The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex
The coronavirus spike protein is a class I virus fusion protein: Structural and functional characterization of the fusion core complex
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DOI：
10.1128/jvi.77.16.8801-8811.2003
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发表时间：
2003-08-01
影响因子：
5.4
通讯作者：
Rottier, PJM
中科院分区：
文献类型：
--
作者：
Bosch, BJ;van der Zee, R;Rottier, PJM
冠状病毒的进入是由病毒刺突(S)糖蛋白介导的。小鼠冠状病毒小鼠肝炎病毒株A59的180 kda寡聚S蛋白被翻译后切割成S1受体结合单元和S2膜融合单元。后者被认为含有一个内部融合肽，并具有两个4,3疏水（七肽）重复区域，称为HR1和HR2。HR2位于膜锚附近，HR1位于其上游约170个氨基酸（aa）处。七肽重复区存在于许多不同病毒的融合蛋白中，是一类病毒融合蛋白的重要特征。我们研究了这些区域在冠状病毒膜融合中的作用。在大肠杆菌中产生HR1和HR2区对应的肽HR1 （96 aa）和HR2 （39 aa）。当混合在一起时，这两种肽被发现组装成一个非常稳定的低聚复合物。无论是在它们自身还是在复合物内，肽都是高度螺旋状的。电子显微镜分析显示，该配合物呈棒状结构，长度约为14.5 nm。有限的蛋白水解结合质谱分析表明，HR1和HR2在络合物中以反平行的方式发生。在天然蛋白中，这种构象会使位于HR1 n端结构域的融合肽与跨膜锚点靠近。通过生物测定，HR2肽被证明是病毒进入细胞以及细胞-细胞融合的有效抑制剂。生化和功能数据均表明，冠状病毒刺突蛋白是一类病毒融合蛋白。
Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure similar to14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.