Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies
Influence of N-linked glycosylation of porcine reproductive and respiratory syndrome virus GP5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies
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DOI：
10.1128/jvi.80.8.3994-4004.2006
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发表时间：
2006-04-01
影响因子：
5.4
通讯作者：
Pattnaik, AK
中科院分区：
文献类型：
--
作者：
Ansari, IH;Kwon, B;Pattnaik, AK
猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus，PRRSV）糖蛋白5（GP 5）是体内最丰富的囊膜糖蛋白，也是体内中和抗体的主要诱导物。三个推定的N-连接糖基化位点（N34、N44和N51）位于GP 5胞外域，其中也存在主要的中和表位。为了确定这些推定位点中的哪些用于糖基化以及聚糖部分在中和抗体应答中的作用，我们产生了一组在这些位点含有氨基酸取代的GP 5突变体。表达的野生型（wt）和突变蛋白的生化研究表明，成熟的GP 5含有高甘露糖型糖部分在所有三个网站。随后将这些突变并入全长cDNA克隆中。我们的数据表明，涉及残基N44的突变不会导致感染性后代的产生，表明N44是感染性最关键的氨基酸残基。携带N34、N51和N34/51突变的病毒生长至比wt PRRSV更低的滴度。在血清中和测定中，突变病毒表现出对wt PRRSV特异性抗体的中和的增强的敏感性。此外，用突变病毒接种猪可诱导显着更高水平的针对突变体和野生型PRRSV的中和抗体，这表明GP 5胞外域中聚糖残基的缺失增强了这些病毒对体外中和的敏感性和免疫原性附近的中和表位。本研究结果对进一步研制保护性强的PRRSV疫苗具有重要意义。
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.