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

1.将猴病毒5型（WR株）F蛋白的Leu-22替换为Pro，获得了一个突变的F蛋白L22 P，该蛋白能诱导非HN依赖的细胞-细胞融合。L22 P的突变分析表明，F2 N-端与融合肽之间的疏水相互作用或HR 1结构域与Cys-rich结构域下游区域之间的静电相互作用影响L22 P的融合活性。2. Se-L22 P是一种突变的F蛋白，其中L22 P的切割位点被仙台病毒F蛋白的切割位点取代。结果表明，只有当用胰蛋白酶处理时，Se-L22 P才能被切割并诱导细胞-细胞融合。我们发现Se-L22 P位于质膜的非筏结构域，而不是筏结构域，在胰蛋白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单克隆抗体（M1-1A）存在下增殖人副流感病毒2型（PIV 2：Toshiba株），选择了表现出非常低致病性（或细胞-细胞融合）的逃逸突变体F13。使用一组通过反向遗传学产生的突变病毒进行分析，已经表明F13 HN蛋白的位置83和186处的突变都是低致细胞病变性所需的，而这些突变不影响病毒-细胞融合。这些结果表明，细胞-细胞融合与病毒-细胞融合的分子机制存在差异，可能与感染细胞中的一种未知细胞因子有关。与PIV 3相反，在神经氨酸酶活性和PIV 2的细胞致病性之间未观察到相关性，表明PIV 2和PIV 3之间的毒力差异可能反映了神经氨酸酶功能的差异。

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.