Envelope glycoprotein interactions in coronavirus assembly.
Envelope glycoprotein interactions in coronavirus assembly.
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DOI：
10.1083/jcb.131.2.339
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发表时间：
1995-10
期刊：
影响因子：
--
通讯作者：
Rottier PJ
中科院分区：
文献类型：
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作者：
Opstelten DJ;Raamsman MJ;Wolfs K;Horzinek MC;Rottier PJ
冠状病毒通过出芽进入中间ER-to-Golgi隔室的光滑膜进行组装。我们研究了病毒膜糖蛋白M和S在病毒粒子包膜形成过程中的关联。通过共免疫沉淀分析，我们证明了小鼠肝炎病毒（MHV）的M和S蛋白在感染细胞中特异性地相互作用形成异多聚物复合物。只有在仔细选择用于从细胞或病毒粒子中溶解它们的洗涤剂时，这些才能被检测到：非离子（NP-40）和离子（脱氧胆酸）洗涤剂的组合被证明是最佳的。脉冲追踪实验表明，新合成的M和S蛋白以不同的动力学参与复合物的形成。M蛋白在合成后立即出现在复合物中，而新合成的S蛋白则经过bbbb20 min的滞后期才出现。新合成的M比S更快地融入病毒颗粒，这表明它与先前存在的S分子相关联。利用牛痘病毒t7驱动M和S的共表达，我们也证明了在没有其他冠状病毒蛋白的情况下M/S复合物的形成。脉冲追踪标记和共免疫沉淀分析显示M和S在前高尔基膜中结合，因为M/S复合物的未糖基化形式迅速出现。由于当ER的蛋白质输出被brefeldin A阻断时，没有检测到M和S的关联，稳定的复合物很可能出现在ER-to- golgi中间区。蔗糖速度梯度分析表明，M/S复合物具有异质性和高阶性，表明它们是通过同型和异型相互作用维持的。M/S复合物与α -甘露糖苷酶II（一种常驻高尔基蛋白）共定位。它们获得高尔基特异性低聚糖修饰，但在细胞表面未检测到。因此，S蛋白本身被运输到质膜，通过与M蛋白的结合被保留在高尔基复合体中。由于冠状病毒在前高尔基膜上发芽，这一结果表明，包膜糖蛋白复合物不能决定出芽的位置。然而，MHV包膜糖蛋白与高阶复合物的自结合表明其在病毒膜蛋白的分选和在病毒组装中驱动病毒脂蛋白外壳形成中的作用。
Coronaviruses are assembled by budding into smooth membranes of the intermediate ER-to-Golgi compartment. We have studied the association of the viral membrane glycoproteins M and S in the formation of the virion envelope. Using coimmunoprecipitation analysis we demonstrated that the M and S proteins of mouse hepatitis virus (MHV) interact specifically forming heteromultimeric complexes in infected cells. These could be detected only when the detergents used for their solubilization from cells or virions were carefully chosen: a combination of nonionic (NP-40) and ionic (deoxycholic acid) detergents proved to be optimal. Pulse-chase experiments revealed that newly made M and S proteins engaged in complex formation with different kinetics. Whereas the M protein appeared in complexes immediately after its synthesis, newly synthesized S protein did so only after a lag phase of > 20 min. Newly made M was incorporated into virus particles faster than S, which suggests that it associates with preexisting S molecules. Using the vaccinia virus T7-driven coexpression of M and S we also demonstrate formation of M/S complexes in the absence of other coronaviral proteins. Pulse-chase labelings and coimmunoprecipitation analyses revealed that M and S associate in pre-Golgi membranes because the unglycosylated form of M appeared in M/S complexes rapidly. Since no association of M and S was detected when protein export from the ER was blocked by brefeldin A, stable complexes most likely arise in the ER-to-Golgi intermediate compartment. Sucrose velocity gradient analysis showed the M/S complexes to be heterogeneous and of higher order, suggesting that they are maintained by homo- and heterotypic interactions. M/S complexes colocalized with alpha-mannosidase II, a resident Golgi protein. They acquired Golgi-specific oligosaccharide modifications but were not detected at the cell surface. Thus, the S protein, which on itself was transported to the plasma membrane, was retained in the Golgi complex by its association with the M protein. Because coronaviruses bud at pre-Golgi membranes, this result implies that the envelope glycoprotein complexes do not determine the site of budding. Yet, the self-association of the MHV envelope glycoproteins into higher order complexes is indicative of its role in the sorting of the viral membrane proteins and in driving the formation of the viral lipoprotein coat in virus assembly.