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Journal of Virology, August 2003, p. 8207-8215, Vol. 77, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.15.8207-8215.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Involvement of Sialoadhesin in Entry of Porcine Reproductive and Respiratory Syndrome Virus into Porcine Alveolar Macrophages

Nathalie Vanderheijden,1 Peter L. Delputte,1 Herman W. Favoreel,1 Joël Vandekerckhove,2 Jozef Van Damme,2 Peter A. van Woensel,3 and Hans J. Nauwynck1*

Laboratory of Virology, Faculty of Veterinary Medicine,1 Department of Biochemistry, Faculty of Medicine and Health Sciences, Flanders Interuniversity Institute of Biotechnology (VIB), Ghent University, B-9000 Ghent, Belgium,2 Intervet International B.V., 5830 AA Boxmeer, The Netherlands3

Received 22 January 2003/ Accepted 6 May 2003

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.

* Corresponding author. Mailing address: Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. Phone: 32 9 264 73 66. Fax: 32 9 264 74 95. E-mail: hans.nauwynck{at}ugent.be.

Journal of Virology, August 2003, p. 8207-8215, Vol. 77, No. 15
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.15.8207-8215.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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