Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV)

In recent years, several entry mediators have been characterized for porcine reproductive and respiratory syndrome virus (PRRSV). Porcine sialoadhesin [pSn, also known as sialic acid-binding immunoglobulin-type lectin (Siglec-1)] and porcine CD163 (pCD163) have been identified as the most important host entry mediators that can fully coordinate PRRSV infection into macrophages. However, recent isolates have not only shown a tropism for sialoadhesin-positive cells, but also for sialoadhesin-negative cells. This observation might be partly explained by the existence of additional receptors that can support PRRSV binding and entry. In the search for new receptors, recently identified porcine Siglecs (Siglec-3, Siglec-5 and Siglec-10), members of the same family as sialoadhesin, were cloned and characterized. Only Siglec-10 was able to significantly improve PRRSV infection and production in a CD163-transfected cell line. Compared with sialoadhesin, Siglec-10 performed equally effectively as a receptor for PRRSV type 2 strain MN-184, but it was less capable of supporting infection with PRRSV type 1 strain LV (Lelystad virus). Siglec-10 was demonstrated to be involved in the endocytosis of PRRSV, confirming the important role of Siglec-10 in the entry process of PRRSV. In conclusion, it can be stated that PRRSV may use several Siglecs to enter macrophages, which may explain the strain differences in the pathogenesis

Demonstration of microchimerism in pregnant sows and effects of congenital PRRSV infection

The presence of foreign cells within the tissue/circulation of an individual is described as microchimerism. The main purpose of the present investigation was to study if microchimerism occurs in healthy sows/fetuses and if porcine reproductive and respiratory syndrome virus (PRRSV) infection influences this phenomenon. Six dams were inoculated intranasally with PRRSV and three non-inoculated dams served as controls. Male DNA was detected in female fetal sera of all dams via PCR. Male DNA was also detected in the maternal circulation. Sex-typing FISH showed the presence of male cells in the female fetal organs and vice versa. PRRSV infection did not influence microchimerism, but might misuse maternal and sibling microchimeric cells to enter fetuses

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

Interaction of the porcine reproductive and respiratory syndrome virus with the macrophage-specific lectin sialoadhesin

