Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer
Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State Is Triggered by Exposure to Low-Molarity Buffer
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DOI：
10.1128/jvi.01813-10
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发表时间：
2011-04-01
影响因子：
5.4
通讯作者：
Peeples, Mark E.
中科院分区：
文献类型：
--
作者：
Chaiwatpongsakorn, Supranee;Epand, Raquel F.;Peeples, Mark E.
副粘病毒融合（F）糖蛋白以亚稳定的预触发形式锚定在病毒体膜上。一旦被触发，F蛋白就会经历一个戏剧性的构象延伸，将其疏水融合肽插入靶细胞膜，然后折叠回自身，将膜聚集在一起并启动融合。与大多数其他副粘病毒不同，呼吸道合胞病毒（RSV）F蛋白本身就足以介导膜融合和病毒感染。为了研究RSV F蛋白的触发机制，我们已经通过用6His标签替换跨膜和胞质尾部结构域来产生可溶性F（sF）蛋白。sF蛋白以完全切割的形式从293T细胞有效分泌。它被中和单克隆抗体识别，通过电子显微镜分析显示为球形，并且不聚集，所有这些都与天然的预触发三聚体一致。在Ni+2柱上纯化sF蛋白，并用含有500 mM NaCl和250 mM咪唑的50 mM磷酸盐缓冲液洗脱。对10 mM缓冲液透析引起sF蛋白触发，形成“帽针”形分子，聚集为roxylate，特征的触发后的形式。进一步的透析实验表明，触发效率与缓冲液摩尔浓度的降低相关。通过稀释降低缓冲液摩尔浓度也导致融合肽的暴露，如通过脂质体缔合所检测的，证实了sF蛋白触发。邻近融合肽的弗林蛋白酶切割位点的突变阻止了脂质体缔合，进一步证实了缔合是通过融合肽进行的。
The paramyxovirus fusion (F) glycoprotein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. Unlike most other paramyxoviruses, the respiratory syncytial virus (RSV) F protein alone is sufficient to mediate membrane fusion and virus infection. To study the triggering mechanism of the RSV F protein, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. The sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopic analysis, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni+2 column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming "hat pin"-shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide, as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, further confirming that association is via the fusion peptide.