Structural and functional characterization of porcine reproductive and respiratory syndrome virus N-glycans.

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is one of the most severe infectious diseases facing the swine industry worldwide. The etiologic agent, PRRS virus (PRRSV), belongs to the order Nidovirales, family Arteriviridae, genus Arterivirus. It is a positive-sense ssRNA virus and has a smooth spherical envelope embedded with seven proteins, four of which are glycoproteins (GP). Since the major envelope proteins GP5 and matrix (M) have short ectodomains, the broadly distributed viral glycans likely cover the virion surface and stretch out as antennae, thus interacting with host cells and contributing to viral infection. Previous studies suggest potential roles of PRRSV envelope protein-linked glycans in virus assembly, virus attachment to target cells, virus neutralization and antigenicity. In particular, sialic acids on GP5 have been shown to bind sialoadhesin on porcine macrophages, mediating virus attachment and internalization. Nevertheless, the complete profile of GP5-linked glycan compositions and structures, and the role of specific glycan moieties in virus infection have yet to be determined. Herein, we purified the North American prototype PRRSV, VR-2332, and analyzed viral glycans in the aspects of composition, structures and functions in virus infection. Endoglycosidase digestion of virus showed that GP5 was the primary protein substrate among all the four envelope glycoproteins, and that the glycans were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers and terminal sialic acids, which was also confirmed by lectin co-precipitation. Based on the structural information, we further demonstrated that GlcNAc and LacNAc oligomer-specific lectins bound to PRRSV and blocked virus attachment, resulting in reduced infection. However, GlcNAc oligomers and LacNAc did not compete with virus to block infection, suggesting that GlcNAc and LacNAc oligomers are not directly involved in virus entry. Finally, removal or alteration of N-glycans from PRRSV envelope proteins did not affect infection, indicating that envelope protein-linked N-glycans are not required for PRRSV infection. In conclusion, GP5 contains most of the PRRSV glycans, which are primarily complex-type N-glycans. GlcNAc and LacNAc oligomers and sialic acids on the PRRSV envelope are accessible for specific recognition that may reduce infection by steric hindrance. Envelope protein-linked N-glycans are not required for PRRSV infection. Our findings provide a glycan database for molecular structural studies of PRRSV and facilitate a better understanding of molecular host-virus interactions.