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

Abstract

The sialic acid-binding lectin sialoadhesin (Sn) is a macrophage-restricted receptor for porcine reproductive and respiratory syndrome virus (PRRSV). To investigate the importance of pSn sialic acid-binding activity for PRRSV infection, an R 116 -to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSn RE , that could not bind sialic acids. PSn, but not pSn RE , allowed PRRSV binding and internalization. These data show that the sialic acid-binding activity of pSn is essential for PRRSV attachment to pSn and thus identifies the variable, N-terminal domain of Sn as a PRRSV binding domain.Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, which belongs, together with the Coronaviridae and Roniviridae, to the order Nidovirales (12,14,19,23). The virus causes reproductive disorders in pregnant sows and boars and respiratory problems in pigs of all ages. 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). Although Sn was shown to be essential for infection of macrophages, other, unidentified, factors are essential for productive infection, since expression of Sn in PRRSV nonpermissive cells, such as PK-15 and CHO K1 cells, allows virus internalization but no virus uncoating, genome release, or production of infectious virus (5, 26). Other putative PRRSV receptors have been described. Macrophage-specific monoclonal antibodies (MAbs) that block or reduce infection of macrophages were shown to be directed, respectively, against a protein of approximately 220 kDa and a 150-kDa protein doublet, but these proteins have not been identified and their exact role in PRRSV infection is not established (31). Recently, another MAb that blocks PRRSV infection of Marc-145 cells was shown to recognize a complex of cytoskeletal proteins (17), and an intact cytoskeleton was shown to be important for efficient infection of Marc-145 cells (2).The PRRSV receptor pSn contains, like other sialoadhesins, an N-terminal variable immunoglobulin (Ig)-like domain, followed by 16 constant Ig domains. The conservation of the critical amino acids of the sialic acid-binding variable Ig-like domain in pSn and the observation that sialic acid-carrying sheep red blood cells agglutinate to porcine Sn-expressing macrophages suggest that porcine Sn is also a sialic acid-binding lectin, but this has not been conclusively demonstrated (7,26). The domain of pSn that is involved...

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

“…PRRSV is then attached and subsequently internalized via clathrin-mediated endocytosis [29] by the macrophage restricted porcine sialoadhesin receptor [44]. Porcine sialoadhesin binds to sialic acids, present on PRRSV viral glycoproteins [14,16]. Following virus internalization, a subsequent pH drop in the endosome is required for virus replication [29].…”

“…Heparan sulphate is not necessary for sialoadhesin to function as a PRRSV internalization receptor, but enhances the interaction of the virus with sialoadhesin. Attachment of PRRSV to sialoadhesin is mediated by sialic acids present on the surface of PRRS virions [14,16]. CD163, a cellular protein in the scavenger receptor cysteine-rich superfamily, has recently been shown also to function as a PRRSV receptor in Marc-145 cells, but its role in PRRSV infection of primary macrophages has not been studied [6].…”

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection

“…Earlier studies showed that pCD163 cooperated with another putative receptor, porcine sialoadhesin (Sn/CD169) (23), to facilitate the PRRSV infection, where the former contributed to the uncoating of PRRSV while the latter promoted the attachment and internalization (invasion) of the virus (40)(41)(42)(43)(44)(45)(46)(47)(48). However, one study showed that Sn gene knockout pigs were infected by PRRSV as usual (49), and a recent study using geneedited pigs demonstrates the indispensability of pCD163 (28).…”

N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus Replication In Vivo

“…The N-linked glycans in GP5 were reported to play a role in M/GP5 complex attachment to pig sialoadhesin (pSn) receptor, thus conferring virus tropism to monocyte/macrophage-derived cells (15,42). The attachment was critically dependent on the sialic acid-binding capacity of the pSn receptor as well as on the sialic acids on the viral GP5 glycoprotein (10,11,25,41). Previous studies stated that ablation of a glycan in GP5 reduced or abolished the growth of PRRSV in MARC-145 cells or porcine alveolar macrophages (PAMs) (2,47).…”