Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.
Fusion of Sendai virus with negatively charged liposomes as studied by pyrene-labelled phospholipid liposomes.
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通过芘标记的磷脂脂质体研究仙台病毒与带负电荷的脂质体的融合。
DOI：
10.1016/0005-2736(86)90527-4
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Lichtenberg,D
中科院分区：
文献类型：
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作者：
Amselem,S;Barenholz,Y;Loyter,A;Nir,S;Lichtenberg,D
仙台病毒颗粒与带负电荷的脂质体融合，但不与由两性离子磷脂制成的囊泡融合。脂质体-病毒的融合过程进行了研究，通过稀释的浓度依赖性准分子形成的荧光团2-芘基十二烷酰磷脂酰胆碱的脂质体中所包含的病毒脂质。在质量作用动力学模型的框架内对数据进行了分析。这提供了探针稀释的最终水平的分析解和整体融合过程的动力学的数值解，在脂质体-病毒粘附、去粘附和融合的速率常数方面。该分析得出以下结论：（1）在中性pH和37°C下，只有15%的病毒颗粒可以与磷脂囊泡融合，尽管所有的病毒粒子都可以与脂质体聚集。聚合、融合和脱粘的速率常数分别为107 M −1· s−1、10− 3 s − 1和10− 2 s −1的数量级。活性病毒的分数随温度而增加。(2)在酸性pH下，“可融合”病毒的分数和融合率均显著增加。融合的最佳pH为3-4，其中大多数病毒颗粒是活性的。在较高的pH值下，越来越多的病毒颗粒变得无活性，可能是由于病毒糖蛋白的电离，而在pH值低于3.0时，融合明显减少，最有可能是由于带负电荷的囊泡的质子化。(3)虽然在pH 7.4和37°C下只有15%的病毒体与脂质体融合，但所有脂质体都失去其内容物（Amselem，S.，Loyter，A. Lichtenberg，D.和Barenholz，Y.（1985）Biochim. Biophys. Acta 820，1-10）。因此，我们建议，释放截留的溶质是由于脂质体病毒聚集，而不是融合。胰蛋白酶消化和病毒颗粒的热灭活不仅抑制融合过程，而且抑制羧基荧光素的释放。这证明了病毒膜蛋白在脂质体-病毒聚集中的强制性作用。(4)由病毒脂质和血凝素/神经氨酸酶（HN）糖蛋白组成的重组囊泡与带负电荷的脂质体融合，类似于完整的病毒体。这表明，与病毒与生物膜的融合不同，病毒粒子与带负电荷的囊泡的融合仅需要HN而不需要融合糖蛋白。
Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: (1) At neutral pH and 37°C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 107M−1· s−1, 10−3s−1and 10−2s−1, respectively. The fraction of active virus increases with temperature. (2) At acidic pH, both the fraction of ‘fusable’ virus and the rate of fusion increase markedly. The optimal pH for fusion is 3–4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. (3) While only 15% of the virions fuse with the liposomes at pH 7.4 and 37°C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1–10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. (4) Reconstituted vesicles made of the viral lipid and the hemagglutinin/ neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein.