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

1.对人副流感病毒2型(PIV2)和猴病毒41(SV41)的F蛋白进行了嵌合分析，确定了PIV2 F蛋白上与HN蛋白在诱导细胞融合过程中功能相互作用的区域。2.SV5株W3A株的F蛋白在没有HN蛋白的情况下诱导BHK细胞融合，而WR株的F蛋白在诱导细胞融合时需要HN蛋白。这种不依赖于HN的融合活性可以通过W3A F2亚基中的Pro-22转移到WR F蛋白上，以突变的F蛋白L22P为代表。3.此外，通过对另一株SV5毒株T1的突变和嵌合分析，确定F1亚基七肽重复序列1区域的Glu-132是参与HN非依赖性融合活性的另一个决定因素。4.突变体L22P在L929细胞中没有诱导细胞融合，因此我们建立了稳定表达L22P的L929细胞系(L22P-L)。L22P-L与BHK细胞表面共培养5.获得了一株能完全抑制L22P介导的细胞融合的抗L22P单抗(mAb21-1)。6.单抗21-1不能与表面定位的WrF蛋白反应。相反，mAb21-1可与L22P发生反应，而与其位置无关。这些观察表明，mAb21-1是一种构象依赖性抗体，其表位可能受到WRF蛋白裂解成F1和F2后可能发生的构象变化的阻碍。或者，在L22P的情况下，这种切割后的构象变化似乎没有正确发生，这可能是其HN独立融合活性的先决条件。

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.