副粘病毒F蛋白HR1和HR2在特异性膜融合中的作用

Function of HR1 and HR2 of F protein in the specific membrane fusion of paramyxoviruses

目的了解副粘病毒融合蛋白(F)分子上的七肽重复序列(HR1、HR2)在特异性膜融合中的作用。方法采用基因定点突变方法,在新城疫病毒(NDV)F与人副流感病毒(hPIV)F基因上创造相同的酶切位点。酶切后采用基因重组方法将F的HR1基因片段相互交换,得到交换HR1的两个嵌合体(chimera),即NDV C-HR1和hPIV C-HR1。用同样的方法又得到交换HR2的两个嵌合体,即NDV C-HR2和hPIV C-HR2。将各种嵌合体DNA与同源及异源HN DNA共转染BHK21细胞后,在真核细胞中表达。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F蛋白的表达效率。结果交换HR1后,NDV C-HR1的融合功能只有野毒株的53.91%,hPIV C-HR1的融合功能达到野毒株的83.15%;交换HR2后,NDV C-HR2的融合功能略高于野毒株,达到野毒株的107.23%,hPIV C-HR2的融合功能却只有野毒株的12.01%。FACS分析表明,交换HR1后的NDV C-HR1和hPIVC-HR1的表达效率均下降。交换HR2后,NDV C-HR2的表达效率没有改变,而hPIV C-HR2的表达效率下降。结论NDV F的HR1对其特异性膜融合具有重要作用,而HR2则没有病毒特异性;hPIV F的HR1和HR2对hPIV的特异性膜融合都有重要作用。

Objective To identify the effects of heptad repeat regions（HR1 and HR2） of fusion protein （F） on the specific membrane fusion of paramyxoviruses. Methods Site-directed mutagenesis was used to create the same enzyme sites on the F gene of Newcastle disease virus（NDV） and human parainfluenza virus（hPIV）. Gene recombination was used to get chimeric F proteins NDV C-HR1 and hPIV C-HR1 by exchanging HR1 ; NDV C-HR2 and hPIV C-HR2 were also obtained by the same technology. All the chimeric F proteins were co-expressed with their homologous or heterogenous hemagglutinin-neuraminidase（HN） in eukaryoeytes. The fusion profiles were assayed by Giemsa staining and reporter gene method; the expression efficiency of F protein was assayed by fluorescence-activated cell sorter（FACS）. Results NDV C-HR1 and hPIV C-HR1 had 53.91% and 83.15% of fusion activities, and NDV C-HR2 and hPIV C-HR2 had 107.23% and 12.01% of fusion activities respectively as compared with their relevant wild types. The FACS analysis indicated that the expression efficacy of all the chimeric F proteine except NDV C-HR2 was lower than those of their relevant wild types. Conclusion HR1 but not HR2 of NDV F was important for specific membrane fusion, both HR1 and HR2 of hPIV F were important for its specific membrane fusion.