Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production

Abstract

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP 2a , GP 3 , GP 4 and GP 5 , the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP 2a and GP 5 proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP 2a protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP 5 protein. In contrast, the oligosaccharide linked to N46 of the GP 5 protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP 2a or of the oligosaccharide attached to N53 of GP 5 did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP 2a and GP 5 for PRRSV assembly and infectivity are discussed.

N-Linked Glycosylation of GP5 of Porcine Reproductive and Respiratory Syndrome Virus Is Critically Important for Virus Replication In Vivo

“…Two previous studies showed that the ablation of N-linked glycan in GP5 decreased the production of virus particles and infectivity of mutant viruses (2,47). However, these investigations did not examine whether the loss of glycosylation per se or other accidental structural alteration of GP5 or ORF5a protein due to artificially introduced amino acid substitutions accounted for the observed defective phenotypes.…”

“…Some viruses lost specific glycosylation sites but propagated well in MARC-145 cells or PAMs (17), which implied that the existence of GP5-associated glycans per se was not vital to the life cycle of the virus. More importantly, an accidental amino acid mutation in the small ORF5a protein might also lead to the growth-defective phenotype of the mutants that showed loss of the glycans in GP5 by replacement of the N-encoding codon of ORF5 overlapping with ORF5a (2,47). In this study, we addressed this issue by introducing specific residues that existed in field PRRSV isolates with a glycan loss to the corresponding position in GP5 of the infectious cDNA clone pAJXM.…”

“…The attachment was critically dependent on the sialic acid-binding capacity of the pSn receptor as well as on the sialic acids on the viral GP5 glycoprotein (10,11,25,41). Previous studies stated that ablation of a glycan in GP5 reduced or abolished the growth of PRRSV in MARC-145 cells or porcine alveolar macrophages (PAMs) (2,47). This conclusion was challenged by the fact that some PRRSV isolates in which N-linked glycosylation was absent at certain sites in GP5 displayed efficient growth in MARC-145 cells and infection in pigs (17,27,43).…”

Cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid

“…ORF1a and ORF1b encode the non-structural proteins, including three proteases and the RNA-dependent RNA polymerase (Snijder & Meulenberg, 1998); ORFs 2-5 encode glycosylated membrane proteins (GP2-GP5) and a small nonglycosylated protein E, ORF6 encodes a non-glycosylated membrane protein (M), and ORF7 encodes the nucleocapsid (N) protein that packages the viral RNA (Dea et al, 2000;Meulenberg et al, 1995). The major components of the envelope are GP5 and M, which form disulfide-linked heterodimers and are essential for particle assembly (Dea et al, 2000;Meulenberg et al, 1995;Wissink et al, 2005), while the minor proteins may play a role in defining tissue tropism (Wissink et al, 2004). All envelope proteins are required for infectivity (Wissink et al, 2005).…”

Cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are non-essential for virus infectivity

“…The role of cysteine 90 of the North American PRRSV N protein still remains to be determined. Using an European PRRSV isolate, Wissink et al (2004) have described a heterotrimeric complex of GP2, GP3 and GP4 and, without evidence, speculated a possible incorporation of E protein with this trimeric complex. Wieringa et al (2004) have recently shown a covalent association of the E protein with the GP2b-GP3-GP4 heterotrimers in EAV, suggesting a possible role of the heteromultimeric complex in the virus entry process.…”