Immune Evasion of Porcine Reproductive and Respiratory Syndrome Virus through Glycan Shielding Involves both Glycoprotein 5 as Well as Glycoprotein 3

Abstract

Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.

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

“…4 illustrate that the potential sites N35, N44, and N51 in GP5 were normally used in wt viruses whereas site N30 (N30-A31-S32) was not. These findings were similar to a recent study in which one potential glycosylation site in GP5 of the PRRSV-01 strain, which contains three glycosylation sites, at positions N30, N33, and N44, was not used for glycosylation addition (43). The specific site (N-X-S/T) is essential for core glycosylation, which has been observed in numerous examples of glycoproteins in either unglycosylated or glycosylated form at low levels (7,8,29).…”

“…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). Similarly, the N-linked glycosylation site (N45) of LDV was abolished by a T substitution but the mutant viruses replicated well in cultured macrophages and mice (5).…”

“…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.…”

“…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).…”