Wander Van Breedam

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.

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.

Characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies

The porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and boars, and respiratory disease in pigs of all ages. Antibodies against several viral envelope proteins are produced upon infection, and the glycoproteins GP4 and GP5 are known targets for virus neutralization. Still, substantial evidence points to the presence of more, yet unidentified neutralizing antibody targets in the PRRSV envelope proteins. The current study aimed to identify and characterize linear antigenic regions (ARs) within the entire set of envelope proteins of the European prototype PRRSV strain Lelystad virus (LV). Seventeen LV-specific antisera were tested in pepscan analysis on GP2, E, GP3, GP4, GP5 and M, resulting in the identification of twenty-one ARs that are capable of inducing antibodies upon infection in pigs. A considerable number of these ARs correspond to previously described epitopes in different European- and North-American-type PRRSV strains. Remarkably, the largest number of ARs was found in GP3, and two ARs in the GP3 ectodomain consistently induced antibodies in a majority of infected pigs. In contrast, all remaining ARs, except for a highly immunogenic epitope in GP4, were only recognized by one or a few infected animals. Sensitivity to antibody-mediated neutralization was tested for a selected number of ARs by in vitro virus-neutralization tests on alveolar macrophages with peptide-purified antibodies. In addition to the known neutralizing epitope in GP4, two ARs in GP2 and one in GP3 turned out to be targets for virus-neutralizing antibodies. No virus-neutralizing antibody targets were found in E, GP5 or M. Since the neutralizing AR in GP3 induced antibodies in a majority of infected pigs, the immunogenicity of this AR was studied more extensively, and it was demonstrated that the corresponding region in GP3 of virus strains other than LV also induces virus-neutralizing antibodies. This study provides new insights into PRRSV antigenicity, and contributes to the knowledge on protective immunity and immune evasion strategies of the virus.

Identification of the CD163 Protein Domains Involved in Infection of the Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Scavenger receptor CD163 is a key entry mediator for porcine reproductive and respiratory syndrome virus (PRRSV). To identify the CD163 protein domains involved in PRRSV infection, deletion mutants and chimeric mutants were created. Infection experiments revealed that scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR 5) is essential for PRRSV infection, while the four N-terminal SRCR domains and the cytoplasmic tail are not required. The remaining CD163 protein domains need to be present but can be replaced by corresponding SRCR domains from CD163-L1, resulting in reduced (SRCR 6 and interdomain regions) or unchanged (SRCR 7 to SRCR 9) infection efficiency. In addition, CD163-specific antibodies recognizing SRCR 5 are able to reduce PRRSV infection.

A Variable Region in GP4 of European-Type Porcine Reproductive and Respiratory Syndrome Virus Induces Neutralizing Antibodies Against Homologous But Not Heterologous Virus Strains

Porcine reproductive and respiratory syndrome virus (PRRSV) can induce severe reproductive failure in sows, and is involved in the porcine respiratory disease complex. The glycoprotein GP4 of the European prototype PRRSV strain Lelystad virus (LV) contains a linear neutralizing epitope that is located in a highly variable region. The current study aimed to evaluate the antibody response against this and other epitopes on GP4 to infection of pigs with European-type PRRSV. It was shown that three virus strains, differing in the region that corresponds to the neutralizing epitope on GP4 of LV, strongly induce antibodies against this area. Antibodies against the epitopes of the different virus strains were purified from polyclonal swine sera, and used in virus-neutralization tests on primary alveolar macrophages. This revealed that antibodies against the variable region in GP4 of different virus strains are able to neutralize infection with homologous but not heterologous virus strains.

GP4-specific neutralizing antibodies might be a driving force in PRRSV evolution

The structural envelope glycoprotein GP4 of European porcine reproductive and respiratory syndrome virus (PRRSV) strains contains a highly variable neutralizing epitope that is susceptible to neutralizing antibody-mediated selective pressure in vitro. In this study, it was analyzed what happens with this neutralizing epitope during infection in vivo in the presence of neutralizing antibodies. A neutralizing antibody-mediated selective pressure was created in 30 pigs by vaccination prior to inoculation with infectious Lelystad virus (LV). Nine viable neutralizing antibody-escape variants were isolated from 9 of these pigs and their neutralizing antibody-escape mutant-identity was confirmed by the acquired resistance to neutralization by autologous neutralizing sera. Six out of 9 neutralizing antibody-escape variants contained aa substitutions in the GP4 neutralizing epitope and had become resistant to neutralization by a monoclonal antibody (mAb) against this epitope. In addition, in all 6 corresponding pigs, antibodies against this epitope were detected early in infection. In contrast to these 6 virus variants, the 3 other antibody-escape variants did not contain aa substitutions in the GP4 neutralizing epitope and were still sensitive to neutralization by the GP4-specific mAb. These antibody-escape variants were isolated from pigs that did not contain antibodies against this epitope early in infection. All these findings together strongly indicate that aa substitutions in the GP4 neutralizing epitope can abrogate antibody recognition, and that neutralizing antibodies might be responsible for the selection of neutralizing antibody-resistant variants with aa substitutions in the neutralizing epitope on GP4. In conclusion, this study indicates that neutralizing antibodies in pigs might be a driving force in the rapid evolution of the neutralizing epitope on GP4 of European PRRSV strains.

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.

Bitter-sweet symphony: glycan–lectin interactions in virus biology

Abstract Glycans are carbohydrate modifications typically found on proteins or lipids, and can act as ligands for glycan-binding proteins called lectins. Glycans and lectins play crucial roles in the function of cells and organs, and in the immune system of animals and humans. Viral pathogens use glycans and lectins that are encoded by their own or the host genome for their replication and spread. Recent advances in glycobiological research indicate that glycans and lectins mediate key interactions at the virus-host interface, controlling viral spread and/or activation of the immune system. This review reflects on glycan–lectin interactions in the context of viral infection and antiviral immunity. A short introduction illustrates the nature of glycans and lectins, and conveys the basic principles of their interactions. Subsequently, examples are discussed highlighting specific glycan–lectin interactions and how they affect the progress of viral infections, either benefiting the host or the virus. Moreover, glycan and lectin variability and their potential biological consequences are discussed. Finally, the review outlines how recent advances in the glycan–lectin field might be transformed into promising new approaches to antiviral therapy.