风疹病毒包膜糖蛋白糖基化作用对细胞融合的影响

目的 探讨JR23株风疹病毒包膜糖蛋白E2和E1的糖基化作用对其引起的特异性细胞融合的影响.方法 采用基因定点突变的方法构建6个突变株,分别改变E2或E1蛋白的糖基化位点结构.采用Giemsa染色法与指示基因法检测各突变株蛋白引起的细胞融合,流式细胞术检测其在细胞表面的表达效率,并用血细胞吸附试验检测其吸附活性.结果 与野毒株蛋白相比较,突变株蛋白E2 N53G、S73I、S131V与E1 T78A、T179A、1211A引起的细胞融合效率分别为62.73%、66.66%、55.12%、66.93%、87.33%、90.18%;除了E2 S131V之外,其他突变株蛋白在细胞表面的表达效率均有不同程度的降低;只有E2 S73I与E1 T78A的血细胞吸附效率略有降低.结论 E2蛋白的3个糖基化位点与E1蛋白的N76位糖基化位点的糖基化对细胞融合有重要作用,E1的N177与N209位点糖基化对细胞融合的影响较小.

Effects of N-linked glycosylation on specific cell fusion in rubella virus

Objective To explore the effects of glycosylation in E2 and E1 protein on specific cell fusion in rubella virus(RV)strain JR23.Methods Site-directed mutagenesis was used tO obtain mutants containing new enzyme sites on the E2 and E1 gene of RV JR23.All the mutants and wild type proteins were expressed in BHK21 cells and treated with acid medium to induce specific cell fusion.The fusion functions were assayed with Giemsa staining and reporter gene method for qualitative and quantitative analysis,respectively.Expression efficieneies of mutant proteins on cell surface were quantified with fluorescence-activated cell sorter(FACS).Hemadsorption assays were performed to detect binding activity of mutant proteins qualitatively and quantitatively.Results Mutant proteins E2 N53G,S73I and S131V had 62.73%,66.66%and 55.12% of fusion activities,and E1 T78A,T179A and T211A had 66.93%,87.33%and 90.18%of fusion activities,respectively,as compared with the wild type protein.The FACS indicated that the expression efficiencies of all the mutant proteins except E2 S131V were lower than that of the wild type protein.Hemadsorption assays demonstrated that binding abilities of E2 S73I and E1 T78A decreased slightly,but that of the other four mutant proteins Wns almost same as the wild type protein. Conclusion Glycosylation on E2 N53,N71,N129 and E1 N76 were important for the specific cell fusion,but E1 N177 and N209 were almost not.