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Analysis of the conformational changes of viral glycoprotein that is involved in inducing syncytium formation.

分析参与诱导合胞体形成的病毒糖蛋白的构象变化。

基本信息

批准号：
12670280
负责人：
TSURUDOME Masato
金额：
$ 2.05万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

As reported previously, the fusion protein (F) of simian virus 5 (SV5) strain WR induces cell fusion only when coexpressed with the hemagglutinin-neuraminidase protein (HN), while the F of SV5 strain W3A can induce cell fusion independently of HN. When Leu-22 in WR F is replaced with the counterpart (Pro-22) in W3A F, the resulting L22P mutant is able to induce the HN-independent cell fusion. In the present study, I have shown that there is difference in conformation between L22P and WR F, in which the epitope in WR F for monoclonal antibody (MAb) 21-1 is cryptic while it is exposed in L22P. Furthermore, a 36-kDa polypeptide was discovered in the L22P-expressing HeLa cells but not in the WR F-expressing HeLa cells, which proved to be derived from the L22P that has been transported to the cell surface. The cell surface-localized L22P but not WR F was internalized into the cell in a cholesterol dependent manner as analyzed by biotin internalization assay. Furthermore, the 36-kDa polypeptide was not generated when the cells were treated with bafilomycin A1. These results suggest that the 36-kDa polypeptide is derived from the L22P which has undergone endocytosis and has been degraded in the lysosome. By heating at 47℃ for 2 min, WR F acquired the HN-independent cell fusion activity and underwent endocytosis, suggesting that an internalization signal is displayed by the WR F that is in the "postfusion" conformation. The heat treatment also resulted in an exposure of the MAb 21-1 epitope in WR F, suggesting that the head/neck domain of F, which harbors the MAb 21-1 epitope, undergoes a conformational change during the induction of cell fusion.

此前报道，猿猴病毒5 （SV5）株WR的融合蛋白(F)只有在与血凝素-神经氨酸酶蛋白（HN）共表达时才能诱导细胞融合，而SV5株W3A的F可以独立于HN诱导细胞融合。将WR F中的Leu-22替换为W3A F中的Pro-22，得到的L22P突变体能够诱导不依赖hn的细胞融合。在本研究中，我发现L22P与WR F的构象存在差异，单克隆抗体（MAb） 21-1在WR F中的表位是隐性的，而在L22P中是暴露的。此外，在表达L22P的HeLa细胞中发现了一个36 kda的多肽，而在表达WR f的HeLa细胞中却没有发现，该多肽被证明是来自于运输到细胞表面的L22P。通过生物素内化实验分析，细胞表面定位的L22P而非WR F以胆固醇依赖的方式内化到细胞中。此外，当细胞用巴菲霉素A1处理时，不产生36kda的多肽。这些结果表明，36 kda的多肽是由L22P产生的，L22P在溶酶体中经历了内吞作用并被降解。经47℃加热2 min， WR F获得了与hn无关的细胞融合活性，并进行了内吞作用，表明WR F显示了“融合后”构象的内化信号。热处理还导致WR F中MAb 21-1表位暴露，这表明在诱导细胞融合过程中，含有MAb 21-1表位的F的头颈结构域发生了构象变化。