Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, 1095 Vienna, Austria.
J Virol. 2011 Sep;85(17):8495-501. doi: 10.1128/JVI.05129-11. Epub 2011 Jun 22.
The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra- and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.
目前的黄病毒膜融合模型基于病毒融合蛋白 E 的二聚体预融合和三聚体融合构象的截断形式的原子结构。这些结构缺乏 E 的两个跨膜结构域（TMD）以及所谓的茎，据信在融合过程中，茎参与 E 三聚体的分子内和分子间拉链反应。为了获得茎在黄病毒膜融合中的功能作用的实验证据，我们使用蜱传脑炎病毒的重组亚病毒颗粒（RSP）进行了一项诱变研究，该病毒具有与整个感染性病毒粒子相似的融合特性，是病毒融合的既定模型。我们在茎部以及在拉链过程中预测与茎相互作用的 E 蛋白的那部分引入了突变，并在体外脂质体融合测定中分析了这些突变对融合肽与靶细胞相互作用、E 蛋白三聚体化、三聚体稳定性和膜融合的影响。我们的数据提供了茎部 N 端保守苯丙氨酸与 E 蛋白结构域 II 中一个口袋之间分子相互作用的证据，这似乎对于将茎定位在允许拉链和形成结构的方向上是必需的，在该结构中，TMD 可以相互作用以实现有效的融合。
