Allison S L, Stiasny K, Stadler K, Mandl C W, Heinz F X
Institute of Virology, University of Vienna, Vienna, Austria.
J Virol. 1999 Jul;73(7):5605-12. doi: 10.1128/JVI.73.7.5605-5612.1999.
Envelope protein E of the flavivirus tick-borne encephalitis virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino-acid-long protein has been shown to differ significantly from that of other viral fusion proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E proteins were examined. Full-length E proteins and proteins lacking the second of two predicted transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E proteins. Sites located within a predicted alpha-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E proteins. A separate site in the stem, also corresponding to a predicted alpha-helix (amino acids 401 to 413), was essential for the conversion of soluble protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and virus assembly.
蜱传脑炎病毒这一黄病毒属病毒的包膜蛋白E介导膜融合，已证明该496个氨基酸长的蛋白N端80%的结构与其他病毒融合蛋白的结构有显著差异。其C端20%即茎-锚定区的结构尚不清楚。它包含对膜锚定、病毒体组装过程中与prM（膜蛋白M的前体）相互作用以及与融合过程相关的低pH诱导结构变化至关重要的序列。为了确定该区域的特定功能元件，构建了一系列C端缺失突变体，并检测了所得截短重组E蛋白的特性。当与prM共表达时，全长E蛋白和缺少两个预测跨膜片段中第二个片段的蛋白以颗粒形式分泌，而两个片段均缺失则导致可溶性同源二聚体E蛋白的分泌。发现位于茎部预测的α螺旋区域（氨基酸431至449）和第一个跨膜区域（氨基酸450至472）内的位点对prM-E异二聚体的稳定性很重要，但对prM介导的可溶性E蛋白的细胞内运输和分泌不是必需的。茎部的另一个位点，也对应于一个预测的α螺旋（氨基酸401至413），对于低pH处理时可溶性蛋白E二聚体转化为同源三聚体形式至关重要，这一过程类似于整个病毒体向融合状态的转变。这种功能图谱将有助于理解膜融合和病毒组装的分子机制。
