Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein
Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein
复制标题
人副流感病毒 3 型血凝素神经氨酸酶蛋白 N 连接糖基化的作用
DOI：
10.1016/j.virusres.2013.03.012
复制
发表时间：
2013-06-01
期刊：
影响因子：
5
通讯作者：
Wang, Zhiyu
中科院分区：
文献类型：
--
作者：
Chu, Fu-lu;Wen, Hong-ling;Wang, Zhiyu
人副流感病毒3型（hPIV-3）是影响婴幼儿的主要呼吸道病原体。hPIV-3血凝素-神经氨酸酶（HN）蛋白是一种多功能蛋白，介导血细胞吸附（HAD）、神经氨酸酶（NA）和融合促进活性，每种活性都会影响HN促进病毒融合和进入的能力。hPIV-3 HN蛋白含有4个潜在的N-连接糖基化位点（N308、N351、N485和N523）。突变HN蛋白的电泳迁移率分析表明，HN蛋白中的N308、N351和N523位点，而不是N485位点，是BHK-21细胞中添加聚糖的靶向位点。这些功能性糖基化位点以各种组合从HN中系统地消除，以形成一组突变体，其中可以分析单个碳水化合物链和碳水化合物链组的作用。去除hPIV-3 HN蛋白上的单个或多个N-聚糖对细胞表面转运、表达和NA活性没有影响。单糖基化位点突变体（G1、G2和G4）不仅削弱了HN蛋白的融合促进活性，而且降低了HN蛋白的HAD活性，这对于所有三个双突变体（G12、G14和G24）和三突变体（G124）来说更加明显。此外，每个突变蛋白保留F-相互作用能力，这是等于野生型蛋白的能力。有趣的是，可以与G12突变蛋白共免疫沉淀或与抗F抗体免疫沉淀的F蛋白没有被有效地切割。对于G14、G24和G124，在免疫共沉淀F蛋白测定中检测到很少切割的F蛋白，并且其总量在细胞裂解物中。hPIV-3 HN和F蛋白在受体结合前后保持相关的机制，HN-受体相互作用的强度调节F蛋白的活化，这可以决定融合的程度。最后，我们证明了单个或多个N-糖基化位点突变在最早阶段抑制融合。综上所述，这些结果表明，hPIV-3 HN的N-糖基化对其受体识别活性、F蛋白切割和融合促进活性至关重要，但对其与同源F蛋白的相互作用和NA活性没有影响。(C)2013爱思唯尔有限公司版权所有。
Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects infants and young children. The hPIV-3 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities, each of which affects the ability of HN to promote viral fusion and entry. The hPIV-3 HN protein contains four potential sites (N308, N351, N485 and N523) for N-linked glycosylation. Electrophoretic mobility analysis of mutated HN proteins indicated that N308, N351 and N523 sites, but not the N485 site in HN protein, were targeted for the addition of glycans in BHK-21 cells. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Removal of individual or multiple N-glycans on the hPIV-3 HN protein had no effects on transport to the cell surface, expression and NA activity. Single glycosylation site mutants (G1, G2 and G4) not only impaired fusion promotion activity but also reduced HAD activity of HN protein, which was even more obvious for all three double mutants (G12, G14 and G24) and the triple mutant (G124). In addition, every mutant protein retained F-interactive capability that was equal to the wild-type protein capability. Interestingly, the F protein that could be co-immunoprecipitated with the G12 mutated protein or immunoprecipitated with anti-F antibody was not efficiently cleaved. For G14, G24 and G124, little cleaved F protein was detected in co-immuoprecipitation F protein assay and its total amounts where in the cell lysates. The mechanism underlying hPIV-3 HN and F protein remained associated before and after receptor engagement and the strength of the HN-receptor interaction modulated the activation of F the protein which could determine the extent of fusion. Finally, we demonstrated that single or multiple N-glycosylation site mutations inhibited fusion at the earliest stages. Taken together, these results indicated that N-glycosylation of hPIV-3 HN is critical to its receptor recognition activity, cleavage of the F protein, and fusion promotion activity, but had no influence on its interaction with the homologous F protein and NA activity. (C) 2013 Elsevier B.V. All rights reserved.