汉坦病毒GM04-38株N-联糖基化位点在细胞融合中作用的初步确定

Defining the function of the N-linked glycosylation site of hantavirus GM04-38 in cell fusion

目的初步确定汉坦病毒GM04—38株N-联糖基化位点在细胞融合中的作用。方法采用基因定点突变技术将汉坦病毒GM04-38株G1片段上N134、N235、N347、N399四位点及G2片段上N928位点共5个位点上的天冬酰胺置换为丙氨酸。转染VeroE6细胞后，间接免疫荧光（IFA）检测蛋白表达，荧光显微镜观察IFA结果；Westernblot检测蛋白的表达情况。采用Giemsa染色法观察细胞融合现象。结果在VetoE6细胞中成功表达出糖蛋白，Westernblot显示除134位点末显示G1片段外，各突变体均表达出G1、G2片段。IFA显示天冬酰胺突变前后各位点均显示荧光信号，呈核周分布。突变后134位点与928位点的突变体融合现象消失，其余各位点突变后融合现象仍然存在。结论G1上的134位点可能与蛋白正确折叠有密切关系，134位点突变极可能导致蛋白的错误折叠，进而无法转运出内质网。G2上的928位点对细胞融合有重要作用，提示汉坦病毒融合肽很有可能位于G2上。

Objective To determine the function of the N-linked glycosylation site of hantavirus GM04-38 in cell fusion preliminarily. Methods Site-directed mutagenesis was used to transform the aspara- gine on N-linked glycosylation site of hantavirus GM04-38 into alanine, according to the position of the sub- stitution, the mutants were named by N134A, N235A, N347A, N399A and N928A, which were then ex- pressed in Vero E6 cells. Western blot and IFA were performed to test the expression of glycosylation protein genes, and Giemsa staining was used to observe cell fusion activities. Results We expressed glycosylation protein successfully in Vero E6 cells. IFA showed the G1 and G2 gene were expressed efficiently, and the fluorescent signal was robust and concentrated in the perinuclear region of the transfected cells. Western blot showed all the mutants expressed G1 and G2 gene except the N134A mutant only expressed G2 gene. After mutation, cell fusion activities were not observed in the N134A and N928A mutant. Conclusion It is likely that N-linked glycosylation at 134 site is crucial in directing correct folding of G1, which made the mutant N134A was retained in the endoplasmic reticulum（ER） and could no longer be recognized by anti-G1 MeAbs. The mutation of the single site on G2 （N928） resulted in a loss of cell fusion, which suggests the 928 site on G2 is crucial for cell fusion and the fusion peptide is likely on G2.