Rizzo Giorgia, Forti Katia, Serroni Anna, Cagiola Monica, Baglivo Sara, Scoccia Eleonora, De Giuseppe Antonio
Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Perugia, Italy.
Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche, Perugia, Italy.
Vet Microbiol. 2016 Dec 25;197:21-26. doi: 10.1016/j.vetmic.2016.10.024. Epub 2016 Oct 25.
The bovine leukaemia virus (BLV) envelope protein (Env) is synthesized as a polyprotein precursor (gp72) proteolytically cleaved into the mature surface (SU) and transmembrane (TM) glycoproteins. The amino-terminal region of SU contains conformational epitopes F, G and H, which require a glycosylated SU to be recognized by monoclonal antibodies (MAbs) and antibodies from BLV-infected cattle. The SU contains eight asparagine (N) residues that are putative N-glycosylation sites. The N129, N203, N230 and N251 appear involved in carbohydrate binding, play an essential role in the in vitro infection. To determine which sites were actually glycosylated, we generated mutated SU forms, where each N-glycosylation site was changed to alanine (A). Subsequently, these N to A mutations were inserted into the env gene to generate Env mutants. The increase of electrophoretic mobility of EnvA256 and EnvA271 derived SU showed that the asparagine residues N256 and N271 were also glycosylated. ELISA revealed that only the N129 oligosaccharide determined the antigenic conformation of SU. The syncytium formation induced by EnvA129 showed that fusogenic capacity was independent of amino-terminal SU glycan conformational structure. Finally, anti-BLV serum inhibited syncytia formation even with the EnvA129 mutant. The latter inhibition was higher than Env, suggesting that the oligosaccharides could be also involved in the glycan shield for viral escape.
牛白血病病毒（BLV）包膜蛋白（Env）以多蛋白前体（gp72）的形式合成，经蛋白水解切割成成熟的表面（SU）糖蛋白和跨膜（TM）糖蛋白。SU的氨基末端区域包含构象表位F、G和H，这些表位需要糖基化的SU才能被单克隆抗体（MAb）和来自BLV感染牛的抗体识别。SU含有8个天冬酰胺（N）残基，它们是假定的N-糖基化位点。N129、N203、N230和N251似乎参与碳水化合物结合，在体外感染中起重要作用。为了确定哪些位点实际发生了糖基化，我们生成了突变的SU形式，其中每个N-糖基化位点都被替换为丙氨酸（A）。随后，将这些N到A的突变插入env基因中以生成Env突变体。EnvA256和EnvA271衍生的SU电泳迁移率的增加表明天冬酰胺残基N256和N271也被糖基化。酶联免疫吸附测定（ELISA）显示只有N129寡糖决定了SU的抗原构象。EnvA129诱导的合胞体形成表明融合能力与SU氨基末端聚糖的构象结构无关。最后，抗BLV血清即使对EnvA129突变体也能抑制合胞体形成。后者的抑制作用高于Env，表明寡糖也可能参与病毒逃逸的聚糖屏蔽。
