Imaging individual retroviral fusion events: From hemifusion to pore formation and growth
Imaging individual retroviral fusion events: From hemifusion to pore formation and growth
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DOI：
10.1073/pnas.0501864102
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发表时间：
2005-06-14
影响因子：
11.1
通讯作者：
Young, JAT
中科院分区：
文献类型：
--
作者：
Melikyan, GB;Barnard, RJO;Young, JAT
病毒融合蛋白通过一系列尚未明确的步骤催化病毒和细胞膜的合并。为了阐明病毒进入的机制，我们对携带禽肉瘤和白血病病毒（ASLV）包膜糖蛋白（Env）的单个病毒粒子与细胞膜的融合进行了成像。病毒颗粒用亲脂性染料和棕榈酰化增强的YFP标记，该YFP被纳入病毒膜的内小叶。当单个病毒粒子与表达同源受体的靶细胞结合时，它们仅在暴露于低而非中性pH环境时才转移脂质和含量。这些数据与提出的ASLV进入的两步机制一致，即受体启动和低pH激活。最重要的是，脂质混合通常发生在形成小融合孔之前，通过棕榈酰化增强的YFP荧光的ph依赖性变化可以快速敏感地检测到融合孔。新生的融合孔是亚稳的，不可逆地关闭，保持小，或完全扩大，允许核衣壳进入细胞质。这些发现强烈暗示，半灌注和小孔隙是ASLV融合的关键中介。在低pH处理前加入一种旨在阻止Env折叠成最终螺旋束构象的肽，可以消除病毒与细胞的融合和感染。因此，我们得出结论，在受体激活后，Env经历了低ph依赖性的重折叠成六螺旋束，并在此过程中依次催化半融合、融合孔打开和扩大。
Viral fusion proteins catalyze merger of viral and cell membranes through a series of steps that have not yet been well defined. To elucidate the mechanism of virus entry, we have imaged fusion between single virions bearing avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) and the cell membrane. Viral particles were labeled with a lipophilic dye and with palmitylated enhanced YFP that was incorporated into the inner leaflet of the viral membrane. When individual virions were bound to target cells expressing cognate receptors, they transferred their lipids and contents only when exposed to low, but not neutral, pH. These data are consistent with the proposed two-step mechanism of ASLV entry that involves receptor-priming followed by low pH activation. Most importantly, lipid mixing commonly occurred before formation of a small fusion pore that was quickly and sensitively detected by pH-dependent changes in palmitylated enhanced YFP fluorescence. Nascent fusion pores were metastable and irreversibly closed, remained small, or fully enlarged, permitting nucleocapsid delivery into the cytosol. These findings strongly imply that hemifusion and a small pore are the key intermediates of ASLV fusion. When added before low pH treatment, a peptide designed to prevent Env from folding into a final helical-bundle conformation abolished virus-cell fusion and infection. Therefore, we conclude that, after receptor-activation, Env undergoes low pH-dependent refolding into a six-helix bundle and, in doing so, sequentially catalyzes hemifusion, fusion pore opening, and enlargement.