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.

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