Burger S R, Remaley A T, Danley J M, Moore J, Muschel R J, Wunner W H, Spitalnik S L
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia.
J Gen Virol. 1991 Feb;72 ( Pt 2):359-67. doi: 10.1099/0022-1317-72-2-359.
The rabies virus glycoprotein (G protein) has several important functions and is a major antigenic stimulus of the host immune system following rabies virus infection or vaccination. We developed a model system for studying the role of N-linked glycosylation in the intracellular transport and antigenicity of this molecule. The full-length cDNA of the G protein of the ERA strain of rabies virus was inserted into the eukaryotic shuttle vector pSG5 and then stably transfected into wild-type Chinese hamster ovary (CHO) cells and mutant CHO cell lines defective in glycosylation. Transfected wild-type CHO cells expressed the G protein (detected by immunofluorescence) on the cell surface in a manner similar to rabies virus-infected cells. The transfected wild-type CHO cells were shown by immunoprecipitation to produce a protein of 67K that comigrated with the fully glycosylated G protein isolated from virus-infected cells or purified virions. Treatment of the transfected cell lines with tunicamycin completely blocked surface expression and resulted in the intracellular accumulation of the G protein, suggesting that the presence of N-linked oligosaccharides is important for transport of this glycoprotein to the plasma membrane. The G protein cDNA was also expressed in the lectin-resistant CHO cell lines Lec 1, Lec 2 and Lec 8. In these cells initial N-linked glycosylation does occur, but later steps in processing of the oligosaccharides are blocked. In each case, the G protein was expressed on the surface of lectin-resistant CHO cells in a similar manner to expression on wild-type CHO cells. This suggests that various different N-linked oligosaccharide structures support intracellular transport of this glycoprotein. Thus, stably transfected CHO cell lines will provide a useful model system for further studies of the role of N-linked glycosylation in trafficking and antigenicity of the rabies virus G protein.
狂犬病病毒糖蛋白（G蛋白）具有多种重要功能，是狂犬病病毒感染或接种疫苗后宿主免疫系统的主要抗原刺激物。我们开发了一个模型系统，用于研究N-连接糖基化在该分子的细胞内运输和抗原性中的作用。将狂犬病病毒ERA株G蛋白的全长cDNA插入真核穿梭载体pSG5中，然后稳定转染到野生型中国仓鼠卵巢（CHO）细胞和糖基化缺陷的突变CHO细胞系中。转染的野生型CHO细胞以类似于狂犬病病毒感染细胞的方式在细胞表面表达G蛋白（通过免疫荧光检测）。免疫沉淀显示，转染的野生型CHO细胞产生一种67K的蛋白质，该蛋白质与从病毒感染细胞或纯化病毒粒子中分离出的完全糖基化的G蛋白迁移率相同。用衣霉素处理转染的细胞系完全阻断了表面表达，并导致G蛋白在细胞内积累，这表明N-连接寡糖的存在对于这种糖蛋白运输到质膜很重要。G蛋白cDNA也在凝集素抗性CHO细胞系Lec 1、Lec 2和Lec 8中表达。在这些细胞中确实发生了初始N-连接糖基化，但寡糖加工的后续步骤被阻断。在每种情况下，G蛋白在凝集素抗性CHO细胞表面的表达方式与在野生型CHO细胞上的表达相似。这表明各种不同的N-连接寡糖结构支持这种糖蛋白的细胞内运输。因此，稳定转染的CHO细胞系将为进一步研究N-连接糖基化在狂犬病病毒G蛋白的运输和抗原性中的作用提供一个有用的模型系统。
