Liu Katherine N, Boxer Steven G
Department of Chemistry, Stanford University, Stanford, California.
Department of Chemistry, Stanford University, Stanford, California.
Biophys J. 2020 May 19;118(10):2426-2433. doi: 10.1016/j.bpj.2020.03.021. Epub 2020 Apr 4.
Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.
宿主脂质组成会影响甲型流感病毒（IAV）进入过程的多个阶段，包括IAV与唾液酸化聚糖的初始结合、病毒包膜与宿主膜的融合，以及形成一个融合孔，病毒基因组通过该融合孔转移到靶细胞中。特别是，已表明靶膜胆固醇优先与病毒受体结合，并改变膜的物理性质，如流动性和曲率。这些性质会影响IAV的结合和融合，这使得难以将胆固醇在IAV融合中的作用与受体结合效应区分开来。在这里，我们开发了一种融合测定法，该方法使用合成的DNA-脂质缀合物作为替代病毒受体，将病毒粒子拴系到靶囊泡上。为避免向病毒膜中添加自猝灭浓度的染料标记脂质可能产生的干扰作用，我们将病毒粒子拴系到脂质标记的靶囊泡上，并使用荧光显微镜检测单个pH触发的IAV膜融合事件。通过这种方法，我们发现靶膜中的胆固醇提高了单粒子IAV脂质混合的效率，而脂质混合的速率与胆固醇组成无关。我们还发现，流感病毒脂质与具有不同胆固醇组成的靶膜混合的单粒子动力学与受体结合无关，这表明胆固醇介导的靶膜内病毒受体的空间聚集不会显著影响IAV半融合。这些结果与靶膜胆固醇通过改变宿主膜曲率来提高脂质混合效率的假设一致。
