j5
j6
j9
(a)
NH4Cl
Chloroquine
Cytochalasin D
100
100
100
100
100
100
58n4p4n5
3n1p3n5
0n0p5n8
100
100
100
100
100
100
56n5p17
3n1p3n9
0n0p7n6
100
100
100
100
100
100
63n8p6n5
3n0p4n3
0n0p4n5
(b)
(c)
Fig. 4. Binding of labelled PRRSV and entry into AMΦ. Binding of PRRSV to the plasma membrane of AMΦ after 1 h incubation
at 4 mC (a) and entry into the cell after 1 h (b) and 3 h (c) incubation at 37 mC are shown.
trations of cytochalasin D of 0n5 µM or less did not inhibit
infection. The inhibitory effect of cytochalasin D was observed
during the early stage of virus replication, similar to the action
of acidotropic agents (Table 2). These results indicate that
PRRSV entry is a microfilament-dependent process.
The entry process visualized by confocal microscopy
Taking advantage of the fact that biotin-tagged virus can
be stained with streptavidin–FITC, the process of PRRSV
entry into AMΦ was visualized by confocal microscopy. After
AMΦ were incubated with biotinylated PRRSV at 4 mC for 60
min, under which conditions maximal virus binding without
penetration was reached, AMΦ were stained with
streptavidin–FITC and observed under the microscope. Fluorescent spots representing bound virus were observed on the
membrane of almost all AMΦ (Fig. 4 a), but with different
degrees of fluorescence intensity on different individual cells.
After a further incubation at 37 mC for a few minutes,
fluorescent spots were widely distributed inside the cells.
Intense fluorescence that was restricted to cytoplasmic
vacuoles was observed after 1–2 h of incubation at 37 mC (Fig.
4 b). The number of fluorescing spots in AMΦ decreased at
3–5 h post-inoculation (Fig. 4 c). After 6–12 h of incubation,
fluorescence was no longer detected in almost all viable AMΦ.
In NH Cl- and chloroquine-treated AMΦ, the kinetics of
%
attachment and endocytosis were not altered. However, at
least 80 % of the cells still showed intense fluorescence in the
cytoplasm after 6–12 h of incubation (Fig. 5).
Co-localization of PRRSV and clathrin
In order to characterize further which components of
microfilaments are involved in uptake of PRRSV, the association between PRRSV and clathrin or caveolae was
examined by double immunofluorescence staining. Clear co-
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H. J. Nauwynck and others
(a)
(b)
(c)
(d)
Fig. 5. Use of FITC-tagged PRRSV to visualize the blockage of PRRSV entry into AMΦ at 16 h post-incubation in the presence
of NH4Cl (a), chloroquine (b) or cytochalasin D (c). A no-compound control (d) was included.
localization was observed between PRRSV and clathrin after a
short period of incubation at 37 mC (5 min) (Fig. 6, arrowheads),
whereas no co-localization was found between PRRSV and
caveolin (data not shown), suggesting that PRRSV uptake is a
clathrin-dependent process. At a later stage of virus entry
(30 min at 37 mC), clathrin was uncoated from endocytotic
vesicles. This can be seen in Fig. 6 (arrows) as endocytotic
vesicles containing virus in the cytoplasm with no co-localized
clathrin.
Discussion
This study shows that entry of PRRSV into AMΦ occurred
by specific binding to the outer cell membrane followed by
uptake into the cell via clathrin-dependent endocytosis.
Furthermore, low pH during the early entry process was
essential for proper virus replication.
The initial interaction between PRRSV and AMΦ appears
to be complex. Binding of PRRSV to AMΦ is dose dependent
DAC
and is competed for by homologous virus. Binding activity
increased gradually during a 60 min incubation at 4 mC.
However, it was impossible to saturate all binding sites. One
explanation for this may be that the number of receptors on
AMΦ exceeds 10% per cell, the maximal concentration of
biotin-labelled virus that can be reached. This number of
receptors is not exceptionally high when other viruses are
considered ; 2n5i10%, 0n4i10% and 60i10% receptors per
susceptible cell were found for murine polyomavirus, adenovirus type 5 and murine leukaemia virus, respectively
(Herrmann et al., 1997 ; Hong et al., 1997 ; Johnson & Rosner,
1986).
After incubation of AMΦ with biotinylated PRRSV and
streptavidin–FITC labelling, different numbers of regularly
distributed fluorescent spots were seen under the microscope
on each individual AMΦ, indicating that the number of areas
on the membrane that contain virus receptors is quite variable.
