Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein
Murine coronavirus membrane fusion is blocked by modification of thiols buried within the spike protein
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DOI：
10.1128/jvi.70.7.4683-4690.1996
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发表时间：
1996-07-01
影响因子：
5.4
通讯作者：
Gallagher, TM
中科院分区：
文献类型：
--
作者：
Gallagher, TM
鼠肝炎病毒（MHV）颗粒的包膜上布满了糖蛋白尖峰，其功能既可以促进病毒颗粒与其细胞受体结合，又可以介导病毒颗粒与细胞膜的融合。在这项研究中，对富含半胱氨酸的刺突进行了化学修饰，以确定这种结构改变是否会影响病毒的进入过程。 Ellman 试剂是一种不透膜的氧化剂，可与暴露的半胱氨酸残基反应，实现大硫代硝基苯甲酸酯部分的共价加成，在 37 摄氏度下与 MHV 的 JHM 株一起孵育。相对于未处理的病毒，1 mM Ellman 试剂在 1 小时后将感染性降低了 2 log(10)。在低于 21 摄氏度的孵育温度下没有观察到这种水平的抑制，这表明病毒体表面蛋白经历热转变，使半胱氨酸残基暴露于试剂的修饰中。开发了定量受体结合和膜融合测定并用于表明 Ellman 试剂特异性抑制 MW JHM 刺突蛋白诱导的膜融合。然而，这种抑制是毒株特异性的，因为密切相关的 MHV 毒株 A59 不受影响。为了确定该菌株特异性的基础，制备了刺突 cDNA，其中部分编码 JHM 或 A59 残基。用牛痘病毒载体表达 cDNA，并在融合测定中测试对 Ellman 试剂的敏感性。结果揭示了 Ellman 试剂介导的抑制严重程度与 JHM 特异性半胱氨酸 (Cys-1163) 的存在之间的相关性。因此，这种半胱氨酸的存在增加了硫醇修饰的尖峰的可用性，最终阻止了融合能力。
The envelopes of murine hepatitis virus (MHV) particles are studded with glycoprotein spikes that function both to promote virion binding to its cellular receptor and to mediate virion-cell membrane fusion. In this study, the cysteine-rich spikes were subjected to chemical modification to determine whether such structural alterations impact the virus entry process. Ellman reagent, a membrane-impermeant oxidizing agent which reacts with exposed cysteine residues to effect covalent addition of large thionitrobenzoate moieties, was incubated at 37 degrees C with the JHM strain of MHV. Relative to untreated virus, 1 mM Ellman reagent reduced infectivity by 2 log(10) after 1 h. This level of inhibition was not observed at incubation temperatures below 21 degrees C, suggesting that virion surface proteins undergo thermal transitions that expose cysteine residues to modification by the reagent. Quantitative receptor binding and membrane fusion assays were developed and used to show that Ellman reagent specifically inhibited membrane fusion induced by the MW JHM spike protein. However, this inhibition was strain specific, because the closely related MHV strain A59 was unaffected. To identify the basis for this strain specificity, spike cDNAs were prepared in which portions encoded either JHM or A59 residues. cDNAs were expressed with vaccinia virus vectors and tested for sensitivity to Ellman reagent in the fusion assays. The results revealed a correlation between the severity of inhibition mediated by Ellman reagent and the presence of a JHM-specific cysteine (Cys-1163). Thus, the presence of this cysteine increases the availability of spikes for a thiol modification that ultimately prevents fusion competence.