Peter Delputte

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

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.

Scavenger receptor CD163, a Jack-of-all-trades and potential target for cell-directed therapy

Scavenger receptor CD163 contains nine scavenger receptor cysteine-rich (SRCR) domains and because of the presence of this ancient and highly conserved protein motif, CD163 belongs to the SRCR superfamily. Expression of CD163 is restricted to cells of the monocyte/macrophage lineage and is tightly regulated, with a general tendency of anti-inflammatory signals to induce CD163 synthesis, while pro-inflammatory signals rather seem to downregulate CD163 expression. The first-identified and most-studied function of CD163 is related to its capacity to bind and internalize haemoglobin-haptoglobin (HbHp) complexes. Later on, its functional repertoire was expanded, with the identification of CD163 as an erythroblast adhesion receptor, a receptor for tumour necrosis factor-like weak inducer of apoptosis (TWEAK), as well as a receptor for distinct pathogens encompassing bacteria and viruses. Interaction of one of these ligands with CD163 might result in receptor-mediated endocytosis, but might as well trigger a signalling cascade leading to the secretion of signalling molecules, which implicates that CD163 also acts as an immunomodulator. Not only the membrane-bound form of CD163 has an immunomodulating capacity, but also soluble CD163, which is generated via ectodomain shedding, is able to exert anti-inflammatory effects. Furthermore, the concentration of this soluble protein is significantly increased under specific pathological conditions, making it a useful marker protein for certain diseases. Finally, its restricted expression pattern and potential to internalize make CD163 an attractive candidate as gateway for cell-directed (immuno)therapy. This review aims to summarize current knowledge on CD163s biology and its different biological functions beyond HbHp scavenging, thereby mainly focussing on the more recently discovered ones. Furthermore, current data supporting the capacity of CD163 to serve as a diagnostic marker in certain diseases and its potential as a target molecule for cell-directed therapy are surveyed.

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 GP5 and that the N protein bound to heparin as a homodimer. GP3, 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-GP5 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.

Sialoadhesin and CD163 join forces during entry of the porcine reproductive and respiratory syndrome virus

The porcine reproductive and respiratory syndrome virus (PRRSV) shows a restricted tropism for subsets of porcine macrophages in vivo. To date, two PRRSV receptors have been identified on primary macrophages, heparan sulphate for binding and sialoadhesin for binding and internalization. However, additional factors are needed because the expression of both receptors in non-permissive cells results in virus internalization but not in virus uncoating and productive infection. Recently, CD163 was described as a PRRSV receptor on Marc-145 cells that renders non-permissive cells susceptible to PRRSV. Therefore, the potential role of CD163 in PRRSV entry in macrophages and its potential interplay with sialoadhesin were studied. Incubation of macrophages at 37 degrees C with either sialoadhesin- or CD163-specific antibodies reduced PRRSV infection by up to 75 %, while infection was completely blocked by a combination of both antibodies. When incubated at 4 degrees C, only sialoadhesin- and not CD163-specific antibodies reduced PRRSV infection. In addition, confocal analysis of PRRSV entry in non-permissive cells expressing only sialoadhesin showed PRRSV internalization but no uncoating. In contrast, when both sialoadhesin and CD163 were expressed, PRRSV was uncoated upon internalization, resulting in productive infection. Virus internalization was not observed when only CD163 was expressed; although, cells became productively infected. Thus, sialoadhesin is confirmed as a PRRSV internalization receptor and CD163 is shown to be involved in PRRSV entry, probably during uncoating. Co-expression of recombinant sialoadhesin and CD163 in non-permissive cells increased virus production 10-100 times compared with cells expressing only CD163, sustaining the requirement of both for efficient PRRSV infection.

Porcine reproductive and respiratory syndrome virus entry into the porcine macrophage

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the late 1980s and rapidly became one of the most significant viral pathogens in the swine industry. In vivo, the virus shows a very narrow cell tropism and targets specific subsets of porcine macrophages. The entry of PRRSV into its host cell is the first crucial step in infection and has been the focus of many fundamental studies. This review provides a comprehensive overview of the current knowledge on PRRSV entry into the porcine macrophage, covering virus binding, internalization and genome release, and integrates these findings into a general model of the entry process.

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.

Genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is a major cause of economic loss for the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) triggers weak and atypical innate immune responses, but key genes and mechanisms by which the virus interferes with the host innate immunity have not yet been elucidated. In this study, genes that control the response of the main target of PRRSV, porcine alveolar macrophages (PAMs), were profiled in vitro with a time-course experiment spanning the first round of virus replication. PAMs were obtained from six piglets and challenged with the Lelystad PRRSV strain, and gene expression was investigated using Affymetrix microarrays and real-time PCR. Of the 1409 differentially expressed transcripts identified by analysis of variance, two, five, 25, 16 and 100 differed from controls by a minimum of 1.5-fold at 1, 3, 6, 9 and 12 h post-infection (p.i.), respectively. A PRRSV infection effect was detectable between 3 and 6 h p.i., and was characterized by a consistent downregulation of gene expression, followed by the start of the host innate immune response at 9 h p.i. The expression of beta interferon 1 (IFN-β), but not of IFN-α, was strongly upregulated, whilst few genes commonly expressed in response to viral infections and/or induced by interferons were found to be differentially expressed. A predominance of anti-apoptotic transcripts (e.g. interleukin-10), a shift towards a T-helper cell type 2 response and a weak upregulation of tumour necrosis factor-α expression were observed within 12 h p.i., reinforcing the hypotheses that PRRSV has developed sophisticated mechanisms to escape the host defence.

Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells

ABSTRACT Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% ± 8% for HS-deficient and 12% ± 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

Porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages

Different viruses have evolved strategies that inhibit apoptosis of the host cell early in infection and/or induce apoptosis in the host cell late in infection. In this study, it was investigated if and when porcine reproductive and respiratory syndrome virus (PRRSV) modulates apoptosis in PRRSV-infected macrophages. The PRRSV replication cycle in macrophages was completed within 12 h post-inoculation (hpi). PRRSV-infected macrophages, treated with staurosporine at 4, 5, 6 and 8 hpi, were significantly protected against staurosporine-induced apoptosis, but PRRSV-infected macrophages, treated with staurosporine at 12 hpi, were not. In contrast, starting from 12 hpi, all PRRSV-infected macrophages died by caspase-dependent apoptosis, which culminated in secondary necrosis. Treatment of PRRSV-infected macrophages with Z-Val-DL-Asp-fluoromethylketone indicated that apoptosis late in infection was not essential for efficient virus release. Anti- and pro-apoptotic activities were also observed in PRRSV-infected Marc-145 cells. In conclusion, this study shows that PRRSV stimulates anti-apoptotic pathways in macrophages early in infection and that PRRSV-infected macrophages die by apoptosis late in infection.

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 R116-to-E mutation was introduced in the predicted sialic acid-binding domain of pSn, resulting in a mutant, pSnRE, that could not bind sialic acids. PSn, but not pSnRE, 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.