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