副粘病毒融合蛋白活性位点中亮氨酸基因突变分析

Mutation Analysis of Leucines in the Active Domain of Paramyxovirus Fusion Protein

为了确定副粘病毒融合蛋白 (F)分子上活性位点中亮氨酸在F的细胞融合作用中的作用 ,弄清F融合细胞的分子机理 ,采用基因定点突变法创造一个酶切位点 ,用酶切反应初步筛选突变株 ,然后用DNA序列分析进一步确定 ,并在真核细胞内进行表达 ,Giemsa染色和指示基因法检测细胞融合功能 ,荧光强度分析 (FACS)检测表达效率。结果表明 ,hPIV3F第 46 0位亮氨酸 (L)和第 474位异亮氨酸 (I)分别突变成丙氨酸 (A) (L46 0A和I474A)时 ,细胞融合功能分别只相当于野毒株的 5 1 2 4%和 48 73% ;而第 46 7位L和第 481位L分别突变成A(L46 7A和L481A)时 ,细胞融合功能则无明显改变 ;当第 46 0和 46 7位L同时突变成A(L46 0A～L46 7A)时 ,细胞融合功能降低了79 13% ;而第 474位I和第 481位L同时突变成A(I474A～L481A)时 ,细胞融合功能无明显改变。NDVF第 481位L和第 488位L分别突变成A(L481A和L488A)时 ,细胞融合功能分别降低了 75 2 2 %和 80 0 4% ;将二者共同突变时 ,则进一步降低 ,只有野毒株的 10 13%。各突变株的表达效率没有改变。说明F分子上与HN相互作用的特异性区域中的亮氨酸在细胞融合中发挥着重要作用 ,即hPIV3F第 46 0位L和第 474位I,NDV第 481和 488位L ,突变后细胞融合作用不同程度下降。

To make sure the effect of leucines in the active domain of fusion protein (F)which interacts with homologous hemagglutinin neuraminidase(HN) of paramyxoviruses and to know the molecular mechanism of cell fusion, site directed mutagenesis was used to obtain mutants by creating a new enzyme site.The mutants were sequenced, and expressed in eukaryocytes. Their fusion activities were assayed by Giemsa staining, reporter gene assay and expression efficiency by fluorescence activated cell sorter(FACS) analysis. The results showed that hPIV3 F mutants of L460A and I474A had 51.24% and 48.73% of fusion activities as wild type, respectively, and L467A and L481A had the same as wild type; NDV F mutants of L481A and L488A had 24.78% and 19.96% of functions as wild type, respectively. Double mutants of L460A L467A had 20.87% of functions as wild type, while I474A L481A kept the same as I474A. Double mutant of NDV F L481A L488A had only 10.13% of activities as wild type. The expression efficiency of each mutant was the same by FACS analysis. These data proved that the leucines in the specific domain of hPIV3 F or NDV F for fusion play an important role in the cell fusion process. If they are mutated, the fusion activity of F will decrease for most of them, although some keep the same as wild type. Some double mutants of adjacent leucines will further decrease F fusion activity.