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

Abstract

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)....

Involvement of proteases in porcine reproductive and respiratory syndrome virus uncoating upon internalization in primary macrophages

“…6). Initial PRRSV attachment to the surface of macrophages is mediated by a cellular heparan sulphate proteoglycan receptor [13,43]. PRRSV is then attached and subsequently internalized via clathrin-mediated endocytosis [29] by the macrophage restricted porcine sialoadhesin receptor [44].…”

“…Two PRRSV receptors on primary macrophages have already been identified. The glycosaminoglycan heparan sulphate is a PRRSV receptor that is involved in PRRSV attachment via its interaction with structural matrix protein and the matrixglycoprotein 5 complex [13,43]. Sialoadhesin, a sialic acid binding immunoglobulin-like lectin [11], also mediates PRRSV attachment to macrophages [15,44].…”

The Human Hyaluronan Receptor for Endocytosis (HARE/Stabilin-2) Is a Systemic Clearance Receptor for Heparin

“…macrophages, vascular smooth muscle cells), none of these mediate substantial clearance of Hep (7)(8)(9). A few reports implicated a role for Kupffer cells in Hep clearance, but were not followed up (10,11).…”

Porcine Arterivirus Attachment to the Macrophage-Specific Receptor Sialoadhesin Is Dependent on the Sialic Acid-Binding Activity of the N-Terminal Immunoglobulin Domain of Sialoadhesin

“…In vivo, the virus has a tropism for a subpopulation of macrophages (10, 11), and only primary pig macrophages and continuous cell lines derived from African green monkey kidney cells, such as Marc-145 cells, allow efficient virus replication in vitro (1,16,28,29). Heparan sulfate is a PRRSV receptor on macrophages that mediates virus attachment, and the viral matrix protein has been shown to be a heparin-binding protein, suggesting its potential role as a viral ligand for heparan sulfate (8,25). Sialoadhesin (Sn) was identified as an essential PRRSV receptor that mediates both attachment and internalization on macrophages (5-7, 9, 26).…”