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

如前所述，猴病毒5型（SV 5）WR株的融合蛋白（F）仅在与血凝素-神经氨酸酶蛋白（HN）共表达时才诱导细胞融合，而SV 5株W3 A的F可独立于HN诱导细胞融合。当WR F中的Leu-22被W3 A F中的对应物（Pro-22）取代时，所得的L22 P突变体能够诱导HN非依赖性细胞融合。在本研究中，我发现L22 P和WRF之间存在构象差异，其中单克隆抗体（MAb）21-1在WRF中的表位是隐蔽的，而在L22 P中是暴露的。此外，在表达L22 P的HeLa细胞中发现了36-kDa的多肽，但在表达WRF的HeLa细胞中没有发现，其被证明来源于已经转运到细胞表面的L22 P。细胞表面定位的L22 P而不是WRF以胆固醇依赖性方式内化到细胞中，如通过生物素内化测定分析的。此外，当用巴弗洛霉素A1处理细胞时，不产生36-kDa多肽。这些结果表明，36-kDa多肽来源于L22 P，L22 P已经历内吞作用并已在溶酶体中降解。在47℃下加热2 min，WRF获得了HN非依赖性细胞融合活性，并经历了内吞作用，表明处于“融合后”构象的WRF显示了内化信号。热处理还导致WRF中MAb 21-1表位的暴露，表明F的头/颈结构域（其含有MAb 21-1表位）在诱导细胞融合期间经历构象变化。

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.