Department of Molecular Physiology and Biological Physics and Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA.
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
J Mol Biol. 2018 Mar 2;430(5):594-601. doi: 10.1016/j.jmb.2018.01.006. Epub 2018 Feb 2.
Influenza viral entry into the host cell cytoplasm is accomplished by a process of membrane fusion mediated by the viral hemagglutinin protein. Hemagglutinin acts in a pH-triggered fashion, inserting a short fusion peptide into the host membrane followed by refolding of a coiled-coil structure to draw the viral envelope and host membranes together. Mutations to this fusion peptide provide an important window into viral fusion mechanisms and protein-membrane interactions. Here, we show that a well-described fusion peptide mutant, G1S, has a phenotype that depends strongly on the viral membrane context. The G1S mutant is well known to cause a "hemifusion" phenotype based on experiments in transfected cells, where cells expressing G1S hemagglutinin can undergo lipid mixing in a pH-triggered fashion similar to virus but will not support fusion pores. We compare fusion by the G1S hemagglutinin mutant expressed either in cells or in influenza virions and show that this hemifusion phenotype occurs in transfected cells but that native virions are able to support full fusion, albeit at a slower rate and 10-100× reduced infectious titer. We explain this with a quantitative model where the G1S mutant, instead of causing an absolute block of fusion, alters the protein stoichiometry required for fusion. This change slightly slows fusion at high hemagglutinin density, as on the viral surface, but at lower hemagglutinin density produces a hemifusion phenotype. The quantitative model thus reproduces the observed virus-cell and cell-cell fusion phenotypes, yielding a unified explanation where membrane context can control the observed viral fusion phenotype.
流感病毒进入宿主细胞质是通过病毒血凝素蛋白介导的膜融合过程实现的。血凝素以 pH 触发的方式起作用，将短融合肽插入宿主膜中，然后卷曲螺旋结构重新折叠，将病毒包膜和宿主膜拉在一起。对该融合肽的突变提供了一个了解病毒融合机制和蛋白-膜相互作用的重要窗口。在这里，我们表明，一种描述良好的融合肽突变体 G1S，其表型强烈依赖于病毒膜环境。众所周知，G1S 突变体会导致“半融合”表型，这是基于转染细胞中的实验得出的，其中表达 G1S 血凝素的细胞可以以类似于病毒的 pH 触发方式发生脂质混合，但不会支持融合孔。我们比较了在细胞或流感病毒中表达的 G1S 血凝素突变体的融合，并表明这种半融合表型发生在转染细胞中，但天然病毒能够支持完全融合，尽管融合速度较慢，感染滴度降低 10-100 倍。我们用一个定量模型来解释这一点，其中 G1S 突变体不是绝对阻止融合，而是改变了融合所需的蛋白比例。这种变化略微减缓了高血凝素密度（如在病毒表面）下的融合，但在较低的血凝素密度下产生了半融合表型。因此，定量模型再现了观察到的病毒-细胞和细胞-细胞融合表型，提供了一个统一的解释，其中膜环境可以控制观察到的病毒融合表型。
