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J Virol, May 1998, p. 4520-4523, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of a Putative Receptor for Porcine
Reproductive and Respiratory Syndrome Virus on Porcine Alveolar
Macrophages
Xiaobo
Duan,
Hans J.
Nauwynck,
Herman W.
Favoreel, and
Maurice B.
Pensaert*
Laboratory of Veterinary Virology, Faculty of
Veterinary Medicine, University of Ghent, B-9820 Merelbeke, Belgium
Received 16 October 1997/Accepted 6 February 1998
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ABSTRACT |
To identify the receptor which may determine the macrophage tropism
of porcine reproductive and respiratory syndrome virus (PRRSV),
monoclonal antibodies (MAbs) against porcine alveolar macrophages (PAM)
were produced. Two MAbs (41D3 and 41D5) which completely blocked PRRSV
infection of PAM were further characterized. It was found that they
reduce the attachment of PRRSV to PAM and immunoprecipitate a 210-kDa
membrane protein from PAM. This protein was detected on the cell
membranes of PAM but not of PRRSV-nonpermissive cells. A colocalization
was found between the reactive sites of MAb 41D3 and PRRSV on PAM
membranes. All PRRSV-infected cells in tissues of experimentally
infected pigs reacted with MAb 41D3. Taken together, all these data
suggest that the identified 210-kDa membrane protein is a putative
receptor for PRRSV on porcine macrophages.
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TEXT |
The porcine reproductive and
respiratory syndrome virus (PRRSV) is a new member of arteriviruses,
which also include equine arteritis virus, murine lactate
dehydrogenase-elevating virus, and simian hemorrhagic fever virus. One
striking feature common to these viruses is that the macrophage is the
main and may be the only type of cell supporting replication in their
respective hosts (10). PRRSV has a restricted tropism for
cells of the monocyte/macrophage lineage both in vivo and in vitro
(1, 3, 7, 11, 12, 14). Of many porcine cell systems tested, only porcine alveolar macrophages (PAM) and some porcine peripheral blood monocytes after cultivation support a productive replication of
PRRSV (1-4, 7, 8, 12, 14).
Our previous observations suggested that certain unique virus receptors
may contribute to the susceptibilities of cells to PRRSV
(4-6). No viral receptors for arteriviruses have been
identified so far. In the present study, we attempted to identify the
PRRSV receptor on PAM by generation of PAM-specific monoclonal
antibodies (MAbs). To increase the chance of obtaining PAM-specific
MAbs, BALB/c mice were made immunologically tolerant with freshly
isolated porcine peripheral blood mononuclear cells (PBMC) before
immunization with PAM. Two MAbs (41D3 and 41D5) which reacted with PAM
and blocked PRRSV infection of PAM were obtained (5). These
two MAbs were affinity purified with goat anti-mouse immunoglobulin G
(IgG)-agarose (Sigma, St. Louis, Mo.) and used in a number of experiments to identify the putative virus receptors on PAM.
We first examined whether preincubation of PAM with affinity-purified
anti-PAM MAbs 41D3 and 41D5 inhibited subsequent virus replication in
the cells. PAM grown in a 96-well plate were incubated with different
amounts of MAbs 41D3 and 41D5 and isotype-matched controls for 60 min
on ice, washed with phosphate-buffered saline, and inoculated with 50 µl of medium containing 100 50% tissue culture infective doses of
PRRSV (Lelystad isolate). After a 48-h incubation at 37°C, the cells
were fixed and stained with swine anti-PRRSV sera according to an
immunoperoxidase monolayer assay as previously described
(13). It was found that as little as 0.6 µg of MAb 41D3
per ml and 5 µg of MAb 41D5 per ml completely protected
105 PAM from PRRSV infection. The protective effect of MAbs
41D3 and 41D5 was concentration dependent (Fig.
1). None of the control MAbs affected
PRRSV replication. MAbs 41D3 and 41D5 exhibited a similar blocking
capacity with PRRSV (94V360), a MARC-145-cell-adapted Belgian isolate
(data not shown).
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FIG. 1.
Ability of MAbs 41D3 and 41D5 to inhibit PRRSV infection
of PAM. Each value is the mean of results from three experiments with
PAM originating from three different
pigs.
