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

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.