Klumperman J, Locker J K, Meijer A, Horzinek M C, Geuze H J, Rottier P J
Department of Cell Biology, Utrecht University, The Netherlands.
J Virol. 1994 Oct;68(10):6523-34. doi: 10.1128/JVI.68.10.6523-6534.1994.
The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.
目前流行的假说是冠状病毒出芽的细胞内位点由其膜蛋白M（以前称为E1）的定位决定。我们通过分析四种不同冠状病毒的出芽位点与其M蛋白的细胞内定位的关系来对此进行测试。在Sac(-)细胞中生长的小鼠肝炎病毒（MHV）和传染性支气管炎病毒（IBV），以及在CrFK细胞中生长的猫传染性腹膜炎病毒（FIPV）和猪传染性胃肠炎病毒（TGEV），均仅出芽到位于粗面内质网和高尔基体复合体之间的光滑壁、管状囊泡膜中，这与先前为MHV确定的所谓出芽区室相同。对感染细胞的间接免疫荧光染色显示，所有四种M蛋白都聚集在核周区域。免疫金显微镜观察将MHV M和IBV M定位在出芽区室中；此外，在高尔基体复合体中出现了密集标记，MHV M主要位于反式高尔基体潴泡和反式高尔基体网状结构中，而IBV M主要位于顺式和中间高尔基体潴泡中。这四种病毒的相应M蛋白在重组痘苗病毒系统中独立表达时，也聚集在核周区域。对代谢标记细胞的定量脉冲追踪分析表明，在每种情况下，大多数M糖蛋白在合成后4小时内携带具有高尔基体特异性修饰的寡糖侧链。免疫电子显微镜观察将重组MHV M和IBV M定位到与冠状病毒感染细胞中的相应蛋白相同的膜上，在高尔基体复合体上具有相同的顺反分布。我们的结果表明，这四种病毒的一些M蛋白被转运到出芽区室之外，并被内在的保留信号差异保留；除了M蛋白外，可能还需要其他病毒和/或细胞因子来决定出芽位点。
