de Haan C A, Smeets M, Vernooij F, Vennema H, Rottier P J
Institute of Virology, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Institute of Biomembranes, Utrecht University, Utrecht, The Netherlands.
J Virol. 1999 Sep;73(9):7441-52. doi: 10.1128/JVI.73.9.7441-7452.1999.
The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.
冠状病毒膜（M）蛋白是病毒粒子组装的关键参与者。其功能之一是介导刺突蛋白掺入病毒包膜。在真核细胞中共表达M蛋白和刺突（S）蛋白的基因后，通过免疫共沉淀和免疫荧光共定位可证明M与S蛋白之间的异型相互作用。利用这些检测方法进行诱变研究，我们已确定了M蛋白中参与M与S形成复合物的结构域。结果表明，M蛋白25个氨基酸残基的腔内暴露氨基末端结构域对M-S相互作用并不重要。15个氨基酸残基的缺失、His标签的插入以及用另一种冠状病毒M蛋白的胞外结构域替换，均不影响M蛋白与S蛋白结合的能力。然而，这种三跨膜蛋白跨膜结构域的变化对复合物形成很敏感。已知不影响蛋白质拓扑结构的情况下，删除前两个或最后两个跨膜结构域会导致复合物形成显著减少，但结合并未完全消除。观察到膜细胞质面的M蛋白部分发生变化会产生各种影响。极端羧基末端尾巴的缺失似乎不干扰M-S复合物的形成。然而，两亲结构域的缺失严重影响M-S相互作用。有趣的是，已知M蛋白氨基末端和极端羧基末端结构域的变化虽不破坏与S的相互作用，但对该蛋白参与病毒粒子形成的能力却是致命的（C.A.M.德哈恩、L.郭、P.S.马斯特斯、H.韦内马和P.J.M.罗蒂尔，《病毒学杂志》72:6838 - 6850，1998）。显然，M蛋白对病毒粒子组装的结构要求与形成M-S复合物所需的要求不同。
