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Studies on the molecular structure and dynamics of the viral proteins that induce membrane fusion

诱导膜融合的病毒蛋白的分子结构和动力学研究

基本信息

批准号：
09670311
负责人：
TSURUDOME Masato
金额：
$ 2.24万
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：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

1. Chimeric analysis of the F proteins of human parainfluenza virus type 2 (PIV2) and simian virus 41 (SV41) was performed, identifying regions on the PIV2 F protein that are involved in the functional interaction with the HN proteins in the induction of cell fusion. 2. The F protein of SV5 strain W3A induced cell fusion in BHK cells in the absence of the HN protein, while that of strain WR required the HN protein in the induction of cell fusion. This HN-independent fusion activity could be transferred to the WR F protein by Pro-22 in the W3A F2 subunits as represented by the mutant F protein, L22P. 3. Furthermore, by mutational and chimeric analyses using another SV5 strain T1, Glu-132 in the heptad repeat 1 domain of the F1 subunit was identified as another determinant involved in the HN-independent fusion activity. 4. The mutant L22P did not induce cell fusion in L929 cells, which enabled us to establish an L929 cell line stably expression L22P (L22P-L). Cocultivation of L22P-L with BHK cell surface. 5. An anti-L22P monoclonal antibody (mAb 21-1), which completely inhibited L22P-mediated cell fusion, was obtained. 6. The mAb 21-1 could not react with surface-localized WR F protein. In contrast the mAb 21-1 could react with L22P irrespective of its location. These observations indicate that the mAb 21-1 is a conformation-dependent antibody whose epitope may be hindered by a putative conformational change which takes place after the WR F protein is cleaved into F1 and F2. Alternatively, in the case of the L22P, this post-cleavage conformational change does not seem to occur correctly which may a prerequisite for its HN-independent fusion activity.

1.对人副流感病毒2型（PIV 2）和猿猴病毒41（SV 41）的F蛋白进行嵌合分析，鉴定PIV 2 F蛋白上参与诱导细胞融合中与HN蛋白功能性相互作用的区域。2.在不存在HN蛋白的情况下，SV 5株W3 A的F蛋白诱导BHK细胞中的细胞融合，而WR株的F蛋白在细胞融合的诱导中需要HN蛋白。这种HN非依赖性融合活性可以通过W3 A F2亚基中的Pro-22转移到WR F蛋白，如突变F蛋白L22 P所代表的。3.此外，通过使用另一种SV 5株T1的突变和嵌合分析，F1亚基的七肽重复1结构域中的Glu-132被鉴定为参与HN非依赖性融合活性的另一个决定因素。4.突变体L22 P在L929细胞中不诱导细胞融合，这使得我们能够建立稳定表达L22 P的L929细胞系（L22 P-L）。L22 P-L与BHK细胞表面的共培养。5.获得了完全抑制L22 P介导的细胞融合的抗L22 P单克隆抗体（mAb 21-1）。6. mAb 21-1不能与表面定位的WRF蛋白反应。相反，mAb 21-1可以与L22 P反应，而不管其位置如何。这些观察结果表明，mAb 21-1是一种构象依赖性抗体，其表位可能受到WR F蛋白裂解成F1和F2后发生的推定构象变化的阻碍。或者，在L22 P的情况下，这种裂解后构象变化似乎没有正确发生，这可能是其HN非依赖性融合活性的先决条件。