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We next tested whether MAbs 41D3 and 41D5 prevented PRRSV binding to
PAM. PAM (2 × 105) were incubated with serial
dilutions of MAbs 41D3 and 41D5 or of isotype-matched control MAbs
18E8, 13D12, 26A8, and 4G3 (9) for 60 min on ice before
being subjected to about 0.5 µg of biotinylated virus. The cells were
stained with streptavidin-fluorescein isothiocyanate (FITC;
Amersham International, Little Chalfont, Buckinghamshire, United
Kingdom) and analyzed by flow cytometry. Pretreatment of PAM with MAbs
41D3 and 41D5 resulted in a 40 to 65% reduction of virus binding
compared to the amount of biotinylated PRRSV bound in the presence of
the negative control IgG1 antibody (Fig. 2). The inhibition of PRRSV attachment to
PAM by MAbs 41D3 and 41D5 was shown to be concentration dependent (Fig.
3).
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FIG. 2.
Flow cytometric analysis of the inhibition of PRRSV
binding to PAM by MAbs 41D3 and 41D5. PAM (2 × 105)
cultivated for 1 day were preincubated with 5 µg of affinity-purified
MAb 41D3 or 41D5 or irrelevant control MAb 18E8 prior to the addition
of 5 µg of biotinylated PRRSV. The fluorescence of cells preincubated
with antibody is shown as a solid-line histogram, the autofluorescence
of cells (without virus) is shown as a shaded histogram, and the
fluorescence of cells without antibodies is shown as a dashed-line
histogram.
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FIG. 3.
Ability of MAbs 41D3 and 41D5 to prevent PRRSV
attachment to PAM. Fifty microliters of various dilutions of
affinity-purified anti-PAM MAbs 41D3 (black bar) and 41D5 (grey bar) or
of the isotype-matched irrelevant MAb 18E8 (white bar) was preincubated
with 2 × 105 PAM before incubation with biotinylated
PRRSV. Fluorescence intensity was measured by flow cytometry, and
inhibition was expressed as a percentage of that of the control
(without MAbs). Each value is the mean of results from three
experiments with PAM originating from three different pigs.
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To examine whether the expression of the MAb 41D3- or 41D5-reactive
antigen(s) on the membranes of different cell types is correlated with
the cells' susceptibility to PRRSV, a variety of cell lines (PK-15,
SK, ST, and MARC-145) and porcine monocyte/macrophage lineage cells
were examined by flow cytometry and fluorescence microscopy. When
membrane immunofluorescence staining of PAM was performed with either
MAb 41D3 or 41D5 as the primary antibody, all cells were positive, but
when staining was performed with irrelevant IgG1 antibodies, all cells
were negative. Fluorescence staining was absent on the membranes of
PRRSV-nonpermissive cells, which include PBMC; porcine peritoneal
macrophages; and ST, SK, and PK-15 cells. Cells from a PRRSV-permissive
cell line, MARC-145 (8), were also negatively stained with
MAbs 41D3 and 41DS. Together with the observation that some PRRSV
isolates need to be adapted to infect MARC-145 cells, our results may
indicate that PRRSV enters MARC-145 cells by a pathway which is
different from that of PAM. When MAb 41D3 or 41D5 was used as the
primary antibody on acetone-fixed cells, PAM cells were strongly
stained but ST, SK, PK-15, and MARC-145 cells showed no staining. A
faint cytoplasmic staining of some PBMC and porcine peritoneal
macrophages was observed. No staining was found in all cell types when
isotype-matched irrelevant antibodies were used as primary antibodies.
When PAM were stained with both MAb 41D3 and fluorescence-labelled
PRRSV and analyzed by confocal microscopy, a clear colocalization was
found on the membranes of PAM (Fig. 4).
This result suggests that MAb 41D3 and PRRSV react with the same
structure(s) on the surfaces of PAM.
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FIG. 4.
Colocalization of MAb 41D3 and biotinylated PRRSV on the
membranes of PAM. PAM were subsequently incubated with biotinylated
PRRSV-streptavidin-FITC and MAb 41D3-goat anti-mouse IgG-Texas red.
Images were produced by superposition of 10 images of different focal
planes which were taken with a confocal laser scanning system with a
distance of 1 µm between each plane. Images were analyzed for Texas
red staining at a 568-nm excitation (A) and for FITC staining at a
488-nm excitation (B).