The porcine reproductive and respiratory syndrome virus (PRRSV) causes the economically most important viral disease in swine industry today. The virus is a major threat to swine health worldwide, since it is both associated with the porcine respiratory disease complex and the cause of severe reproductive problems. The virus can persist in animals for extended periods of time, partly due to a hampered immune reponse upon infection. PRRSV also evolves rapidly and shows a high variability in vivo, further complicating accurate diagnosis and disease control. Today, twenty years after the discovery of PRRSV, effective antiviral strategies are still lacking and there are still no vaccines available that are safe, effective and broadly applicable, while the demand for these is higher than ever. Clearly, rational development of new profylactic and therapeutic strategies is needed to tackle the virus. However, to reach this, it is crucial to advance our fundamental understanding of the PRRSV biology. In this context, detailed knowledge on the entry of the virus in its target cell, the porcine macrophage, seems particularly interesting: therapeutics or vaccine-induced immune mechanisms that can selectively interfere with this crucial step in the infection process may efficiently inhibit primary infection and viral spread. Chapter 1 of this dissertation discusses glycan-lectin interactions in virus biology and gives an introduction on PRRSV. The first section of this chapter reflects on glycosylation of cells and viruses and explores glycan-lectin interactions in the context of viral infections. A brief introduction on glycosylation and lectins is given and some specific glycan-lectin interactions are highlighted and situated within the larger framework of viral infection and immunity. Distinction is made between interactions that benefit the host and interactions that benefit the virus. In addition, factors that contribute to glycan and lectin variation, and that ultimately govern glycan-lectin interactions, are briefly discussed. The second part of chapter 1 focuses on PRRSV. The viral taxonomy is given and the genome organization, the virion structure and the viral cell tropism are briefly discussed. Furthermore, this section explores the PRRSV replication cycle, thereby mainly focusing on the entry of PRRSV into its host cell, the porcine macrophage. In conclusion, PRRSV-associated disease and immunity are briefly discussed. In Chapter 2, the aims of this thesis are formulated. Recent studies identified the macrophage-specific protein sialoadhesin as an important entry receptor for PRRSV. In addition, it was shown that sialylated glycans on the virion surface are crucial for the infectivity of the virus towards macrophages. However, the exact nature of the interaction between the PRRSV virion and sialoadhesin has remained unknown. The main goal of this thesis was to further characterize the interaction between PRRSV and sialoadhesin. The first study in this thesis (Chapter 3) mainly focused on the receptor side of the PRRSV-sialoadhesin interaction. Just like murine (mSn) and human (hSn) sialoadhesin, the porcine sialoadhesin (pSn) shows clear sialic acid binding activity. A first objective was to verify if the R116 amino acid (aa) in the N-terminal V-set domain of pSn is important for its sialic acid binding capacity, as has been shown for mSn and hSn. A second objective was to determine if the sialic acid binding capacity of pSn is necessary for pSn-mediated PRRSV binding and internalization. To investigate the importance of the R116 aa for pSn sialic acid binding activity, an R116-to-E mutation was introduced in the predicted sialic acid-binding domain of a recombinant pSn via site-directed mutagenesis, resulting in a mutant protein pSnRE. Subsequent tests confirmed that this mutation results in the loss of sialic acid binding capacity without significantly altering the overall protein structure. Using the recombinant proteins, it was found that cells expressing pSn efficiently bind and internalize PRRSV virions, while cells expressing pSnRE bind the virus less efficiently (remnant binding via heparan sulphate on the cell surface) and do not internalize it. These data point out that the R116 aa of pSn is crucial for its sialic acid binding activity and that the sialic acid binding activity is essential for pSn to function as a PRRSV receptor. The second study taken up in this thesis (Chapter 4) focused on the viral side of the PRRSV-pSn interaction. The aim was to identify viral glycoproteins that may function as binding partners for pSn and to characterize the interaction between these ligands and pSn. For this purpose, a soluble form of pSn was constructed and validated. The soluble pSn could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of pSn as an important PRRSV receptor on macrophages. The soluble pSn was subsequently used for ligand fishing in lysates of purified PRRSV. Although sialic acids were found on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for pSn. The interaction was found to be dependent on the sialic acid binding capacity of pSn and on the presence of sialic acids on the GP5 protein. A final question was if PRRSV-specific antibodies, that are directed against structural PRRSV components that are directly implicated in interaction with the pSn receptor or that can indirectly modulate the interaction between PRRSV ligands and pSn, are invariably able to influence infection of macrophages. The third study in this thesis (Chapter 5) describes the production of PRRSV-specific hybridomas and an extensive characterization of the monoclonal antibodies (mAbs) they produce. A GP4-specific mAb, directed against a well-characterized neutralizing epitope in the GP4 ectodomain, exerted a strong neutralizing effect on PRRSV infection. In contrast, different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested show virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the “major neutralizing epitope” suggested for American type PRRSV. Hence, presence of a viral protein/epitope on the virion surface does not automatically imply that antibodies directed against this epitope can influence infection of macrophages. Chapter 6 recapitulates and critically reviews the findings presented in this thesis. Based on literature and the data obtained in this thesis, a model of the early steps in PRRSV infection of the porcine macrophage is proposed. Initial contact of the virus with the macrophage occurs via heparan sulphate glycosaminoglycans on the cell surface. Subsequently, the virus engages pSn in a more stable interaction. Interaction of the virus with this receptor involves binding of the viral M/GP5 complex to the N-terminal part of pSn. The sialic acid binding domain at the N-terminus of pSn and sialic acids on the virion surface are critical for this interaction. Attachment of the virus to pSn is followed by the uptake of the virus-receptor complex via a process of clathrin-mediated endocytosis. Upon internalization, the viral genome is released into the cytoplasm. This last stage of the entry occurs when the virus is present in the early endosome and is critically dependent on acidification of the endosome and on scavenger receptor CD163. The role of CD163 in genome release may require interaction with the viral GP2 and GP4 glycoproteins and relies on a functional CD163 SRCR domain 5. Also the cellular protease cathepsin E and an as yet unidentified trypsin-like serine protease have been implicated in this process. The above model describes the main entry pathway of PRRSV into the porcine macrophage. Nevertheless, it cannot be excluded that PRRSV also uses alternative entry pathways, independent of or partly overlapping the main entry pathway described here. Further research is needed to address this issue. The chapter concludes with a short discussion on the potential and pitfalls of profylactic and antiviral strategies that specifically target the PRRSV-pSn interaction

