Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH
Glycine 29 Is Critical for Conformational Changes of the Spike Glycoprotein of Mouse Hepatitis Virus A59 Triggered by either Receptor Binding or High pH
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甘氨酸 29 对于由受体结合或高 pH 值触发的小鼠肝炎病毒 A59 刺突糖蛋白的构象变化至关重要
DOI：
10.1128/jvi.01046-19
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发表时间：
2019-08
影响因子：
5.4
通讯作者：
Qian Zhaohui
中科院分区：
文献类型：
--
作者：
Mi Dan;Ou Xiuyuan;Li Pei;Peng Guiqing;Liu Yan;Guo Ruixuan;Mu Zhixia;Li Fang;Holmes Kathryn;Qian Zhaohui
MHV S蛋白与受体mCEACAM 1a的结合引发S蛋白的构象变化，导致六螺旋束的形成以及病毒和细胞膜的融合。然而，受体结合后S蛋白的构象变化启动的机制尚未确定。在这项研究中，我们发现，虽然G29，在受体结合界面的边缘和结构转折的中心后，β1折叠的S蛋白，与D或T的取代引发自发的构象变化的S蛋白和pH非依赖性RIS，G29 P突变显着阻碍由受体结合或pH 8.0引发的S蛋白的构象变化。我们的理由是，这种结构的转变可能是至关重要的S蛋白的构象变化，改变这种结构的转变可能会引发S蛋白的构象变化，导致膜融合。小鼠肝炎病毒（MHV）利用其S蛋白的N端结构域（NTD）与宿主受体小鼠癌胚抗原相关细胞粘附分子1a（mCEACAM 1a）结合并介导病毒侵入。我们以前对MHV NTD/mCEACAM 1a复合物的晶体结构研究（G. Peng，L.孙，K. R. Rajashankar，Z. Qian等人，Proc Natl Acad Sci U S A 108：10696-10701，2011，https：//doi.org/10.1073/pnas.1104306108）揭示了NTD中有14个残基与受体相互作用。然而，它们对受体结合和病毒进入的贡献尚未得到充分研究。在这里，我们分析了13个14接触残基的诱变，并确定I22是必不可少的受体结合和病毒进入。出乎意料的是，我们发现G29对于由受体结合或高pH触发的S蛋白的构象变化是关键的。用A、D、F、K、M和T在不同程度上替换G29，引起S1从S蛋白的自发解离，导致HEK 293 T细胞中高pH触发的受体非依赖性合胞体（RIS）形成的增强，与野生型（WT）相比。相比之下，用具有严格构象的倾向于转向的残基P替换G29，阻碍了病毒进入和由受体结合或pH 8.0触发的S蛋白的构象变化，这表明围绕G29的结构转向及其灵活性是至关重要的。最后，稳定的NTD的G29 P几乎没有影响的pH值无关的RIS诱导的Y320 A突变的C-末端结构域（CTD）的S1亚基，表明有可能是一个不存在的NTD和CTD之间的串扰在S蛋白的构象变化。我们的研究将有助于更好地理解S蛋白的构象变化是如何触发的机制。重要性MHV S蛋白与受体mCEACAM 1a的结合引发S蛋白的构象变化，导致六螺旋束的形成以及病毒和细胞膜的融合。然而，受体结合后S蛋白的构象变化启动的机制尚未确定。在这项研究中，我们发现，虽然G29，在受体结合界面的边缘和结构转折的中心后，β1折叠的S蛋白，与D或T的取代引发自发的构象变化的S蛋白和pH非依赖性RIS，G29 P突变显着阻碍由受体结合或pH 8.0引发的S蛋白的构象变化。我们的理由是，这种结构的转变可能是至关重要的S蛋白的构象变化，改变这种结构的转变可能会引发S蛋白的构象变化，导致膜融合。
Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion. ABSTRACT Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of the viral spike (S) protein to bind the host receptor mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) and mediate virus entry. Our previous crystal structure study of the MHV NTD/mCEACAM1a complex (G. Peng, D. Sun, K. R. Rajashankar, Z. Qian, et al., Proc Natl Acad Sci U S A 108:10696–10701, 2011, https://doi.org/10.1073/pnas.1104306108) reveals that there are 14 residues in the NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis and identified I22 as being essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of the S protein triggered by either receptor binding or high pH. Replacement of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from the S protein, resulting in an enhancement of high-pH-triggered receptor-independent syncytium (RIS) formation in HEK293T cells, compared to the wild type (WT). In contrast, replacement of G29 with P, a turn-prone residue with a strict conformation, hindered virus entry and conformational changes of the S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of the NTD by G29P had almost no effect on pH-independent RIS induced by the Y320A mutation in the C-terminal domain (CTD) of the S1 subunit, indicating that there might be an absence of cross talk between the NTD and CTD during conformational changes of the S protein. Our study will aid in better understanding the mechanism of how conformational changes of the S protein are triggered. IMPORTANCE Binding of the MHV S protein to the receptor mCEACAM1a triggers conformational changes of S proteins, leading to the formation of a six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of the S protein is initiated after receptor binding has not been determined. In this study, we showed that while replacement of G29, a residue at the edge of the receptor binding interface and the center of the structural turn after the β1-sheet of the S protein, with D or T triggered spontaneous conformational changes of the S protein and pH-independent RIS, the G29P mutation significantly impeded the conformational changes of S proteins triggered by either receptor binding or pH 8.0. We reason that this structural turn might be critical for conformational changes of the S protein and that altering this structural turn could initiate conformational changes of the S protein, leading to membrane fusion.
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DOI：
10.1074/jbc.m400759200
发表时间：
2004-05-14
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Tripet B;Howard MW;Jobling M;Holmes RK;Holmes KV;Hodges RS
通讯作者：
Hodges RS
影响因子：
16.8
作者：
Walls AC;Tortorici MA;Frenz B;Snijder J;Li W;Rey FA;DiMaio F;Bosch BJ;Veesler D
通讯作者：
Veesler D
影响因子：
5.4
作者：
Kleine-Weber H;Elzayat MT;Wang L;Graham BS;Müller MA;Drosten C;Pöhlmann S;Hoffmann M
通讯作者：
Hoffmann M
影响因子：
5.4
作者：
Wu, Kailang;Chen, Lang;Li, Fang
通讯作者：
Li, Fang
影响因子：
6.7
作者：
Di Giovine P;Settembre EC;Bhargava AK;Luftig MA;Lou H;Cohen GH;Eisenberg RJ;Krummenacher C;Carfi A
通讯作者：
Carfi A