Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59
Identification of H209 as Essential for pH 8-Triggered Receptor-Independent Syncytium Formation by S Protein of Mouse Hepatitis Virus A59
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通过小鼠肝炎病毒 A59 的 S 蛋白鉴定 H209 对于 pH 8 触发的受体非依赖性合胞体形成至关重要
DOI：
10.1128/jvi.00209-18
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发表时间：
2018-06-01
影响因子：
5.4
通讯作者：
Qian, Zhaohui
中科院分区：
文献类型：
--
作者：
Li, Pei;Shan, Yiwei;Qian, Zhaohui
摘要小鼠冠状病毒小鼠肝炎病毒A59株的S蛋白(S)以小鼠癌胚抗原相关细胞黏附分子1a为细胞进入受体，但在37℃、无受体的情况下，仅用pH 8.0即可触发S蛋白的构象变化，其作用机制尚未确定。在这里，我们发现MHV-A59 S蛋白在37℃下被pH 8.0触发在293T细胞上诱导非受体依赖的合胞体(RIS)的形成，并且由pH 8.0触发的S蛋白的构象变化与由受体结合触发的构象变化非常相似。我们系统地突变了S蛋白中的15个组氨酸残基，发现H209对于pH 8.0触发的RIS的形成是必不可少的，而H179、H441、H643和H759也在这一过程中发挥着重要作用。用ALA取代H209对受体结合没有影响，但在小鼠17Cl.1细胞中，突变体H209A MHV-A59表现出延迟的生长动力学，当与野生型病毒混合时很容易被野生型病毒竞争，表明H209A突变导致了病毒适应性的缺陷。最后，H209A突变显著提高了S蛋白在融合前构象的热稳定性，这可能提高了膜融合所需的S蛋白构象变化的能垒，从而降低了细胞培养中的病毒适合度。因此，MHV-A59可能进化为降低其S蛋白的稳定性，以增加病毒适合性。重要的包膜病毒通过病毒和细胞膜的融合进入细胞，这个过程是通过病毒被膜蛋白和它们的宿主受体之间的相互作用来调节的。在前融合构象中，病毒包膜蛋白是亚稳定的，对融合构象的激活受到严格的调控，因为过早激活会导致病毒感染性的丧失。病毒被膜蛋白的稳定性对其活性和病毒适合性有很大影响。在这里，我们报告了类似于埃博拉病毒糖蛋白的A82V突变，在小鼠冠状病毒MHV-A59的S糖蛋白中，209位的组氨酸残基显著影响S蛋白的热稳定性，决定了S蛋白在37℃时是否可以被pH 8.0单独激活或通过受体结合激活，并影响病毒在细胞培养中的适合性。因此，MHV-A59的尖峰糖蛋白已经进化成在209位保留组氨酸，以优化病毒的适应性。
ABSTRACT The spike glycoprotein (S) of murine coronavirus mouse hepatitis virus (MHV) strain A59 uses murine carcinoembryonic antigen-related cell adhesion molecule 1a as its receptor for cell entry, but S protein can also be triggered in the absence of receptor by pH 8.0 alone at 37°C. The mechanism by which conformational changes of this S glycoprotein can be triggered by pH 8.0 has not yet been determined. Here, we show that MHV-A59 S protein is triggered by pH 8.0 at 37°C to induce receptor-independent syncytium (RIS) formation on 293T cells, and that the conformational changes in S proteins triggered by pH 8.0 are very similar to those triggered by receptor binding. We systemically mutated each of 15 histidine residues in S protein and found that H209 is essential for pH 8.0-triggered RIS formation, while H179, H441, H643, and H759 also play important roles in this process. Replacement of H209 with Ala had no effect on receptor binding, but in murine 17Cl.1 cells mutant H209A MHV-A59 showed delayed growth kinetics and was readily outcompeted by wild-type virus when mixed together, indicating that the H209A mutation caused a defect in virus fitness. Finally, the H209A mutation significantly increased the thermostability of S protein in its prefusion conformation, which may raise the energy barrier for conformational change of S protein required for membrane fusion and lead to a decrease in virus fitness in cell culture. Thus, MHV-A59 may have evolved to lower the stability of its S protein in order to increase virus fitness. IMPORTANCE Enveloped viruses enter cells through fusion of viral and cellular membranes, and the process is mediated by interactions between viral envelope proteins and their host receptors. In the prefusion conformation, viral envelope proteins are metastable, and activation to the fusion conformation is tightly regulated, since premature activation would lead to loss of viral infectivity. The stability of viral envelope proteins greatly influences their activation and virus fitness. Here, we report that, similar to the A82V mutation in Ebola glycoprotein, in the S glycoprotein of murine coronavirus MHV-A59, the histidine residue at position of 209 significantly affects the thermal stability of the S protein, determines whether S protein can be activated at 37°C by either pH 8.0 alone or by receptor binding, and affects viral fitness in cell culture. Thus, the spike glycoprotein of MHV-A59 has evolved to retain histidine at position 209 to optimize virus fitness.