Domanska Marta K, Dunning Rebecca A, Dryden Kelly A, Zawada Katarzyna E, Yeager Mark, Kasson Peter M
Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia.
Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia.
Biophys J. 2015 Nov 3;109(9):1917-24. doi: 10.1016/j.bpj.2015.09.014.
Influenza virus delivers its genome to the host cytoplasm via a process of membrane fusion mediated by the viral hemagglutinin protein. Optimal fusion likely requires multiple hemagglutinin trimers, so the spatial distribution of hemagglutinin on the viral envelope may influence fusion mechanism. We have previously shown that moderate depletion of cholesterol from the influenza viral envelope accelerates fusion kinetics even though it decreases fusion efficiency, both in a reversible manner. Here, we use electron cryo-microscopy to measure how the hemagglutinin lateral density in the viral envelope changes with cholesterol extraction. We extract this information by measuring the radial distribution function of electron density in >4000 viral images per sample, assigning hemagglutinin density by comparing images with and without anti-HA Fab bound. On average, hemagglutinin trimers move closer together: we estimate that the typical trimer-trimer spacing reduces from 94 to 84 Å when ∼90% of cholesterol is removed from the viral membrane. Upon restoration of viral envelope cholesterol, this spacing once again expands. This finding can qualitatively explain the observed changes to fusion kinetics: contemporary models from single-virus microscopy are that fusion requires the engagement of several hemagglutinin trimers in close proximity. If removing cholesterol increases the lateral density of hemagglutinin, this should result in an increase in the rate of fusion.
流感病毒通过由病毒血凝素蛋白介导的膜融合过程将其基因组传递到宿主细胞质中。最佳融合可能需要多个血凝素三聚体，因此血凝素在病毒包膜上的空间分布可能会影响融合机制。我们之前已经表明，从流感病毒包膜中适度去除胆固醇会加速融合动力学，尽管它会以可逆的方式降低融合效率。在这里，我们使用电子冷冻显微镜来测量病毒包膜中血凝素的侧向密度如何随胆固醇提取而变化。我们通过测量每个样本中 >4000 个病毒图像的电子密度径向分布函数来提取此信息，通过比较有和没有结合抗 HA Fab 的图像来确定血凝素密度。平均而言，血凝素三聚体彼此靠得更近：我们估计当从病毒膜中去除约 90% 的胆固醇时，典型的三聚体 - 三聚体间距从 94 Å 减小到 84 Å。当病毒包膜胆固醇恢复时，这种间距再次扩大。这一发现可以定性地解释观察到的融合动力学变化：单病毒显微镜的当代模型表明，融合需要几个紧密相邻的血凝素三聚体参与。如果去除胆固醇会增加血凝素的侧向密度，这应该会导致融合速率增加。
