Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the family Arteriviridae within the order Nidovirales which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus. The viral genome is a linear, positive-stranded RNA molecule of approximately 15.0 kb in length and possesses a cap structure at the 5Ј end and a poly(A) tail at the 3Ј end. Eight open reading frames (ORFs) are in the viral genome (9, 34). The first two open reading frames (ORF1a and ORF1ab) encode viral nonstructural (NS) polyproteins that are involved in polyprotein processing and genome transcription and replication (47). The viral structural proteins, encoded in ORFs 2 to 7, are expressed from six subgenomic capped and polyadenylated mRNAs that are synthesized as a 3Ј-coterminal nested set of mRNAs with a common leader sequence at the 5Ј end.The major viral envelope protein is glycoprotein 5 (GP5), which is encoded in ORF5 of the viral genome (29,35,36). GP5 is a glycosylated transmembrane protein of approximately 25 kDa (10,16,35). It has a putative N-terminal signal peptide and possesses three potential N-linked glycosylation sites which are located in a small ectodomain comprising the first 40 residues of the mature protein (28,35). In EAV and LDV, the major envelope glycoprotein forms a disulfide-l...

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

“…PRRSV-neutralizing antibody titers in serum samples were determined using fluorescent focus neutralization (FFN) as described previously (2,17,43). Briefly, serum samples were heat inactivated for 30 min at 56°C and 2-fold diluted.…”

“…In addition, the N-glycan might also be employed by the virus to shield against the neutralizing antibodies (2, 17). Viruses carrying an N-linked glycosylation site mutation at N35 or N51 in GP5 were more sensitive to neutralization by antibodies (2,43). The influence of the N44-linked glycan of GP5 on host immune response remains elusive.…”

“…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).…”

“…On the other hand, the N-linked glycosylation in GP5 may be associated with the antigenicity of the neutralization epitopes located in the ectodomain. The removal of the N-linked glycosylation site at N34 or N51 from GP5 led to an increase in neutralization level as tested using the mutant viruses (2,17,43). In addition, the N-glycan might also be employed by the virus to shield against the neutralizing antibodies (2, 17).…”

N-Linked Glycosylation Status of Classical Swine Fever Virus Strain Brescia E2 Glycoprotein Influences Virulence in Swine

“…However, the significance of viral envelope protein glycosylation in virus replication, pathogenesis, and virulence in the natural host is unknown. Recently it has been shown that glycosylation of porcine respiratory and reproductive syndrome virus GP5 affects virus infectivity, antigenicity, and ability to induce neu-tralizing antibodies in swine (2). Loss of N-linked glycosylation from the hemagglutinin-neuraminidase (HN) protein from Newcastle disease virus (NDV), a bird pathogen, results in attenuation of the virus in chickens (25).…”