The porcine reproductive and respiratory syndrome virus requires trafficking through CD163-positive early endosomes, but not late endosomes, for productive infection

The porcine reproductive and respiratory syndrome virus (PRRSV) enters its target cell via clathrin-mediated endocytosis. Using dominant-negative Rab5 and Rab7 mutants, we show that upon internalization, PRRSV enters early endosomes but does not continue through the endocytic pathway to late endosomes. This was confirmed via colocalization experiments visualizing PRRSV and markers for different compartments of the endocytic pathway. Furthermore, it was shown that PRRSV colocalizes with its internalization receptor, sialoadhesin, on the cell surface and beneath the plasma membrane, while CD163 and PRRSV only meet in early endosomes

IFN-α treatment enhances porcine Arterivirus infection of monocytes via upregulation of the porcine Arterivirus receptor sialoadhesin

The Arterivirus porcine reproductive and respiratory syndrome virus ( PRRSV) has a specific tropism for a subset of differentiated macrophages. Porcine sialoadhesin was identified as a PRRSV internalization receptor that is, similarly to sialoadhesins from other species, only expressed on subsets of macrophages. Sialoadhesin is not expressed or only expressed at low levels on monocytes, which might explain why monocytes are largely refractory to PRRSV infection. Different molecules have been identified that regulate human, mouse, or rat sialoadhesin expression in in vitro cultivated monocytes and macrophages, but the effect of these varies greatly between species. In this study, we observed that interferon-alpha ( IFN-alpha) induces sialoadhesin expression on monocytes to levels similar as those on macrophages and that it increases sialoadhesin on macrophages. IFN-alpha-induced sialoadhesin expression was shown to be functional using a red blood cell ( RBC) binding assay. Furthermore, a 2 or 3 day IFN-alpha pretreatment of monocytes caused a 20-fold increase in the numbers of PRRSV-infected monocytes and increased production of infectious virus. We conclude that IFN-alpha, although it is a potent antiviral molecule, upregulated sialoadhesin infection on in vitro cultivated monocytes, which results in enhanced PRRSV infection of monocytes

Porcine, murine and human sialoadhesin (Sn/Siglec-1/CD169): portals for porcine reproductive and respiratory syndrome virus entry into target cells

Porcine sialoadhesin (pSn; a sialic acid-binding lectin) and porcine CD163 (pCD163) are molecules that facilitate infectious entry of porcine reproductive and respiratory syndrome virus (PRRSV) into alveolar macrophages. In this study, it was shown that murine Sn (mSn) and human Sn (hSn), like pSn, can promote PRRSV infection of pCD163-expressing cells. Intact sialic acid-binding domains are crucial, since non-sialic acid-binding mutants of pSn, mSn and hSn did not promote infection. Endodomain-deletion mutants of pSn, mSn and hSn promoted PRRSV infection less efficiently, but also showed markedly reduced expression levels, making further research into the potential role of the Sn endodomain in PRRSV receptor activity necessary. These data further complement our knowledge on Sn as an important PRRSV receptor, and suggest - in combination with other published data - that species differences in the main PRRSV entry mediators Sn and CD163 do not account for the strict host species specificity displayed by the virus

Demonstration of microchimerism in pregnant sows and fetuses and putative microchimerism involvement in the pathogenesis of congenital PRRSV infection

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