Zheng Z, Maidji E, Tugizov S, Pereira L
Department of Stomatology, School of Dentistry, University of California, San Francisco 94143-0512, USA.
J Virol. 1996 Nov;70(11):8029-40. doi: 10.1128/JVI.70.11.8029-8040.1996.
Human cytomegalovirus glycoprotein B (gB) plays a role in the fusion of the virion envelope with the host cell membrane and in syncytium formation in infected cells. Hydrophobic sequences at the carboxyl terminus, amino acids (aa) 714 to 771, anchor gB in the lipid bilayer, but the unusual length of this domain suggests that it may serve another role in gB structure. To explore the function(s) of this region, we deleted aa 717 to 747 (gB deltaI mutation), aa 751 to 771 (gB deltaII mutation), and aa 717 to 772 (gB deltaI-II mutation) and constructed a substitution mutation, Lys-748 to Val (Lys748Val)-Asn749Ala-Pro750Ile (gB KNPm). Mutated forms of gB were expressed in U373 glioblastoma cells and subjected to analysis by flow cytometry, confocal microscopy, and immunoprecipitation. Mutations gB deltaI-II and gB deltaII alone caused secretion of gB into the medium, confirming that aa 751 to 771 function as a membrane anchor. In contrast, mutations gB deltaI and gB KNPm blocked cell surface expression and arrested gB transport in the endoplasmic reticulum (ER). Detailed examination of gB deltaI and gB KNPm with a panel of monoclonal antibodies showed that the mutated forms were indistinguishable from wild-type gB in conformation and formed oligomers; however, they remained sensitive to endoglycosidase H and did not undergo endoproteolytic cleavage. Analysis of protein complexes formed by gB and molecular chaperones in the ER showed that calnexin and calreticulin, lectin-like chaperones, bound equal amounts of uncleaved wild-type gB, gB deltaI, and gB KNPm, but the glucose-regulated proteins 78 (BiP) and 94 formed stable complexes only with the mutated forms, causing their retention in the ER. Our studies show that aa 714 to 750 are key residues in the architecture of gB molecules and that the ER chaperones, which facilitate gB folding and monitor the quality of glycoproteins, detect subtle changes in folding intermediates that are conferred by mutations in this region.
人巨细胞病毒糖蛋白B（gB）在病毒体包膜与宿主细胞膜融合以及感染细胞的合胞体形成过程中发挥作用。其羧基末端的疏水序列，即氨基酸（aa）714至771，将gB锚定在脂质双层中，但该结构域的异常长度表明它可能在gB结构中发挥其他作用。为了探究该区域的功能，我们删除了aa 717至747（gB deltaI突变）、aa 751至771（gB deltaII突变）以及aa 717至772（gB deltaI-II突变），并构建了一个替代突变，即赖氨酸-748突变为缬氨酸（Lys748Val）-天冬酰胺749突变为丙氨酸-脯氨酸750突变为异亮氨酸（gB KNPm）。gB的突变形式在U373胶质母细胞瘤细胞中表达，并通过流式细胞术、共聚焦显微镜和免疫沉淀进行分析。单独的gB deltaI-II和gB deltaII突变导致gB分泌到培养基中，证实aa 751至771作为膜锚定起作用。相反，gB deltaI和gB KNPm突变阻断了细胞表面表达，并使gB在内质网（ER）中的转运停滞。用一组单克隆抗体对gB deltaI和gB KNPm进行详细检查表明，突变形式在构象上与野生型gB无法区分，并形成寡聚体；然而，它们对内切糖苷酶H仍然敏感，并且未经历内切蛋白水解切割。对gB与ER中的分子伴侣形成的蛋白质复合物进行分析表明，凝集素样伴侣钙连蛋白和钙网蛋白结合等量的未切割野生型gB、gB deltaI和gB KNPm，但葡萄糖调节蛋白78（BiP）和94仅与突变形式形成稳定复合物，导致它们滞留在ER中。我们的研究表明，aa 714至750是gB分子结构中的关键残基，并且促进gB折叠并监测糖蛋白质量的ER伴侣能够检测到该区域突变赋予的折叠中间体的细微变化。
