马传染性贫血病毒囊膜基因gp90V3区糖化回复突变感染性克隆的构建

Construction of an Infectious Clone within the V3 Region of gp90 Surface Protein of EIAV by N-glycosylation Revers-mutations

为阐明我国马传染性贫血病毒(Equine infectious anemia virus,EIAV)弱毒疫苗致弱及免疫保护的分子机制,作者分析了疫苗株及其亲本强毒株共34个囊膜基因序列。结果发现疫苗株在膜基因gp90 V3区的潜在N-连接糖基化位点出现稳定的碱基替换,使该位点消失。为研究囊膜糖基化的作用,以疫苗株全长感染性克隆pLG-FD3-8为亲本,利用反向遗传技术对该突变位点进行糖基化序列回复操作,构建全基因感染性克隆pLGFDg5。将pLGFDg5转染驴胎皮肤细胞(FDD),通过逆转录酶活性和RT-PCR方法评价其感染性。结果表明,将pLGFDg5在FDD细胞中盲传3代后,可在细胞培养上清中检测到逆转录酶活性,用RT-PCR检测到EIAV保守基因片段,在电镜下可见典型的EIAV粒子。在FDD细胞上的病毒复制动力学分析显示,与其亲本克隆pLGFD3-8的衍生病毒pLGFD3-V相比,回复突变克隆衍生的pLGFDg5-V复制速度较慢,获得较低的病毒载量。体外抗体中和试验表明,与其亲本pLGFD3-V相比,引入潜在的糖基化位点g5降低了衍生病毒对中和抗体的敏感性。这一结果为N-连接糖基化在我国马传贫弱毒疫苗致弱机理的作用研究提供了重要依据。

The Chinese equine infectious anemia virus （EIAV） donkey-leukocyte attenuated vaccine （DLV） provides a unique natural model system, by which the mechanism of virulent attenuation and the immunological control of lentivirus replication can be studied. In this study, we compared gp90 sequences of the env gene from both virulent and attenuated EIAV strains and found that all theses sequences lost the potential N-linked glycosylation sites in the principal neutralization domain of the V3 region of the attenuated envelope gp90. To determine whether the loss of this potential N linked glycosylation site also changes the neutralization sensitivity of the vaccine strain, this glycosylation site was restored in the infectious clone pLGFDgS. The derived virus, pLGFDg5V, demonstrated a decreased replication rate in vitro when compared with the parental vaccine strain. More importantly, the reverse mutation of the glycosylation site in the V3 region significantly enhanced the resistance of pLGFDgSV to serum neutralizing antibodies, which can recognize parental viruses. This study improves the understanding of the mechanism that the Chinese EIAV vaccine provides solid immunity to the infection of virulent strains.