Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH
Membrane interactions of the tick-borne encephalitis virus fusion protein E at low pH
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DOI：
10.1128/jvi.76.8.3784-3790.2002
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发表时间：
2002-04-01
影响因子：
5.4
通讯作者：
Heinz, FX
中科院分区：
文献类型：
--
作者：
Stiasny, K;Allison, SL;Heinz, FX
黄病毒蜱传脑炎病毒的膜融合是由内体的轻度酸性pH触发的，并由11类病毒融合蛋白包膜蛋白E介导。低ph触发诱导低聚重排，其中天然E同型二聚体的亚基解离，单体亚基然后重新结合成同型三聚体。在这里，我们提供的证据表明膜结合是由中间单体形式的E介导的，由低ph诱导的二聚体解离产生。脂质体共浮实验表明，只有当脂质体在酸化时存在时，才会与靶膜发生关联，而在低pH下预处理病毒粒子，在没有膜的情况下，会导致病毒粒子失去稳定附着在脂质体上的能力。对于可切割的交联剂乙二醇酸酯（琥珀酰琥珀酸），研究表明，截断的可溶性形式的E蛋白（sE）只有在二聚体在低pH下自由解离时才能与膜结合，并且结合可以被识别融合肽的单克隆抗体阻断，该融合肽位于E单体的远端，但埋在天然二聚体中。令人惊讶的是，对膜相关sE蛋白的分析显示它们形成了三聚体。这是出乎意料的，因为该蛋白在c端茎锚区缺乏一个序列元件，这被证明是在没有靶膜的情况下三聚化所必需的。因此可以得出结论，膜结合促进了三聚体形式硒的形成。它的稳定性显然仅由外结构域之间的接触维持，而不依赖于先前假设的茎锚区序列元素。
Membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic pH of the endosome and is mediated by envelope protein E, a class 11 viral fusion protein. The low-pH trigger induces an oligomeric rearrangement in which the subunits of the native E homodimers dissociate and the monomeric subunits then reassociate into homotrimers. Here we provide evidence that membrane binding is mediated by the intermediate monomeric form of E, generated by low-pH-induced dissociation of the dimer. Liposome coflotation experiments revealed that association with target membranes occurred only when liposomes were present at the time of acidification, whereas pretreating virions at low pH in the absence of membranes resulted in the loss of their ability to stably attach to liposomes. With the cleavable cross-linker ethylene glycolbis(succinimidylsuccinate), it was shown that a truncated soluble form of the E protein (sE) could bind to membranes only when the dimers were free to dissociate at low pH, and binding could be blocked by a monoclonal antibody that recognizes the fusion peptide, which is at the distal tip of the E monomer but is buried in the native dimer. Surprisingly, analysis of the membrane-associated sE proteins revealed that they had formed trimers. This was unexpected because this protein lacks a sequence element in the C-terminal stem-anchor region, which was shown to be essential for trimerization in the absence of a target membrane. It can therefore be concluded that the formation of a trimeric form of sE is facilitated by membrane binding. Its stability is apparently maintained by contacts between the ectodomains only and is not dependent on sequence elements in the stem-anchor region as previously assumed.