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. 2003 Aug;77(15):8207-15.
doi: 10.1128/jvi.77.15.8207-8215.2003.

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Nathalie Vanderheijden  1 , Peter L Delputte, Herman W Favoreel, Joël Vandekerckhove, Jozef Van Damme, Peter A van Woensel, Hans J Nauwynck
Affiliations

Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages

Nathalie Vanderheijden et al. J Virol. 2003 Aug.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) shows a very restricted tropism for cells of the monocyte/macrophage lineage. It enters cells via receptor-mediated endocytosis. A monoclonal antibody (MAb) that is able to block PRRSV infection of porcine alveolar macrophages (PAM) and that recognizes a 210-kDa protein (p210) was described previously (MAb41D3) (X. Duan, H. Nauwynck, H. Favoreel, and M. Pensaert, J. Virol. 72:4520-4523, 1998). In the present study, the p210 protein was purified from PAM by immunoaffinity using MAb41D3 and was subjected to internal peptide sequencing after tryptic digestion. Amino acid sequence identities ranging from 56 to 91% with mouse sialoadhesin, a macrophage-restricted receptor, were obtained with four p210 peptides. Using these peptide data, the full p210 cDNA sequence (5,193 bp) was subsequently determined. It shared 69 and 78% amino acid identity, respectively, with mouse and human sialoadhesins. Swine (PK-15) cells resistant to viral entry were transfected with the cloned p210 cDNA and inoculated with European or American PRRSV strains. Internalized virus particles were detected only in PK-15 cells expressing the recombinant sialoadhesin, demonstrating that this glycoprotein mediated uptake of both types of strains. However, nucleocapsid disintegration, like that observed in infected Marc-145 cells as a result of virus uncoating after fusion of the virus with the endocytic vesicle membrane, was not observed, suggesting a block in the fusion process. The ability of porcine sialoadhesin to mediate endocytosis was demonstrated by specific internalization of MAb41D3 into PAM. Altogether, these results show that sialoadhesin is involved in the entry process of PRRSV in PAM.

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Figures

FIG.1.
FIG.1.
Alignment of the amino acid sequence of porcine sialoadhesin with its homologous sequences in human and mouse (SWISS-PROT accession numbers, Q9BZZ2 and Q62230, respectively). The predicted hydrophobic leader peptides and transmembrane regions are underlined. The conserved cysteine residues characteristic of Siglecs are shaded. Residues that interact with sialic acid in mouse sialoadhesin are boxed in white in the consensus sequence. The six sequenced peptides are also boxed. Residues that are identical between the three proteins are indicated by stars and are given below in the consensus sequence. Gaps are indicated by a dot.
FIG. 2.
FIG. 2.
Porcine sialoadhesin immunoprecipitated from alveolar macrophages is modified with N-linked glycans. Alveolar macrophages were surface-labeled with biotin, and lysates were subjected to immunoprecipitation with MAb41D3. Immunoprecipitates were either treated with N-glycosidase F (lanes 2 and 3) or untreated (lanes 1 and 4) and run under reducing (lanes 1 and 2) or nonreducing (lanes 3 and 4) conditions on SDS-7% polyacrylamide gels. After transfer to polyvinylidene difluoride membrane, proteins on Western blots were detected with streptavidin-horseradish peroxidase, and this was followed by staining with 3,3′-diaminobenzidine as the substrate. M, apparent molecular mass markers (kilodaltons).
FIG. 3.
FIG. 3.
Colocalization of PRRSV nucleocapsid protein and recombinant porcine sialoadhesin in rPK cells. rPK cells were infected with PRRSV (LV strain), and 20 h postinoculation, cells were fixed and a double immunofluorescence staining was performed to detect the nucleocapsid protein (A) and the recombinant porcine sialoadhesin (B). (C) Overlay of panels A and B. Bar: 75 μm.
FIG. 4.
FIG. 4.
Detection of the viral nucleocapsid at different time points after inoculation of rPK (A, B, and C) and Marc-145 (D, E, and F) cells with PRRSV. (A and D) 1 h, (B and E) 3 h, and (C and F), 12 h postinoculation. After the selected time points, cells were fixed, stained for the nucleocapsid, and analyzed by confocal microscopy. Bar: 16 μm.
FIG. 5.
FIG. 5.
Porcine sialoadhesin mediates endocytosis of MAb41D3. Alveolar macrophages were incubated for 1 h at 37°C with MAb41D3 (A) or with control MAb MIL-2 anti-CD14 (B). After fixation, washings, and permeabilization, cells were incubated with FITC-conjugated goat polyclonal anti-mouse immunoglobulins and analyzed by confocal microscopy. (C) As a control, cells were fixed, incubated for 1 h at 37°C with MAb41D3, washed, permeabilized, and incubated with the same conjugated polyclonal antiserum as above. (D) Macrophages were incubated at 4°C with MAb41D3 before being shifted to 37°C to allow endocytosis. At different time points (0, 30, 60, 90, and 180 min), cells were shifted back to 4°C, stained with FITC-labeled goat anti-mouse antibodies, and analyzed by flow cytometry. Each value reflects the means ± standard deviation (error bars) of three experiments. (E to G) Macrophages were incubated with MAb41D3 and incubated at 37°C for 10 min before fixation and permeabilization. Cells were analyzed by confocal microscopy after performing a double immunofluorescence staining to detect porcine sialoadhesin (E) and clathrin (F). (G) Overlay of panels E and F. Symbols: Arrowheads, colocalization; arrows, no colocalization. Bar: 10 μm.
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