Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion
Biochemical Analysis of Coronavirus Spike Glycoprotein Conformational Intermediates during Membrane Fusion
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DOI：
10.1128/jvi.00785-19
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发表时间：
2019-10-01
影响因子：
5.4
通讯作者：
Matsuyama, Shutoku
中科院分区：
文献类型：
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作者：
Kawase, Miyuki;Kataoka, Michiyo;Matsuyama, Shutoku
在包膜病毒表面上表达的融合蛋白介导病毒和细胞膜的融合以促进病毒感染。病毒融合蛋白的融合前和融合后结构已被表征，但它们之间的构象变化仍然知之甚少。在这里，我们研究了鼠冠状病毒融合蛋白的中间构象，称为刺突蛋白，它必须在受体结合后被细胞蛋白酶切割。蛋白酶消化产物的蛋白质印迹分析显示，两个亚基（67和69 kDa）从一个单一的刺突蛋白（180 kDa）产生。这两个亚基被认为是来自构象变化的副产物，并用于探测刺突蛋白的中间构象。与七肽重复序列（HR）肽的相互作用表明，这些亚基分别采用包装和非包装构象，二维电泳显示三聚体组装。基于生化观察，我们提出了刺突蛋白中间结构的不对称三聚体模型。受体结合诱导三聚体的膜结合潜力，其中至少一个HR基序形成一个密集的发夹结构，而膜融合亚基被受体结合亚基覆盖，从而防止刺突蛋白形成典型的同源三聚体prehairpin结构预测的当前模型的I类病毒融合蛋白。随后的蛋白水解诱导同时包装剩余的未包装的HR后，在中心轴组装的三个HR，以产生一个六螺旋束。我们的模型提出了一个膜融合的包膜病毒的关键机制。重要性最近的研究使用单粒子冷冻电子显微镜（cryoEM）揭示了激活病毒融合蛋白在启动阶段的机制。然而，表征从融合前到融合后结构的后续触发阶段的基础过渡是困难的，因为单粒子cryoEM排除不稳定的结构，出现异质形状。因此，需要基于群体的生化分析来捕获不稳定蛋白质的特征。在这里，我们分析了冠状病毒融合蛋白在激活过程中的蛋白酶消化产物;它们的大小似乎直接受构象状态的影响。我们提出了一个模型的病毒融合蛋白在中间状态，其中涉及一个紧凑的结构和构象的变化，克服空间位阻内的三个融合蛋白亚基。
A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and postfusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric prehairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.IMPORTANCE Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to postfusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.