Wu Bing, Liu Xiaoli, Wang Zhiyu
Department of Virology, School of Public Health, Shandong University, 44 Wenhua Xilu Road, Jinan, China.
Intervirology. 2009;52(2):68-77. doi: 10.1159/000214861. Epub 2009 Apr 28.
To explore the effects of glycosylation in glycoprotein on membrane fusion in rubella virus strain JR23.
The N-linked glycosylation sites in glycoproteins were mutated individually or in combination. Total expression and cell surface expression efficiencies of mutant proteins were assayed with Western blot and FACS. The fusion functions were assayed with Giemsa staining and reporter gene method. Binding activity of mutant proteins was detected with hemadsorption assays.
We observed that total expression levels of all the mutant proteins in cells were unchanged, but the cell surface expression efficiencies of all the mutant proteins except E2 S131V were lower than wild-type protein. When effects of reduced cell surface expression were eliminated, mutant proteins N53G, S73I, S131V and T78A had lower fusion activities than wild-type protein, and binding abilities of E2 S73I and E1 T78A decreased slightly. But in all the combined mutants, no cell fusion was detected, and only a minor hemadsorption was observed in N53G-S131V, N53G-T78A and N53G-T211A.
Glycosylation of glycoproteins were involved in cell surface expression synergistically. Glycosylation on E2 N53, N71, N129 and E1 N76 altered the specific membrane fusion, whereas no effects were detected on E1 N177 and N209 individually.
探讨风疹病毒JR23株糖蛋白糖基化对膜融合的影响。
对糖蛋白中的N - 糖基化位点进行单独或组合突变。采用蛋白质免疫印迹法（Western blot）和荧光激活细胞分选术（FACS）检测突变蛋白的总表达及细胞表面表达效率。采用吉姆萨染色和报告基因法检测融合功能。采用血细胞吸附试验检测突变蛋白的结合活性。
我们观察到细胞中所有突变蛋白的总表达水平未发生变化，但除E2 S131V外，所有突变蛋白的细胞表面表达效率均低于野生型蛋白。当消除细胞表面表达降低的影响后，突变蛋白N53G、S73I、S131V和T78A的融合活性低于野生型蛋白，且E2 S73I和E1 T78A的结合能力略有下降。但在所有组合突变体中，未检测到细胞融合，仅在N53G - S131V、N53G - T78A和N53G - T211A中观察到轻微的血细胞吸附。
糖蛋白的糖基化协同参与细胞表面表达。E2的N53、N71、N129和E1的N76位点的糖基化改变了特异性膜融合，而单独对E1的N177和N209位点的糖基化未检测到影响。
