STABLE EXPRESSION OF RABIES VIRUS GLYCOPROTEIN IN CHINESE-HAMSTER OVARY CELLS
STABLE EXPRESSION OF RABIES VIRUS GLYCOPROTEIN IN CHINESE-HAMSTER OVARY CELLS
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DOI：
10.1099/0022-1317-72-2-359
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发表时间：
1991-02-01
影响因子：
3.8
通讯作者：
SPITALNIK, SL
中科院分区：
文献类型：
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作者：
BURGER, SR;REMALEY, AT;SPITALNIK, SL
狂犬病毒糖蛋白（G蛋白）具有多种重要功能，是狂犬病毒感染或疫苗接种后宿主免疫系统的主要抗原刺激。我们开发了一个模型系统来研究n -链糖基化在细胞内运输和该分子的抗原性中的作用。将狂犬病毒ERA株G蛋白全长cDNA插入真核穿梭载体pSG5中，稳定转染野生型中国仓鼠卵巢（CHO）细胞和糖基化缺陷突变型CHO细胞系。转染的野生型CHO细胞在细胞表面表达G蛋白（免疫荧光检测），表达方式与狂犬病毒感染细胞相似。通过免疫沉淀显示转染的野生型CHO细胞产生67K蛋白，该蛋白与从病毒感染细胞或纯化病毒粒子中分离的完全糖基化的G蛋白相结合。用tunicamycin处理转染细胞系完全阻断了表面表达，导致G蛋白在细胞内积累，这表明n -连接寡糖的存在对该糖蛋白转运到质膜很重要。G蛋白cDNA在抗凝集素CHO细胞系Lec 1、Lec 2和Lec 8中也有表达。在这些细胞中，最初的n -连接糖基化确实发生，但低聚糖加工的后期步骤被阻断。在每种情况下，G蛋白都以与野生型CHO细胞相似的方式在抗凝集素CHO细胞表面表达。这表明多种不同的n链寡糖结构支持这种糖蛋白的细胞内转运。因此，稳定转染的CHO细胞系将为进一步研究n链糖基化在狂犬病毒G蛋白转运和抗原性中的作用提供一个有用的模型系统。
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 oligosaccarides 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.