Bos E C, Heijnen L, Luytjes W, Spaan W J
Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Virology. 1995 Dec 20;214(2):453-63. doi: 10.1006/viro.1995.0056.
The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino acids that are important for cleavage of S, and we show that cleavage is not strictly required for fusion. However, the level of cleavage seems to influence the fusion kinetics. After introduction of an arginine at position P2 to mimick the MHV-JHM cleavage site, full cleavage of the spike protein was obtained. Further, we analyzed the effect of mutations in the transmembrane (TM) domain of the S protein. Maturation and cell surface expression of the mutant proteins were not affected, and all proteins became acylated. The mutant in which the predicted transmembrane domain was shortened did not induce syncytia. From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines, one was unable to induce syncytia, another showed delayed syncytia formation, and the third mutant induced syncytia as did the wild-type protein. The potential role of the transmembrane domain in fusion is discussed.
鼠冠状病毒A59株（MHV - A59）的刺突（S）蛋白是一种诱导膜融合的I型膜蛋白。在本研究中，我们分析了S蛋白中两个结构域在融合过程中的作用。180 kDa的成熟S蛋白在转运到质膜的过程中会部分裂解为两个90 kDa的亚基。我们鉴定出了几个对S蛋白裂解很重要的氨基酸，并且表明裂解对于融合并非严格必需。然而，裂解水平似乎会影响融合动力学。在P2位置引入精氨酸以模拟MHV - JHM裂解位点后，刺突蛋白实现了完全裂解。此外，我们分析了S蛋白跨膜（TM）结构域中突变的影响。突变蛋白的成熟和细胞表面表达未受影响，并且所有蛋白都发生了酰化。预测的跨膜结构域被缩短的突变体未诱导形成多核体。在一组TM结构域中几个保守半胱氨酸被丝氨酸取代的突变体中，一个无法诱导形成多核体，另一个显示多核体形成延迟，第三个突变体诱导形成多核体的情况与野生型蛋白相同。本文讨论了跨膜结构域在融合中的潜在作用。
