Harmsen M C, Wilschut J, Scherphof G, Hulstaert C, Hoekstra D
Eur J Biochem. 1985 Jun 18;149(3):591-9. doi: 10.1111/j.1432-1033.1985.tb08966.x.
Sendai virus membranes were reconstituted by detergent dialysis, using the non-ionic detergents Triton X-100 and octyl glucoside. Membrane reassembly was determined by measuring the surface-density-dependent efficiency of resonance energy transfer between two fluorescent phospholipid analogues, which were co-reconstituted with the viral envelopes. The functional incorporation of the viral proteins was established by monitoring the ability of the reconstitution products to fuse with erythrocyte membranes, utilizing assays based on either resonance energy transfer or on relief of fluorescence selfquenching. The persistent adherence of residual Triton X-100 with the reconstituted membrane was revealed by an artificial detergent-effect on the resonance energy transfer efficiency and the occurrence of hemolysis of human erythrocytes under conditions where fusion does not occur. Properly reconstituted Sendai virus envelopes were obtained with octyl glucoside. The fusion activity of the viral envelopes was dependent on the initial concentration of octyl glucoside used to disrupt the virus and the rate of detergent removal. Rapid removal of detergent by dialysis against large volumes of dialysis buffer (ratio 1:850) or by gel filtration produced reconstituted membranes capable of inducing hemagglutination but significant fusion activity was not detected. By decreasing the volume ratio of dialysate versus dialysis buffer to 1:250 or 1:25, fusogenic viral envelopes were obtained. The initial fusion kinetics of the reconstituted viral membrane and the parent virus were different in that both the onset and the initial rate of fusion of the reconstituted membranes were faster, whereas the extents to which both particles eventually fused with the target membrane were similar. The differences in the initial fusion kinetics lead us to suggest that the details of the fusion mechanism between Sendai virus and the target membrane involve factors other than the mere presence of glycoproteins F and HN in the viral bilayer. Finally, the results also indicate that determination of the viral fusion activity in a direct manner, rather than by an indirect assay, such as hemolysis, is imperative for a proper evaluation of the functional properties retained upon viral reconstitution.
使用非离子去污剂Triton X - 100和辛基葡糖苷通过去污剂透析法重建仙台病毒膜。通过测量与病毒包膜共重建的两种荧光磷脂类似物之间共振能量转移的表面密度依赖性效率来确定膜的重新组装。通过监测重建产物与红细胞膜融合的能力来确定病毒蛋白的功能性掺入，该能力利用基于共振能量转移或荧光自猝灭缓解的测定法。通过对共振能量转移效率的人工去污剂效应以及在不发生融合的条件下人红细胞溶血的发生，揭示了残留的Triton X - 100与重建膜的持续粘附。用辛基葡糖苷获得了适当重建的仙台病毒包膜。病毒包膜的融合活性取决于用于破坏病毒的辛基葡糖苷的初始浓度和去污剂去除速率。通过对大量透析缓冲液（比例1:850）进行透析或通过凝胶过滤快速去除去污剂，产生了能够诱导血细胞凝集但未检测到明显融合活性的重建膜。通过将透析液与透析缓冲液的体积比降低到1:250或1:25，获得了具有融合活性的病毒包膜。重建病毒膜和亲本病毒的初始融合动力学不同，在于重建膜的融合起始和初始速率都更快，而两种颗粒最终与靶膜融合的程度相似。初始融合动力学的差异使我们认为，仙台病毒与靶膜之间融合机制的细节涉及病毒双层中糖蛋白F和HN之外的其他因素。最后，结果还表明，以直接方式而非通过间接测定法（如溶血）来确定病毒融合活性对于正确评估病毒重建后保留的功能特性至关重要。