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In order to determine if PRRSV-antigen-positive cells in the tissues of
experimentally infected pigs also contain the MAb 41D3 antigen, we
performed a double immunofluorescence staining using sections of
various tissues from experimentally PRRSV-infected pigs and uninfected
controls (3). It was found that all the cells which
contained PRRSV antigens in lungs, thymus, tonsils, spleen, and lymph
nodes from the experimentally PRRSV-infected pigs also stained with MAb
41D3 (Fig. 5). No double staining was observed in the tissue sections from normal pigs or when
isotype-matched irrelevant antibodies were used.
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FIG. 5.
MAb 41D3 recognizes PRRSV-antigen-positive cells in
experimentally infected pigs. A section of lung tissue from an
experimentally PRRSV-infected pig was double stained with MAb
41D3-goat anti-mouse IgG-Texas red and swine anti-PRRSV IgG-FITC.
Images for Texas red staining (568-nm excitation) (A) and for FITC
staining (488-nm excitation) (B) were observed with a confocal laser
scanning system.
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We predicted from the above-described data that MAbs 41D3 and 41D5
block PRRSV infection of PAM by binding to molecules which are used for
virus attachment on the surfaces of PAM. To identify these molecules,
PAM cell membrane proteins were labelled by using a protein
biotinylation kit (Amersham International). Biotinylated cell lysate
was precleared with insoluble protein A (Sigma), and proteins were
immunoprecipitated with MAbs 41D3 and 41D5 or various control MAbs.
Standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis was
performed with a 4- to 20%-gradient minigel system (Bio-Rad
Laboratories, Hercules, Calif.). The separated proteins were blotted
onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories) and
detected with an enhanced chemiluminescence Western blotting detection
reagent containing horseradish peroxidase substrate (Amersham
International). A biotin-labelled protein with a molecular mass of
approximately 210 kDa was specifically precipitated by MAbs 41D3 and
41D5. Faint background bands and two unspecific bands with molecular
masses of 49 and 25 kDa were also seen in the absence of MAbs or after
immunoprecipitation with control MAbs (Fig.
6). The specificities of MAbs 41D3 and 41D5 for this 210-kDa protein were further examined by a Western blotting assay in which the cell lysates prepared from
PRRSV-nonpermissive cells (PK-15) were used as a negative control. A
specific protein band with a molecular mass of about 210 kDa was
detected only in the cell lysates of PAM with purified MAbs 41D3 and
41D5 and not in the cell lysates of PK-15 or with control MAbs.
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FIG. 6.
MAbs 41D3 and 41D5 immunoprecipitate a 210-kDa protein
from biotinylated PAM cell membrane preparations. PAM membrane proteins
were labelled with biotin and prepared, and an immunoprecipitation
assay was performed. MAbs 41D3 and 41D5 (lanes 1 and 2, respectively)
specifically precipitate a 210-kDa protein. Isotype-matched irrelevant
MAbs 18E8, 13D12, and 4G3 (lanes 3 to 5, respectively) and a control
with no first antibody (lane 6) were used as negative controls. MAb
26A8 (lane 7), which reacts with PAM and recognizes an abundant 40-kDa
PAM membrane protein but has no effect upon PRRSV infection, was used
as a positive control. Molecular mass standards (in thousands) are
noted at the left.
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In summary, we have isolated two anti-PAM MAbs which reduce PRRSV
attachment and block PRRSV infection. Both MAbs recognize a 210-kDa
membrane protein on PAM. This 210-kDa protein is expressed only on the
cell membranes of PRRSV-permissive PAM and not on those of
nonpermissive cells, which suggests that this protein may determine the
unique cell and tissue specificity of the virus. All these data show
that this protein appears to fulfil the criteria for a PRRSV receptor.
The characterization of the 210-kDa protein by amino acid analysis of
immunoaffinity-purified 210-kDa peptides or by cDNA cloning may further
help to clarify its role in the regulation of the tropism of the virus.
| |
ACKNOWLEDGMENTS |
We thank Patrick Van Oostveldt (Faculty of Agricultural and Applied
Biological Sciences, Ghent, Belgium) for his help with confocal image
analysis and G. Charlier (National Veterinary Research Institute,
Brussels, Belgium) for the electromicroscopic quantification of
purified PRRSV.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Veterinary Virology, Faculty of Veterinary Medicine, University of
Ghent, Salisburylaan 133, B-9820 Merelbeke, Belgium. Phone: 32 9 264 73 66. Fax: 32 9 264 74 95. E-mail:
maurice.pensaert{at}allserv.rug.ac.be.
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J Virol, May 1998, p. 4520-4523, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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