Nebraska Center for Virology and School of Veterinary Medicine and Biomedical Sciences, University of Nebraska—Lincoln, Lincoln, NE, USA.
J Virol. 2011 Jun;85(11):5555-64. doi: 10.1128/JVI.00189-11. Epub 2011 Mar 16.
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.
猪繁殖与呼吸综合征病毒（PRRSV）中和抗体（NAb）的被动给药可以有效地保护猪免受 PRRSV 感染。然而，在 PRRSV 感染后，猪通常会产生微弱和延迟的 NAb 反应。这种微弱的 NAb 反应的一个主要原因是糖基化屏蔽现象，涉及主要糖蛋白 GP5 携带一个主要中和表位。我们在这里描述了一种 II 型 PRRSV 田间分离株（PRRSV-01），它对中和作用高度敏感，并在体内诱导出非典型的快速、强大的 NAb 反应。序列分析表明，PRRSV-01 缺乏两个通常存在于野生型（wt）PRRSV 株中的糖基化位点，这两个糖基化位点分别位于其两种包膜糖蛋白中的一个，一个位于 GP3（位置 131），另一个位于 GP5（位置 51）。为了确定这些缺失的糖基化位点对 PRRSV-01 明显的中和表型的影响，通过用 PRRSV-01 的结构基因替换 II 型 PRRSV 株 FL12 cDNA 感染性克隆的结构基因，生成了嵌合病毒（FL01）。通过定点突变，分别或同时在 GP3 和 GP5 中重新引入 N-糖基化位点。在 GP3 或 GP5 中重新引入 N-糖基化位点可恢复体内和体外糖基化屏蔽能力，当这些位点同时在两种糖蛋白中重新引入时，具有累加效应。尽管这些糖基化位点的缺失似乎是自然发生的（可能是通过细胞培养传代），但 PRRSV-01 病毒在体内复制时很快恢复了这些糖基化位点，这表明这些位点受到了强烈的选择压力。总的来说，我们的数据表明，位于 GP3 中的一个 N-聚糖部分参与了糖基化屏蔽干扰。