The fact that AMΦ are a heterogeneous population of cells
may be the reason for the variation in membrane expression of
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Entry of PRRSV into porcine alveolar macrophages
0
5
30
(a)
(b)
Fig. 6. Co-localization of PRRSV with clathrin. AMΦ were incubated with FITC-labelled PRRSV for different times (0, 5 and 30
min) at 37 mC before fixation, permeabilization and staining of clathrin with Texas red. (a) FITC-labelled PRRSV was excited at
488 nm and (b) Texas red-labelled anti-clathrin was excited at 568 nm (arrowheads, co-localization ; arrows, no
co-localization).
the virus receptor. Therefore, variable expression of virus
receptors on the surface of each AMΦ may be one of the
important factors that determines the rate of virus entry into
cells and the susceptibility of cells to PRRSV infection. This
may also be the reason for the observation that AMΦ with
different densities and in different states of differentiation and
activation have variable susceptibility to PRRSV (Choi et al.,
1994 ; Duan et al., 1997 b).
The presence of a functional cell-surface receptor for PRRSV
has been recognized as an important factor determining the
highly restricted macrophage tropism of this virus (Duan et al.,
1997 b, 1998 a). However, it was observed that MAbs that
recognize a 210 kDa protein and block PRRSV infection
cannot prevent the binding of PRRSV to AMΦ completely
(Duan et al., 1998 a). This suggests that PRRSV–AMΦ binding
is mediated by the interaction of one ligand with different
receptors or, alternatively, by the interaction of multiple viral
ligands with their respective receptors.
Following binding to cell-surface receptors, PRRS virions
undergo receptor-mediated endocytosis through clathrincoated pits and vesicles and are delivered intact into endosomes. These endocytotic events were clearly demonstrated
by confocal microscopy. When AMΦ were infected with
labelled virus and visualized under the microscope, labelled
virus particles were found inside the cells after incubation at
37 mC. Intensely fluorescing spots were observed after 1–2 h
of incubation, suggesting that virions accumulate in distinct
vacuoles in the cytoplasm. Degradation of viral particles took
place at 6–12 h post-incubation, since no fluorescence of
labelled virus could be observed and newly synthesized viral
proteins were detected after this time. This entry process is
microfilament dependent, since cytochalasin D blocked virus
replication. Cytochalasin D is a microfilament-disrupting
compound that inhibits clathrin-mediated endocytosis, phagocytosis and macropinocytosis in AMΦ. Clathrin-mediated
endocytosis of PRRSV was proven directly by co-localization
of PRRSV and clathrin at an early stage of entry. At a later
stage of entry, clathrin was uncoated from the endocytotic
vesicles. Many cell-surface receptors are known to interact
with clathrin in coated pits and to mediate endocytosis (Marsh
& Pelchen-Matthews, 1994). It is thought that, after PRRSV
binding, the virus receptors are concentrated in clathrin-coated
pits to form coated vesicles, which may lead to the formation
of virus-containing endosomes.
It was found that low pH is essential for proper PRRSV
replication. Although there is no direct evidence to explain
why low pH is critical for PRRSV replication, it is generally
believed that acidification of virus-carrying endosomes may
induce conformational changes in the envelope glycoproteins
or capsid structures that trigger membrane fusion or penetration reactions (Marsh & Pelchen-Matthews, 1994), as for
SFV (Garoff et al., 1994) and influenza virus (Skehel et al., 1995).
Whether similar mechanisms are involved in the uncoating of
PRRSV capsid structures remains to be studied.
The pH-dependent endocytotic pathway of PRRSV entry
into MARC-145 cells that has been described previously
(Kreutz & Ackermann, 1996) closely resembles that described
in AMΦ in the present study. NH Cl, chloroquine and
%
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H. J. Nauwynck and others
cytochalasin all blocked the entry process in both cell types
and the concentrations of these compounds required were
similar. The time during which NH Cl and chloroquine were
%
effective was quite different, however. In MARC-145 cells,
virus replication was inhibited when acidotropic agents were
added during the first 30 min post-inoculation, whereas in
AMΦ this was extended until 4 h post-inoculation. This
difference may be attributable to the m.o.i. and the cell type.
Indications of this were found with influenza virus, another
virus that infects cells using endocytotic entry and a pH drop.
With an increase of the m.o.i. of influenza virus in CHO cells,
it became more difficult to block infection by inhibiting the
acidification of the endosomes (Martin & Helenius, 1991). The
chance that one virus penetrates quickly increases with a larger
m.o.i., and this was suggested as a possible explanation for this
observation. A similar effect of the m.o.i. might explain the
shorter effective time-window reported for PRRSV by Kreutz
& Ackermann (1996). The m.o.i. used by these authors was
one-hundred times higher than that used in the present study.
The cell type may also be important. As for PRRSV, it has been
shown for influenza virus that virus penetration into cells of
continuous cell lines is faster than in primary macrophages
(Martin & Helenius, 1991 ; Meyer & Horisberger, 1984 ; Koff &
Knight, 1979).
Although attachment and endocytosis of PRRSV was
observed in almost all inoculated AMΦ, viral antigens were
only produced in 30 % of these cells. This discrepancy may be
attributable to a blockage in the virus replication cycle at the
level of fusion, transcription or translation. Further studies will
be performed to clarify this specific point.
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Received 12 May 1998 ; Accepted 28 September 1998
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