Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen, Netherlands.
J Phys Condens Matter. 2018 May 23;30(20):204005. doi: 10.1088/1361-648X/aabc21. Epub 2018 Apr 6.
The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.
流感 A 病毒感染的第一步是与宿主细胞膜接触，随后病毒与宿主细胞膜融合。靶双层的组成对融合效率和时间都有复杂的影响。在这里，采用体外单颗粒方法研究这种效应。使用全内反射荧光（TIRF）显微镜和微流控流动池，可视化单个病毒粒子的半融合。在改变靶双层胆固醇含量和唾液酸供体的情况下，研究半融合效率和动力学。测试的胆固醇比为 0%、10%、20%和 40%。使用 GD1a 和 GYPA 作为唾液酸供体。胆固醇比和唾液酸供体都被证明对半融合效率有显著影响。此外，GD1a 和 GYPA 条件之间的比较表明，半融合时间的胆固醇依赖性严重受唾液酸供体的影响。只有 GD1a 显示出随着靶双层胆固醇浓度的增加，半融合效率和时间呈明显增加趋势，最大速率为 GD1A 和 40%胆固醇。总的来说，我们的结果表明，根据实验中所需的半融合时间和效率，应仔细选择唾液酸供体和靶双层组成。
