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Difference in molecular mechanism between envelope-cell fusion and cell-cell fusion.

包膜细胞融合和细胞间融合分子机制的差异。

基本信息

批准号：
15590414
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：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590414/
关键词：
simian virus 5 human parainfuenza virus type 2 membrane fusion HN protein F protein monoclonal antibody neuraminidase reverse genetics パラミクソウイルスパラインフルエンザ2型ウイルス Escape mutant 細胞膜-細胞膜融合構造変化多核巨細胞分子モデリング負電荷間相互作用モノクロナール抗体

项目摘要

1.By replacing Leu-22 of the F protein of simian virus 5 (WR strain) with Pro, a mutant F protein, L22P, was obtained which induced the HN-independent cell-cell fusion. Mutational analysis of L22P has suggested that either the hydrophobic interaction between the F2 N-terminus and fusion peptide or the electrostatic interaction between the HR1 domain and a region downstream of the Cys-rich domain affects the fusing activity of L22P.2.Se-L22P is a mutant F protein, in which the cleavage site of L22P is replaced with that of the Sendai virus F protein. As the result, Se-L22P can be cleaved and can induce cell-cell fusion only when treated with trypsin. We found that Se-L22P located in the nonraft domains, but not in the raft domains, of the plasma membrane undergoes a conformational change upon treatment with trypsin and that cell-cell fusion is induced after this conformational change.3.By propagating human parainfluenza virus type 2 (PIV2:Toshiba strain) in the presence of an anti-HN monoclonal antibody (M1-1A), an escape mutant, F13, was selected which exhibited very low pathogenicity (or cell-cell fusion). Analysis employing a set of mutant viruses that were produced by reverse genetics, it has been shown that mutations at positions 83 and 186 of the F13 HN protein are both required for the low cytopathogenicity, whereas these mutations do not affect virus-cell fusion. These results suggest that there is difference in molecular mechanism between cell-cell fusion and virus-cell fusion and that an unknown cellular factor in the infected cell is involved. In contrast to PIV3, no correlation was observed between the neuraminidase activity and the cytopathogenicity of PIV2, suggesting that the difference in virulence between PIV2 and PIV3 may reflect the difference in neuraminidase function.

1.用Pro取代猴病毒5号(WR株)F蛋白的Leu-22，获得了诱导HN非依赖性细胞-细胞融合的突变型F蛋白L22P。L22P的突变分析表明，F2N末端与融合肽之间的疏水相互作用或HR1结构域与富含半胱氨酸的下游区域之间的静电相互作用影响了L22P的融合活性。Se-L22P是一种突变的F蛋白，其切割位点被仙台病毒F蛋白取代。结果表明，只有在胰酶作用下，Se-L22P才能被切割并诱导细胞-细胞融合。我们发现，Se-L22P位于质膜的非RAFT结构域，而不是RAFT结构域，经胰酶处理后发生构象变化，并在这种构象变化后诱导细胞-细胞融合。3.通过在抗HN单抗(M1-1A)存在下增殖人副流感病毒2型(PIV2：Toshiba株)，筛选出一株逃逸突变株F13，其致病性很低(或细胞-细胞融合)。利用一组由反向遗传学产生的突变病毒进行的分析表明，F13 HN蛋白第83位和186位的突变都是低细胞致病性所必需的，而这些突变不影响病毒与细胞的融合。这些结果表明，细胞-细胞融合和病毒-细胞融合在分子机制上存在差异，可能涉及感染细胞中未知的细胞因子。与PIV3相比，PIV2的神经氨酸酶活性与细胞致病性之间没有相关性，提示PIV2和PIV3毒力的差异可能反映了神经氨酸酶功能的不同。