Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells

Therrien, Dominic; St‐Pierre, Yves; Dea, S.

doi:10.1007/s007050070112

Cited by 16 publications

(11 citation statements)

References 43 publications

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“…Cells were then washed with PBS alone (open circles), with 2500 mg heparin ml "1 (squares) to remove virus attached to the macrophages via heparan sulphate, with 25 mg mAb 41D3 ml "1 to remove virus attached to Sn (diamonds) or with 2500 mg heparin ml "1 and 25 mg Sn-specific mAb 41D3 ml , and since our data also indicate that at that time point all the virus is attached to Sn, we conclude that at least some of the virus that is removed from Sn by washing with mAb 41D3 attaches again to the heparan sulphate receptor. Together, these data show that virus attaches first to heparan sulphate followed by an interaction with Sn, making PRRSV binding resistant to washing with heparin, but sensitive to washing with both heparin and mAb 41D3.Previous reports have shown that PRRSV can attach to several cell lines, but that these cannot be infected (Therrien et al, 2000). Since most cells contain heparan sulphate on their cell surface, but not the macrophage restricted protein Sn, we hypothesized that heparan sulphate accounts for the observed attachment.…”

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confidence: 74%

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Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin

Delputte

Costers

Nauwynck

2005

Journal of General Virology

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Heparan sulphate and sialoadhesin were previously identified on porcine macrophages as receptors for porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the exact role and cooperation of heparan sulphate and sialoadhesin during PRRSV attachment and internalization was analysed. It was observed that both heparan sulphate and sialoadhesin mediate PRRSV attachment and that only these two receptors are involved in attachment. Analysis of attachment kinetics of PRRSV to macrophages revealed that early attachment is mediated mainly via an interaction with heparan sulphate, followed by a gradual increase in interaction with sialoadhesin. By using wild-type CHO and CHO deficient in heparan sulphate expression, it was shown that heparan sulphate alone is sufficient to mediate PRRSV attachment, but not entry, and that heparan sulphate is not necessary for sialoadhesin to function as a PRRSV internalization receptor, but enhances the interaction of the virus with sialoadhesin.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae, which is grouped together with the Coronaviridae and the Roniviridae in the order of the Nidovirales (Enjuanes et al., 2000;Mayo, 2002). A characteristic for the Arteriviridae is that they all share a marked in vivo tropism for cells of the monocyte/macrophage lineage (Plagemann & Moennig, 1992;Snijder & Meulenberg, 1998). In vivo, PRRSV infects a subpopulation of differentiated macrophages, which can be identified by expression of sialoadhesin (Sn) (Duan et al., 1998; Vanderheijden et al., 2003). Besides differentiated macrophages, porcine testicular germ cells (spermatids and spermatocids) show limited susceptibility to PRRSV infection (Sur et al., 1997). In vitro, porcine alveolar macrophages (PAM), some cultivated peripheral blood monocytes and the African green monkey kidney cell-line MA-104, and cells derived thereof, such as Marc-145 and CL-2621, can be used to grow the virus (Kim et al., 1993;Voicu et al., 1994;Duan et al., 1997).At present, two PRRSV receptors are identified on porcine macrophages, the in vivo target cell. Heparan sulphate was identified as a receptor on macrophages for both European and American strains, and the viral matrix protein on itself, or as a complex with GP 5 , was identified as a heparinbinding protein (Delputte et al., 2002). The macrophagespecific protein Sn was identified as a receptor that mediates PRRSV internalization of European and American strains (Vanderheijden et al., 2003). mAb 41D3, specific for Sn, is able to block infection completely, and cells that are not permissive for PRRSV infection internalize the virus upon expression of a recombinant Sn (Duan et al., 1998; Vanderheijden et al., 2003). Although these cells can internalize the virus, they are not productively infected. The virus apparently remains in the endosome and the viral genome is not released in the cytoplasm, indicating that a cellular factor, which is essential for infection, is lacking in these cells (Vanderheijd...

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mentioning

confidence: 74%

“…Previous reports have shown that PRRSV can attach to several cell lines, but that these cannot be infected (Therrien et al, 2000). Since most cells contain heparan sulphate on their cell surface, but not the macrophage restricted protein Sn, we hypothesized that heparan sulphate accounts for the observed attachment.…”

mentioning

confidence: 91%

Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin

Delputte

Costers

Nauwynck

2005

Journal of General Virology

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“…The full cDNA sequence of the porcine sialoadhesin coding sequence was amplified by RT-PCR using PAM RNA and cloned into an eukaryotic expression vector to yield plasmid pcDNAp210. Porcine (PK-15) cells, resistant to viral entry (31,54), were transfected with the pcDNAp210 plasmid and selected for Geneticin resistance. Generation of a cell line originating from a single positive cell was not achieved, but instead, we used a noncloned, Geneticin-resistant cell population (designated rPK) in which about 6% of the cells expressed the recombinant p210 on the plasma membrane and in the cytoplasm, as determined by an indirect immunofluorescence assay using MAb41D3.…”

Section: Resultsmentioning

confidence: 99%

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

Vanderheijden¹,

Delputte²,

Favoreel³

et al. 2003

J Virol

247

242

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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.Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory problems in pigs and severe reproductive failure in sows, leading to late-term abortions or the birth of stillborn and weak pigs. It is a member of the Arteriviridae family (order Nidovirales), which so far includes three other members: Equine arteritis virus, Mouse lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (6, 35). These enveloped, single-stranded RNA viruses share morphological and genomic similarities, can establish a persistent infection in their natural host, and have a predilection for cells of the monocyte/macrophage lineage. Of the porcine cells tested, only porcine alveolar macrophages (PAM) and some cultivated peripheral blood monocytes support productive replication of PRRSV in vitro (19,60). Additional studies showed that the virus also infects macrophages in the spleen, tonsils, lymph nodes, liver, Peyer's patches, and thymus, whereas peritoneal macrophages, freshly isolated blood monocytes, and progenitor cells in the bone marrow are refractory to infection (18,19,51). A few nonmacrophage cells are also susceptible to PRRSV, including porcine testicular germ cells (spermatids and spermatocytes) (52), the African green monkey kidney cell line MA-104, and cells derived...

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“…Since infection with other PRRSV strains, both American and European types, was also reduced by heparin, it may be concluded that binding to heparan sulfate GAG is generally used by PRRSV to attach to PAM. Other studies have demonstrated that PRRSV can bind to different cell types, though these cells cannot be infected (18,36). The presence of hepa- ran sulfate GAG on these cells may be responsible for the binding of PRRSV.…”

Section: Discussionmentioning

confidence: 96%

Involvement of the Matrix Protein in Attachment of Porcine Reproductive and Respiratory Syndrome Virus to a Heparinlike Receptor on Porcine Alveolar Macrophages

Delputte

Vanderheijden

Nauwynck

et al. 2002

J Virol

169

138

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The porcine reproductive and respiratory syndrome virus (PRRSV) has a very restricted tropism for well-differentiated cells of the monocyte-macrophage lineage, which is probably determined by specific receptors on these cells. In this study, the importance of heparinlike molecules on porcine alveolar macrophages (PAM) for PRRSV infection was determined. Heparin interacted with the virus and reduced infection of PAM up to 92 or 88% for the American and European types of PRRSV, respectively. Other glycosaminoglycans, similar to heparin, had no significant effect on infection while heparinase treatment of PAM resulted in a significant reduction of the infection. Analysis of infection kinetics showed that PRRSV attachment to heparan sulfate occurs early in infection. A heparin-sensitive binding step was observed which converted completely into a heparin-resistant binding after 120 min at 4°C. Using heparin-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it was observed that the structural matrix (M) and nucleocapsid (N) proteins attached to heparin. Nonreducing SDS-PAGE revealed that M bound to heparin mainly as a complex with glycoprotein GP 5 and that the N protein bound to heparin as a homodimer. GP 3 , which was identified as a minor structural protein of European types of PRRSV, did not bind to heparin. Since the N protein is not exposed on the virion surface, it was concluded that the structural M protein and the M-GP 5 complex contribute to PRRSV attachment on a heparinlike receptor on PAM. This is the first report that identifies a PRRSV ligand for a cell surface heparinlike receptor on PAM.Porcine reproductive and respiratory syndrome (PRRS) was first described in 1987 in North America as a new syndrome in pigs, characterized by reproductive failure in sows and respiratory problems in pigs of all ages (5). The causative agent of the disease was identified in 1991 in The Netherlands as a virus (35) and is now designated PRRS virus (PRRSV). PRRSV has been classified as a member of the Arteriviridae, a family of enveloped, positive-strand RNA viruses which also includes Equine arteritis virus, Lactate dehydrogenase-elevating virus (LDV), and Simian hemorrhagic fever virus (4,29).PRRSV is 45 to 70 nm in diameter and consists of a nucleocapsid core surrounded by an envelope. The virus contains three major structural proteins which together represent approximately 90 to 95% of the structural protein content: the nucleocapsid protein N (15 kDa), which makes up the nucleocapsid; a nonglycosylated membrane protein M (18 to 19 kDa); and a glycosylated membrane protein GP 5 (24.5 kDa) (21,23,34). The N protein is incorporated in virions mainly as a disulfide-linked dimer with an M r of 28 kDa (23). The M and GP 5 proteins are incorporated in virions mainly as a disulfide-linked heterodimer or as a disulfide-linked multimer with an approximate M r of, respectively, 40 and 87 kDa (21, 23). The M and N proteins are also present as single proteins in purified virions (23)....

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Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells

Cited by 16 publications

References 43 publications

Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin

Analysis of porcine reproductive and respiratory syndrome virus attachment and internalization: distinctive roles for heparan sulphate and sialoadhesin

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

Involvement of the Matrix Protein in Attachment of Porcine Reproductive and Respiratory Syndrome Virus to a Heparinlike Receptor on Porcine Alveolar Macrophages

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